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    Thermo Fisher qrt pcr reactions
    Administration of IONIS-APOL1 Rx prevents IFN-γ–induced proteinuria. Female APOL1 G0– and G1–transgenic mice ( n = 3–4) were treated with 50 mg/kg IONIS-APOL1 Rx or control ASO 1 time per week for 4 weeks and challenged with a single dose of IFN-γ (1.125 × 10 7 U/kg) or vehicle (PBS). Study endpoints were evaluated 48 hours after IFN-γ challenge. ( A ) Urine was collected prior to sacrifice 48 hours after IFN-γ challenge, and urinary albumin was measured by ELISA and normalized to urine creatinine. ( B ) Kidney Irf1 expression was measured by <t>qRT-PCR</t> and normalized to Cyp expression. ( C ) Plasma ALT levels were measured using a clinical chemistry analyzer. ( D ) Kidney and ( E ) liver APOL1 expression were measured by qRT-PCR and normalized to Cyp expression. All data are presented as mean ± SD. Two-way ANOVA with Tukey’s multiple comparisons test, * P
    Qrt Pcr Reactions, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Antisense oligonucleotide treatment ameliorates IFN-γ–induced proteinuria in APOL1-transgenic mice"

    Article Title: Antisense oligonucleotide treatment ameliorates IFN-γ–induced proteinuria in APOL1-transgenic mice

    Journal: JCI Insight

    doi: 10.1172/jci.insight.126124

    Administration of IONIS-APOL1 Rx prevents IFN-γ–induced proteinuria. Female APOL1 G0– and G1–transgenic mice ( n = 3–4) were treated with 50 mg/kg IONIS-APOL1 Rx or control ASO 1 time per week for 4 weeks and challenged with a single dose of IFN-γ (1.125 × 10 7 U/kg) or vehicle (PBS). Study endpoints were evaluated 48 hours after IFN-γ challenge. ( A ) Urine was collected prior to sacrifice 48 hours after IFN-γ challenge, and urinary albumin was measured by ELISA and normalized to urine creatinine. ( B ) Kidney Irf1 expression was measured by qRT-PCR and normalized to Cyp expression. ( C ) Plasma ALT levels were measured using a clinical chemistry analyzer. ( D ) Kidney and ( E ) liver APOL1 expression were measured by qRT-PCR and normalized to Cyp expression. All data are presented as mean ± SD. Two-way ANOVA with Tukey’s multiple comparisons test, * P
    Figure Legend Snippet: Administration of IONIS-APOL1 Rx prevents IFN-γ–induced proteinuria. Female APOL1 G0– and G1–transgenic mice ( n = 3–4) were treated with 50 mg/kg IONIS-APOL1 Rx or control ASO 1 time per week for 4 weeks and challenged with a single dose of IFN-γ (1.125 × 10 7 U/kg) or vehicle (PBS). Study endpoints were evaluated 48 hours after IFN-γ challenge. ( A ) Urine was collected prior to sacrifice 48 hours after IFN-γ challenge, and urinary albumin was measured by ELISA and normalized to urine creatinine. ( B ) Kidney Irf1 expression was measured by qRT-PCR and normalized to Cyp expression. ( C ) Plasma ALT levels were measured using a clinical chemistry analyzer. ( D ) Kidney and ( E ) liver APOL1 expression were measured by qRT-PCR and normalized to Cyp expression. All data are presented as mean ± SD. Two-way ANOVA with Tukey’s multiple comparisons test, * P

    Techniques Used: Transgenic Assay, Mouse Assay, Allele-specific Oligonucleotide, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

    Hepatocyte-targeted IONIS-APOL1 Rx provides incomplete protection against IFN-γ–induced proteinuria. Female APOL1 G1–transgenic mice ( n = 4) were treated with IONIS-APOL1 Rx , hepatocyte-targeted IONIS-APOL1 Rx , or control ASO 1 time per week for 4 weeks and challenged with a single dose of IFN-γ (1.125 × 10 7 U/kg) or vehicle (PBS). Study endpoints were evaluated 48 hours after IFN-γ challenge. ( A ) Kidney and ( B ) liver APOL1 expression were measured by qRT-PCR and normalized to Cyp expression. ( C ) Urine was collected prior to sacrifice 48 hours after IFN-γ challenge, and urinary albumin was measured by ELISA and normalized to urine creatinine. ( D ) Urinary shed cells were collected from PBS-challenged mice, and APOL1 expression was measured by qRT-PCR and normalized to 36B4/Rplp0 expression. All data are presented as mean ± SD. Statistics were performed ( A – C ) by comparing each APOL1 ASO–treated PBS- or IFN-γ–challenged group to the respective control ASO group. Two-way ANOVA with Tukey’s multiple comparisons test, * P
    Figure Legend Snippet: Hepatocyte-targeted IONIS-APOL1 Rx provides incomplete protection against IFN-γ–induced proteinuria. Female APOL1 G1–transgenic mice ( n = 4) were treated with IONIS-APOL1 Rx , hepatocyte-targeted IONIS-APOL1 Rx , or control ASO 1 time per week for 4 weeks and challenged with a single dose of IFN-γ (1.125 × 10 7 U/kg) or vehicle (PBS). Study endpoints were evaluated 48 hours after IFN-γ challenge. ( A ) Kidney and ( B ) liver APOL1 expression were measured by qRT-PCR and normalized to Cyp expression. ( C ) Urine was collected prior to sacrifice 48 hours after IFN-γ challenge, and urinary albumin was measured by ELISA and normalized to urine creatinine. ( D ) Urinary shed cells were collected from PBS-challenged mice, and APOL1 expression was measured by qRT-PCR and normalized to 36B4/Rplp0 expression. All data are presented as mean ± SD. Statistics were performed ( A – C ) by comparing each APOL1 ASO–treated PBS- or IFN-γ–challenged group to the respective control ASO group. Two-way ANOVA with Tukey’s multiple comparisons test, * P

    Techniques Used: Transgenic Assay, Mouse Assay, Allele-specific Oligonucleotide, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    IFN-γ induces kidney APOL1 expression in APOL1 G0 and G1 mice, but proteinuria only in G1 mice. Female APOL1 G0– and G1–transgenic and WT littermate mice ( n = 3–4) were challenged with a single dose of IFN-γ (1.125 × 10 7 U/kg) or vehicle (PBS). ( A ) Urine was collected 24, 48, and 72 hours after IFN-γ challenge, and urinary albumin was measured by ELISA and normalized to urine creatinine. ( B ) Kidney Irf1 expression was measured by qRT-PCR 24 and 72 hours after IFN-γ challenge and normalized to Cyp expression. ( C ) Kidney and ( D ) liver APOL1 expression were measured by qRT-PCR 24 and 72 hours after IFN-γ challenge and normalized to Cyp expression. ( E ) Liver Irf1 expression was measured by qRT-PCR 24 and 72 hours after IFN-γ challenge and normalized to Cyp expression. All data are presented as mean ± SD. Two-way ANOVA with Tukey’s multiple comparisons test, *** P
    Figure Legend Snippet: IFN-γ induces kidney APOL1 expression in APOL1 G0 and G1 mice, but proteinuria only in G1 mice. Female APOL1 G0– and G1–transgenic and WT littermate mice ( n = 3–4) were challenged with a single dose of IFN-γ (1.125 × 10 7 U/kg) or vehicle (PBS). ( A ) Urine was collected 24, 48, and 72 hours after IFN-γ challenge, and urinary albumin was measured by ELISA and normalized to urine creatinine. ( B ) Kidney Irf1 expression was measured by qRT-PCR 24 and 72 hours after IFN-γ challenge and normalized to Cyp expression. ( C ) Kidney and ( D ) liver APOL1 expression were measured by qRT-PCR 24 and 72 hours after IFN-γ challenge and normalized to Cyp expression. ( E ) Liver Irf1 expression was measured by qRT-PCR 24 and 72 hours after IFN-γ challenge and normalized to Cyp expression. All data are presented as mean ± SD. Two-way ANOVA with Tukey’s multiple comparisons test, *** P

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

    IONIS-APOL1 Rx is an APOL1 ASO that reduces APOL1 mRNA levels in vitro and in vivo. ( A ) APOL1 expression in A431 cells in vitro ( n = 4) was measured by qRT-PCR after 72 hours free uptake of IONIS-APOL1 Rx or control ASO and normalized to GAPDH expression. IONIS-APOL1 Rx significantly reduced APOL1 expression compared with control ASO. ( B ) A431 cells were treated with IONIS-APOL1 Rx for 72 hours by free uptake ( n = 4) and expression of APOL1 , APOL2 , and APOL3 were measured by qRT-PCR followed by normalization to GAPDH expression. ( C and D ) APOL1 G0–transgenic mice were treated with IONIS-APOL1 Rx 1 time per week for 4 weeks and sacrificed 48 hours after the last dose ( n = 3–4). ( C ) Liver and kidney APOL1 expression was measured by qRT-PCR and normalized to Cyp expression. IONIS-APOL1 Rx significantly reduced APOL1 mRNA expression compared with PBS control. ( D ) Plasma APOL1 levels were measured by ELISA and are shown relative to PBS control levels. IONIS-APOL1 Rx significantly reduced plasma APOL1 compared with PBS control. All data are presented as mean ± SD. Two-way ANOVA with Bonferroni’s multiple comparisons test for A and one-way ANOVA with Dunnett’s multiple comparison’s test for C and D , * P
    Figure Legend Snippet: IONIS-APOL1 Rx is an APOL1 ASO that reduces APOL1 mRNA levels in vitro and in vivo. ( A ) APOL1 expression in A431 cells in vitro ( n = 4) was measured by qRT-PCR after 72 hours free uptake of IONIS-APOL1 Rx or control ASO and normalized to GAPDH expression. IONIS-APOL1 Rx significantly reduced APOL1 expression compared with control ASO. ( B ) A431 cells were treated with IONIS-APOL1 Rx for 72 hours by free uptake ( n = 4) and expression of APOL1 , APOL2 , and APOL3 were measured by qRT-PCR followed by normalization to GAPDH expression. ( C and D ) APOL1 G0–transgenic mice were treated with IONIS-APOL1 Rx 1 time per week for 4 weeks and sacrificed 48 hours after the last dose ( n = 3–4). ( C ) Liver and kidney APOL1 expression was measured by qRT-PCR and normalized to Cyp expression. IONIS-APOL1 Rx significantly reduced APOL1 mRNA expression compared with PBS control. ( D ) Plasma APOL1 levels were measured by ELISA and are shown relative to PBS control levels. IONIS-APOL1 Rx significantly reduced plasma APOL1 compared with PBS control. All data are presented as mean ± SD. Two-way ANOVA with Bonferroni’s multiple comparisons test for A and one-way ANOVA with Dunnett’s multiple comparison’s test for C and D , * P

    Techniques Used: Allele-specific Oligonucleotide, In Vitro, In Vivo, Expressing, Quantitative RT-PCR, Transgenic Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Genomic APOL1 -transgenic mice express APOL1 in similar tissues as those observed in humans and at similar relative physiological levels. ( A ) Schematic of the human APOL1 -containing fosmid fragment used to generate APOL1 G0– and G1–transgenic mice. Arrows mark the protein coding sequence location of the 2 point mutations that constitute the G1 genotype. ( B ) Relative APOL1 expression levels in livers, kidneys, hearts, and lungs of transgenic mice ( n = 2–4) were measured by qRT-PCR and normalized to total RNA. Data from 2 different founder lines are shown for each genotype. ( C ) APOL1 plasma levels in transgenic mice ( n = 4) and human pooled plasma ( n = 2) were measured by ELISA. ( D ) Urine albumin levels of APOL1 -transgenic and WT littermate mice ( n = 4–8) were measured by albumin ELISA and normalized to urine creatinine levels. ( E ) FISH was used to evaluate APOL1 mRNA expression in podocytes ( Nphs1 ), mesangial cells ( Des ), and endothelial cells ( Pecam ) in APOL1 G1 mouse kidneys ( n = 4). Representative images shown (scale bar: 20 μm). ( F ) Plasma ALT levels of APOL1 -transgenic and WT littermate mice ( n = 8) were measured using a clinical chemistry analyzer. Two-way ANOVA with Bonferroni’s multiple comparisons test, * P
    Figure Legend Snippet: Genomic APOL1 -transgenic mice express APOL1 in similar tissues as those observed in humans and at similar relative physiological levels. ( A ) Schematic of the human APOL1 -containing fosmid fragment used to generate APOL1 G0– and G1–transgenic mice. Arrows mark the protein coding sequence location of the 2 point mutations that constitute the G1 genotype. ( B ) Relative APOL1 expression levels in livers, kidneys, hearts, and lungs of transgenic mice ( n = 2–4) were measured by qRT-PCR and normalized to total RNA. Data from 2 different founder lines are shown for each genotype. ( C ) APOL1 plasma levels in transgenic mice ( n = 4) and human pooled plasma ( n = 2) were measured by ELISA. ( D ) Urine albumin levels of APOL1 -transgenic and WT littermate mice ( n = 4–8) were measured by albumin ELISA and normalized to urine creatinine levels. ( E ) FISH was used to evaluate APOL1 mRNA expression in podocytes ( Nphs1 ), mesangial cells ( Des ), and endothelial cells ( Pecam ) in APOL1 G1 mouse kidneys ( n = 4). Representative images shown (scale bar: 20 μm). ( F ) Plasma ALT levels of APOL1 -transgenic and WT littermate mice ( n = 8) were measured using a clinical chemistry analyzer. Two-way ANOVA with Bonferroni’s multiple comparisons test, * P

    Techniques Used: Transgenic Assay, Mouse Assay, Sequencing, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Fluorescence In Situ Hybridization

    2) Product Images from "Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells"

    Article Title: Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.79

    Inhibition of single miRNA has no effect on entinostat-mediated downregulation of erbB2 and erbB3 in breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells were transfected with either control miRNA inhibitor no. 1 or the indicated miRNA inhibitor (60 nmol/l each). After 24 h, the cells were then untreated or treated with entinostat (1.5 μ mol/l) for another 24 h. ( a ) Half of the cells were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-125a, miR-125b, and miR-205 were measured by qRT-PCR using TaqMan miRNA assays. RNU6B was used as an internal control. Bars , S.D. Data shows the representative of three independent experiments. ( b ) Another half of the cells were collected and subjected to western blot analyses with specific antibody directed against erbB2, erbB3, or β -actin
    Figure Legend Snippet: Inhibition of single miRNA has no effect on entinostat-mediated downregulation of erbB2 and erbB3 in breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells were transfected with either control miRNA inhibitor no. 1 or the indicated miRNA inhibitor (60 nmol/l each). After 24 h, the cells were then untreated or treated with entinostat (1.5 μ mol/l) for another 24 h. ( a ) Half of the cells were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-125a, miR-125b, and miR-205 were measured by qRT-PCR using TaqMan miRNA assays. RNU6B was used as an internal control. Bars , S.D. Data shows the representative of three independent experiments. ( b ) Another half of the cells were collected and subjected to western blot analyses with specific antibody directed against erbB2, erbB3, or β -actin

    Techniques Used: Inhibition, Multiple Displacement Amplification, Transfection, RNA Extraction, Expressing, Quantitative RT-PCR, Western Blot

    Simultaneous inhibition of two miRNAs reduces entinostat-mediated downregulation of erbB2 and erbB3 in breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells were transfected with combinations of either the two control miRNA inhibitors no. 1 and no. 2 or the two indicated miRNA inhibitors (60 nmol/l each). After 24 h, the cells were then untreated or treated with entinostat (1.5 μ mol/l) for another 24 h. ( a ) Half of the cells were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-125a, miR-125b, and miR-205 were measured by qRT-PCR using TaqMan miRNA assays. RNU6B was used as an internal control. ( b ) Another half of the cells were collected and subjected to western blot analyses with specific antibody directed against erbB2, erbB3, or β -actin. The bar graph underneath was obtained by densitometry analysis. The relative signal intensities of erbB2 or erbB3 were measured by the Bio-Rad Gel Documentation System. Bars, S.D. The data are representative of three independent experiments
    Figure Legend Snippet: Simultaneous inhibition of two miRNAs reduces entinostat-mediated downregulation of erbB2 and erbB3 in breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells were transfected with combinations of either the two control miRNA inhibitors no. 1 and no. 2 or the two indicated miRNA inhibitors (60 nmol/l each). After 24 h, the cells were then untreated or treated with entinostat (1.5 μ mol/l) for another 24 h. ( a ) Half of the cells were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-125a, miR-125b, and miR-205 were measured by qRT-PCR using TaqMan miRNA assays. RNU6B was used as an internal control. ( b ) Another half of the cells were collected and subjected to western blot analyses with specific antibody directed against erbB2, erbB3, or β -actin. The bar graph underneath was obtained by densitometry analysis. The relative signal intensities of erbB2 or erbB3 were measured by the Bio-Rad Gel Documentation System. Bars, S.D. The data are representative of three independent experiments

    Techniques Used: Inhibition, Multiple Displacement Amplification, Transfection, RNA Extraction, Expressing, Quantitative RT-PCR, Western Blot

    SAHA and panobinostat inhibit proliferation and induce apoptosis in erbB2-overexpressing breast cancer cells associated with the reduction of mRNA levels and protein expression of erbB2/erbB3 . ( a ) MDA-MB-453 or BT474 cells were plated onto 96-well plates. After 24 h incubation, cells were grown in either control medium, or the same medium containing indicated concentrations of SAHA or panobinostat for another 72 h. The percentages of surviving cells relative to controls, defined as 100% survival, were determined by reduction of MTS. Bars, S.D. The data are representative of three independent experiments. ( b ) MDA-MB-453 cells were treated with SAHA (300 nmol/l) or panobinostat (8 nmol/l) for 24 h. BT474 cells were treated with SAHA (1.5 μ mol/l) or panobinostat (20 nmol/l) for 24 h. All cells were collected and subjected to western blot analyses with specific antibody directed against erbB2, erbB3, PARP, or β -actin (top) and a specific apoptotic ELISA (bottom). Bars, S.D. The data are representative of three independent experiments. ( c ) MDA-MB-453 or BT474 cells were treated as described in ( b ) for 16 h. All cells were collected and subjected to total RNA extraction. First-strand cDNA was synthesized using a reverse transcription kit from Applied Biosystems. The mRNA levels of erbB2 and erbB3 were measured by qRT-PCR. Bars, S.D. The data are representative of three independent experiments. * P
    Figure Legend Snippet: SAHA and panobinostat inhibit proliferation and induce apoptosis in erbB2-overexpressing breast cancer cells associated with the reduction of mRNA levels and protein expression of erbB2/erbB3 . ( a ) MDA-MB-453 or BT474 cells were plated onto 96-well plates. After 24 h incubation, cells were grown in either control medium, or the same medium containing indicated concentrations of SAHA or panobinostat for another 72 h. The percentages of surviving cells relative to controls, defined as 100% survival, were determined by reduction of MTS. Bars, S.D. The data are representative of three independent experiments. ( b ) MDA-MB-453 cells were treated with SAHA (300 nmol/l) or panobinostat (8 nmol/l) for 24 h. BT474 cells were treated with SAHA (1.5 μ mol/l) or panobinostat (20 nmol/l) for 24 h. All cells were collected and subjected to western blot analyses with specific antibody directed against erbB2, erbB3, PARP, or β -actin (top) and a specific apoptotic ELISA (bottom). Bars, S.D. The data are representative of three independent experiments. ( c ) MDA-MB-453 or BT474 cells were treated as described in ( b ) for 16 h. All cells were collected and subjected to total RNA extraction. First-strand cDNA was synthesized using a reverse transcription kit from Applied Biosystems. The mRNA levels of erbB2 and erbB3 were measured by qRT-PCR. Bars, S.D. The data are representative of three independent experiments. * P

    Techniques Used: Expressing, Multiple Displacement Amplification, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, RNA Extraction, Synthesized, Quantitative RT-PCR

    Entinostat upregulates the expression levels of miR-125a, miR-125b, and miR-205 in erbB2-overexpressing breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells untreated or treated with entinostat (0.5 and 2 μ mol/l, respectively) for 8, 16, 24 h were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-125a, miR-125b, and miR-205 were measured by qRT-PCR using TaqMan miRNA assays. All results were normalized with the internal control RNU6B. Bars, S.D. The data are representative of three independent experiments
    Figure Legend Snippet: Entinostat upregulates the expression levels of miR-125a, miR-125b, and miR-205 in erbB2-overexpressing breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells untreated or treated with entinostat (0.5 and 2 μ mol/l, respectively) for 8, 16, 24 h were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-125a, miR-125b, and miR-205 were measured by qRT-PCR using TaqMan miRNA assays. All results were normalized with the internal control RNU6B. Bars, S.D. The data are representative of three independent experiments

    Techniques Used: Expressing, Multiple Displacement Amplification, RNA Extraction, Quantitative RT-PCR

    Treatment with entinostat does not affect mRNA levels of both erbB2 and erbB3 in breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells untreated or treated with entinostat (ent) at indicated concentrations for 24 h were subjected to total RNA extraction. ( a ) First-strand cDNA was synthesized using a reverse transcription kit from Applied Biosystems. The partial coding sequence of erbB2 , erbB3 , or β-actin was amplified with specific primers. The PCR products were separated on a 2% agarose gel containing ethidium bromide and visualized under a UV light. ( b ) The mRNA levels of erbB2 and erbB3 were measured by qRT-PCR. Bars, S.D. The data are representative of three independent experiments
    Figure Legend Snippet: Treatment with entinostat does not affect mRNA levels of both erbB2 and erbB3 in breast cancer cells. MDA-MB-453 (MDA-453) and BT474 cells untreated or treated with entinostat (ent) at indicated concentrations for 24 h were subjected to total RNA extraction. ( a ) First-strand cDNA was synthesized using a reverse transcription kit from Applied Biosystems. The partial coding sequence of erbB2 , erbB3 , or β-actin was amplified with specific primers. The PCR products were separated on a 2% agarose gel containing ethidium bromide and visualized under a UV light. ( b ) The mRNA levels of erbB2 and erbB3 were measured by qRT-PCR. Bars, S.D. The data are representative of three independent experiments

    Techniques Used: Multiple Displacement Amplification, RNA Extraction, Synthesized, Sequencing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR

    3) Product Images from "Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis"

    Article Title: Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-14-136

    Effects of heat on the alternative splicing of the clock genes. Ten-day-old Col-0 plants grown on MS-agar plates under LDs were transferred to 37°C under continuous light conditions. Whole plant materials were harvested at the indicated ZT points. The levels of the RNA splice variants of CCA1 (A) , PRR7 (B) , PRR9 (C) , TOC1 (D) , ELF3 (E) , and ZTL (F) genes were determined by qRT-PCR. Biological triplicates were averaged. Bars indicate the standard error of the mean. Note that the expression data at 23°C are identical to those in Figure 7 .
    Figure Legend Snippet: Effects of heat on the alternative splicing of the clock genes. Ten-day-old Col-0 plants grown on MS-agar plates under LDs were transferred to 37°C under continuous light conditions. Whole plant materials were harvested at the indicated ZT points. The levels of the RNA splice variants of CCA1 (A) , PRR7 (B) , PRR9 (C) , TOC1 (D) , ELF3 (E) , and ZTL (F) genes were determined by qRT-PCR. Biological triplicates were averaged. Bars indicate the standard error of the mean. Note that the expression data at 23°C are identical to those in Figure 7 .

    Techniques Used: Mass Spectrometry, Quantitative RT-PCR, Expressing

    Effects of low temperatures on the alternative splicing of the clock genes. Ten-day-old Col-0 plants grown on MS-agar plates under LDs were transferred to 4°C under continuous light conditions. Whole plant materials were harvested at the indicated ZT points. The levels of the RNA splice variants of CCA1 (A) , PRR7 (B) , PRR9 (C) , TOC1 (D) , ELF3 (E) , and ZTL (F) genes were determined by qRT-PCR. Biological triplicates were averaged. Bars indicate the standard error of the mean.
    Figure Legend Snippet: Effects of low temperatures on the alternative splicing of the clock genes. Ten-day-old Col-0 plants grown on MS-agar plates under LDs were transferred to 4°C under continuous light conditions. Whole plant materials were harvested at the indicated ZT points. The levels of the RNA splice variants of CCA1 (A) , PRR7 (B) , PRR9 (C) , TOC1 (D) , ELF3 (E) , and ZTL (F) genes were determined by qRT-PCR. Biological triplicates were averaged. Bars indicate the standard error of the mean.

    Techniques Used: Mass Spectrometry, Quantitative RT-PCR

    Effects of high salinity on the alternative splicing of the clock genes. Ten-day-old Col-0 plants grown on MS-agar plates under LDs were transferred to hydroponic MS medium containing 200 mM NaCl. The levels of the RNA splice variants of CCA1 (A) , PRR7 (B) , PRR9 (C) , TOC1 (D) , ELF3 (E) , and ZTL (F) genes were determined by qRT-PCR. Biological triplicates were averaged. Bars indicate the standard error of the mean.
    Figure Legend Snippet: Effects of high salinity on the alternative splicing of the clock genes. Ten-day-old Col-0 plants grown on MS-agar plates under LDs were transferred to hydroponic MS medium containing 200 mM NaCl. The levels of the RNA splice variants of CCA1 (A) , PRR7 (B) , PRR9 (C) , TOC1 (D) , ELF3 (E) , and ZTL (F) genes were determined by qRT-PCR. Biological triplicates were averaged. Bars indicate the standard error of the mean.

    Techniques Used: Mass Spectrometry, Quantitative RT-PCR

    The fates of RNA splice variants. Steady-state levels of the β transcripts were determined by qRT-PCR in Col-0 plants after CHX treatments (left panels) and in the upf1 - 5 and upf3 - 1 mutants (right panels). Biological triplicates were averaged and statistically treated using Student t -test (* P
    Figure Legend Snippet: The fates of RNA splice variants. Steady-state levels of the β transcripts were determined by qRT-PCR in Col-0 plants after CHX treatments (left panels) and in the upf1 - 5 and upf3 - 1 mutants (right panels). Biological triplicates were averaged and statistically treated using Student t -test (* P

    Techniques Used: Quantitative RT-PCR

    Effects of photoperiod on the alternative splicing of the clock genes. Ten-day-old Col-0 plants grown on MS-agar plates under either LDs or SDs were harvested at the indicated ZT points for the extraction of total RNA samples. The levels of the RNA splice variants of CCA1 (A) , PRR7 (B) , PRR9 (C) , TOC1 (D) , ELF3 (E) , and ZTL (F) genes were determined by qRT-PCR. Biological triplicates were averaged. Bars indicate the standard error of the mean.
    Figure Legend Snippet: Effects of photoperiod on the alternative splicing of the clock genes. Ten-day-old Col-0 plants grown on MS-agar plates under either LDs or SDs were harvested at the indicated ZT points for the extraction of total RNA samples. The levels of the RNA splice variants of CCA1 (A) , PRR7 (B) , PRR9 (C) , TOC1 (D) , ELF3 (E) , and ZTL (F) genes were determined by qRT-PCR. Biological triplicates were averaged. Bars indicate the standard error of the mean.

    Techniques Used: Mass Spectrometry, Quantitative RT-PCR

    4) Product Images from "A novel mechanism for the transcriptional regulation of Wnt signaling in development"

    Article Title: A novel mechanism for the transcriptional regulation of Wnt signaling in development

    Journal: Genes & Development

    doi: 10.1101/gad.17227011

    Vax2-dependent expression of dnTcf7l2 and transcriptional corepressors. ( A ) In situ hybridization reveals that the Vax2 ( left ) and dnTcf7l2 1b ( middle ) mRNAs are coexpressed in the wild-type ventral retina at E13.5. ( Right ) dnTcf7l2 expression is lost in this region in the Vax2 −/− mutant retina. (D) Dorsal; (V) ventral. ( B ) Truncated Tcf7l2 mRNAs 1b and 1c are down-regulated in Vax2 −/− eyes, as determined by qRT–PCR. Expression of the full-length Tcf7l2 mRNA is unchanged, as determined using primers spanning exons 1–3 and exons 5–6. Fold change is the ratio between the relative mRNA expression normalized to Gapdh in the Vax2 −/− mutant versus wild-type eyes. ( C ) Tle1 , Hdac2 , Gsk3β , Apc , and Ctbp1 mRNAs are expressed in the wild-type (WT) retina at E13.5, with strongest expression in the RGC layer, as shown by in situ hybridization. Their expression is lost in the Vax2 −/− ventral retina. (D) Dorsal; (V) ventral. ( D , left , top ) Wnt signaling is not active in the E13.5 wild-type retina, except in the dorsal RPE, as shown by X-Gal staining of retinal sections from a BATgal reporter mouse. ( Left , bottom ) The BATgal reporter is derepressed in the ventral neural retina of a Vax2 −/− /BATgal mouse. A schematic of the BATgal reporter transgene containing seven Tcf/Lef consensus binding sites fused to the siamois promoter and the lacZ gene (Maretto et al. 2003) in repressed ( right , top ) and activated ( right , bottom ) states. ( E ) Vax2 binds to the potential regulatory regions of the Wnt antagonists (A–E), as shown by ChIP analysis. Gel panels containing ChIP-PCR products are turned 90° from running direction, as indicated by the arrow. Candidate regulatory regions A–E are located at the indicated positions and contain the indicated clusters of potential Vax2 (homeodomain)-binding sites. A′–E′ indicate negative control regions surrounding each gene.
    Figure Legend Snippet: Vax2-dependent expression of dnTcf7l2 and transcriptional corepressors. ( A ) In situ hybridization reveals that the Vax2 ( left ) and dnTcf7l2 1b ( middle ) mRNAs are coexpressed in the wild-type ventral retina at E13.5. ( Right ) dnTcf7l2 expression is lost in this region in the Vax2 −/− mutant retina. (D) Dorsal; (V) ventral. ( B ) Truncated Tcf7l2 mRNAs 1b and 1c are down-regulated in Vax2 −/− eyes, as determined by qRT–PCR. Expression of the full-length Tcf7l2 mRNA is unchanged, as determined using primers spanning exons 1–3 and exons 5–6. Fold change is the ratio between the relative mRNA expression normalized to Gapdh in the Vax2 −/− mutant versus wild-type eyes. ( C ) Tle1 , Hdac2 , Gsk3β , Apc , and Ctbp1 mRNAs are expressed in the wild-type (WT) retina at E13.5, with strongest expression in the RGC layer, as shown by in situ hybridization. Their expression is lost in the Vax2 −/− ventral retina. (D) Dorsal; (V) ventral. ( D , left , top ) Wnt signaling is not active in the E13.5 wild-type retina, except in the dorsal RPE, as shown by X-Gal staining of retinal sections from a BATgal reporter mouse. ( Left , bottom ) The BATgal reporter is derepressed in the ventral neural retina of a Vax2 −/− /BATgal mouse. A schematic of the BATgal reporter transgene containing seven Tcf/Lef consensus binding sites fused to the siamois promoter and the lacZ gene (Maretto et al. 2003) in repressed ( right , top ) and activated ( right , bottom ) states. ( E ) Vax2 binds to the potential regulatory regions of the Wnt antagonists (A–E), as shown by ChIP analysis. Gel panels containing ChIP-PCR products are turned 90° from running direction, as indicated by the arrow. Candidate regulatory regions A–E are located at the indicated positions and contain the indicated clusters of potential Vax2 (homeodomain)-binding sites. A′–E′ indicate negative control regions surrounding each gene.

    Techniques Used: Expressing, In Situ Hybridization, Mutagenesis, Quantitative RT-PCR, Staining, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control

    Vax2 activates endogenous dnTCF7L2 and represses Wnt signaling. ( A ) Expression of full-length TCF7L2 mRNA (exons 5–6) is unaffected by overexpressing mouse Vax2 in HEK293 cells, as determined by qRT–PCR. In contrast, the truncated TCF7L2 mRNAs 1b and 1e are up-regulated by full-length Vax2, but not by Vax2 lacking the activation SAFEPY motif (Vax2ΔAD). Fold change is the ratio between expression normalized to GAPDH in cells transfected with pCS2-Vax2 versus cells transfected with empty pCS2 vector. Error bars are mean ± SD ( n = 3). ( B ) Vax2 up-regulates the enhancer activity of the F1R1 and F2R2 Vax2-bound regions in a luciferase reporter assay. The Vista Browser plot shows sequence conservation of the dnTcf7l2 regulatory regions between humans and chickens. The alternative Tcf7l2 first exons 1b and 1c lie immediately downstream from the Vax2-bound regions (blue squares). Fold induction is the ratio between the normalized luciferase activity of the insert containing reporter construct activated with Vax2 and that activated with empty expression vector. Error bars are mean ± SD ( n = 3). ( C ) Both dnTcf7l2 and Vax2 are strong repressors of β-catenin activation of Wnt targets. While full-length Vax2 is able to repress the β-catenin-activated Wnt reporter with a potency comparable with dnTcf7l2, Vax2 lacking the SAFEPY activation motif (Vax2ΔAD) is not. Fold induction is the ratio between the normalized luciferase activity of the TOPflash and FOPflash reporter constructs. Error bars are mean ± SD ( n = 3). ( D ) Vax2 is unable to repress an activated Notch-responsive CBF1-pGL2 reporter containing eight CBF1-binding sites. The reporter was activated by overexpressing the Notch intracellular domain (NICD). Fold induction is the ratio between the normalized luciferase activity of the wild-type and mutated reporter. Error bars are mean ± SD ( n = 3).
    Figure Legend Snippet: Vax2 activates endogenous dnTCF7L2 and represses Wnt signaling. ( A ) Expression of full-length TCF7L2 mRNA (exons 5–6) is unaffected by overexpressing mouse Vax2 in HEK293 cells, as determined by qRT–PCR. In contrast, the truncated TCF7L2 mRNAs 1b and 1e are up-regulated by full-length Vax2, but not by Vax2 lacking the activation SAFEPY motif (Vax2ΔAD). Fold change is the ratio between expression normalized to GAPDH in cells transfected with pCS2-Vax2 versus cells transfected with empty pCS2 vector. Error bars are mean ± SD ( n = 3). ( B ) Vax2 up-regulates the enhancer activity of the F1R1 and F2R2 Vax2-bound regions in a luciferase reporter assay. The Vista Browser plot shows sequence conservation of the dnTcf7l2 regulatory regions between humans and chickens. The alternative Tcf7l2 first exons 1b and 1c lie immediately downstream from the Vax2-bound regions (blue squares). Fold induction is the ratio between the normalized luciferase activity of the insert containing reporter construct activated with Vax2 and that activated with empty expression vector. Error bars are mean ± SD ( n = 3). ( C ) Both dnTcf7l2 and Vax2 are strong repressors of β-catenin activation of Wnt targets. While full-length Vax2 is able to repress the β-catenin-activated Wnt reporter with a potency comparable with dnTcf7l2, Vax2 lacking the SAFEPY activation motif (Vax2ΔAD) is not. Fold induction is the ratio between the normalized luciferase activity of the TOPflash and FOPflash reporter constructs. Error bars are mean ± SD ( n = 3). ( D ) Vax2 is unable to repress an activated Notch-responsive CBF1-pGL2 reporter containing eight CBF1-binding sites. The reporter was activated by overexpressing the Notch intracellular domain (NICD). Fold induction is the ratio between the normalized luciferase activity of the wild-type and mutated reporter. Error bars are mean ± SD ( n = 3).

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

    Truncated Tcf7l2 mRNAs originate in Tcf7l2 intron 5. ( A ) 5′ RACE reveals the existence of four alternative Tcf7l2 first exons in Tcf7l2 intron 5. Exons 1–17 of the mouse Tcf7l2 ). Alternative exons 4, 13, 14, 15, and 16 are marked with asterisks. Alternative first exons of the truncated Tcf7l2 mRNAs are shown in blue. Black arrows depict primers used in the qRT–PCR experiments, and blue arrows depict alternative TSSs for exons 1b–e. ( B ) qRT–PCR analysis of the expression of the alternative Tcf7l2 mRNAs 1b, 1c, 1d, and 1e during embryogenesis, relative to Hprt. Error bars are mean ± SD ( n = 3). ( C ) Northern analysis of Tcf7l2 mRNAs at E13.5. ( Left two lanes) A probe against exons 1–3 detects only the full-length Tcf7l2 mRNAs (∼4.1 kb) in both the E13.5 head and body. ( Right two lanes) A probe against exons 6–10 detects the full-length mRNAs in both the head and body, and also the truncated Tcf7l2 mRNAs (∼3.4 kb, arrow), but only in the E13.5 head. ( D ) Whole-mount in situ hybridization with a probe against exon 1b reveals a strong diencephalic expression (D) of the alternative Tcf7l2 mRNA 1b at E11.5. ( E ) In situ hybridization on a coronal section through an E13.5 head reveals the expression of the Tcf7l2 mRNA isoform 1b in ganglion cells of the developing retina (RGC), the optic stalk (OS), and diencephalon (D).
    Figure Legend Snippet: Truncated Tcf7l2 mRNAs originate in Tcf7l2 intron 5. ( A ) 5′ RACE reveals the existence of four alternative Tcf7l2 first exons in Tcf7l2 intron 5. Exons 1–17 of the mouse Tcf7l2 ). Alternative exons 4, 13, 14, 15, and 16 are marked with asterisks. Alternative first exons of the truncated Tcf7l2 mRNAs are shown in blue. Black arrows depict primers used in the qRT–PCR experiments, and blue arrows depict alternative TSSs for exons 1b–e. ( B ) qRT–PCR analysis of the expression of the alternative Tcf7l2 mRNAs 1b, 1c, 1d, and 1e during embryogenesis, relative to Hprt. Error bars are mean ± SD ( n = 3). ( C ) Northern analysis of Tcf7l2 mRNAs at E13.5. ( Left two lanes) A probe against exons 1–3 detects only the full-length Tcf7l2 mRNAs (∼4.1 kb) in both the E13.5 head and body. ( Right two lanes) A probe against exons 6–10 detects the full-length mRNAs in both the head and body, and also the truncated Tcf7l2 mRNAs (∼3.4 kb, arrow), but only in the E13.5 head. ( D ) Whole-mount in situ hybridization with a probe against exon 1b reveals a strong diencephalic expression (D) of the alternative Tcf7l2 mRNA 1b at E11.5. ( E ) In situ hybridization on a coronal section through an E13.5 head reveals the expression of the Tcf7l2 mRNA isoform 1b in ganglion cells of the developing retina (RGC), the optic stalk (OS), and diencephalon (D).

    Techniques Used: Quantitative RT-PCR, Expressing, Northern Blot, In Situ Hybridization

    DnTcf7l2 is essential for Xenopus forebrain development. ( A ) A 5′ RACE experiment in X. laevis reveals the existence of an alternative XTcf7l2 first exon in intron 5, which contains an ATG start codon in-frame with Ser 180 in XTcf7l2 exon 6. The positions of the translation-blocking (MO-ATG) and splicing-blocking (MO-SPL) morpholinos are indicated. Arrows represent the primers used for qRT–PCR. ( B ) In situ hybridization analysis of dnTcf7l2 expression at stages 13, 15, and 28 of X. laevis development. (A) Anterior; (P) posterior; (D) dorsal; (V) ventral; (bp) blastopore. ( C ) Knockdown of XdnTcf7l2 with MO-ATG or MO-SPL leads to embryos with severely truncated anterior head regions at stage 40. Phenotype ranges from moderate (gray bars), showing small head and small eyes, to strong (black bars), where no head and no eyes are present.
    Figure Legend Snippet: DnTcf7l2 is essential for Xenopus forebrain development. ( A ) A 5′ RACE experiment in X. laevis reveals the existence of an alternative XTcf7l2 first exon in intron 5, which contains an ATG start codon in-frame with Ser 180 in XTcf7l2 exon 6. The positions of the translation-blocking (MO-ATG) and splicing-blocking (MO-SPL) morpholinos are indicated. Arrows represent the primers used for qRT–PCR. ( B ) In situ hybridization analysis of dnTcf7l2 expression at stages 13, 15, and 28 of X. laevis development. (A) Anterior; (P) posterior; (D) dorsal; (V) ventral; (bp) blastopore. ( C ) Knockdown of XdnTcf7l2 with MO-ATG or MO-SPL leads to embryos with severely truncated anterior head regions at stage 40. Phenotype ranges from moderate (gray bars), showing small head and small eyes, to strong (black bars), where no head and no eyes are present.

    Techniques Used: Blocking Assay, Quantitative RT-PCR, In Situ Hybridization, Expressing

    5) Product Images from "TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis"

    Article Title: TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis

    Journal: Cell & Bioscience

    doi: 10.1186/s13578-018-0221-7

    TP53TG1 regulated PTEN expression through miR-18a in NSCLC cells. a PTEN expression was assessed by western blot in A549 cells transfected with miR-18a mimics or anti-miR-18a. b Western blot assay of PTEN expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1. Dual-luciferase reporter assay was performed by transfecting PTEN-WT vector into A549 cells together with miR-18 mimics or miR-18a mimics + pcDNA-TP53TG1 ( c ), and anti-miR-18a or anti-miR-18a + si-TP53TG1#1 ( d ). e qRT-PCR assay of PTEN expression in 40 pairs of NSCLC tumor samples. f qRT-PCR assay of PTEN expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. g The correlation analysis between TP53TG1 and PTEN expression in NSCLC tumor specimen. Each experiment is repeated three times. * P
    Figure Legend Snippet: TP53TG1 regulated PTEN expression through miR-18a in NSCLC cells. a PTEN expression was assessed by western blot in A549 cells transfected with miR-18a mimics or anti-miR-18a. b Western blot assay of PTEN expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1. Dual-luciferase reporter assay was performed by transfecting PTEN-WT vector into A549 cells together with miR-18 mimics or miR-18a mimics + pcDNA-TP53TG1 ( c ), and anti-miR-18a or anti-miR-18a + si-TP53TG1#1 ( d ). e qRT-PCR assay of PTEN expression in 40 pairs of NSCLC tumor samples. f qRT-PCR assay of PTEN expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. g The correlation analysis between TP53TG1 and PTEN expression in NSCLC tumor specimen. Each experiment is repeated three times. * P

    Techniques Used: Expressing, Western Blot, Transfection, Luciferase, Reporter Assay, Plasmid Preparation, Quantitative RT-PCR

    Overexpression of TP53TG1 sensitized NSCLC cells to cisplatin in vivo. About 2.0 × 10 7 SUNE2 cells stably transfected with lenti-control or lenti-TP53TG1 were subcutaneously inoculated into the nude mice, followed by intraperitoneal injection of PBS or cisplatin. Mice were euthanized to remove tumor masses at 32 days after inoculation. a The tumor volumes were measured with a caliper at indicated time points. b The representative photographs and average weights of resected tumors. c qRT-PCR analysis of TP53TG1, miR-18a and PTEN mRNA levels in excised tumor tissues. d Western blot assay of PTEN and cleaved caspase-3 levels in excised tumor tissues. Each experiment is repeated three times. * P
    Figure Legend Snippet: Overexpression of TP53TG1 sensitized NSCLC cells to cisplatin in vivo. About 2.0 × 10 7 SUNE2 cells stably transfected with lenti-control or lenti-TP53TG1 were subcutaneously inoculated into the nude mice, followed by intraperitoneal injection of PBS or cisplatin. Mice were euthanized to remove tumor masses at 32 days after inoculation. a The tumor volumes were measured with a caliper at indicated time points. b The representative photographs and average weights of resected tumors. c qRT-PCR analysis of TP53TG1, miR-18a and PTEN mRNA levels in excised tumor tissues. d Western blot assay of PTEN and cleaved caspase-3 levels in excised tumor tissues. Each experiment is repeated three times. * P

    Techniques Used: Over Expression, In Vivo, Stable Transfection, Transfection, Mouse Assay, Injection, Quantitative RT-PCR, Western Blot

    TP53TG1 was associated with cisplatin sensitivity in NSCLC cells. a A549 and A549/DDP cells were exposed to different concentrations of cisplatin (1, 10, 20, 40, 80, 160 μM) for 48 h, followed by the determination of cell viability and the calculation of IC50 of cisplatin by MTT assay. b A549/DDP cells were transfected with pcDNA-TP53TG1 and A549 cells were introduced with two individual TP53TG1 siRNAs (si-TP53TG1#1 and si-TP53TG1#2), followed by the detection of TP53TG1 expression by qRT-PCR assay. c pcDNA-TP53TG1-transfected A549/DDP cells were treated with various concentrations of cisplatin for 48 h, and IC50 of cisplatin and cell proliferation capacity were measured by MTT. d si-TP53TG1#1- or si-TP53TG1#2-transfected A549 cells were treated with different doses of cisplatin for 48 h, and IC50 of cisplatin and cell proliferation capacity were monitored by MTT. e Cell apoptosis was evaluated by flow cytometry in pcDNA-TP53TG1-transfected A549/DDP cells after exposed to 60 μM of cisplatin for 48 h. f The apoptotic rate was analyzed by flow cytometry in si-TP53TG1#1- or si-TP53TG1#2-transfected A549 cells after treated with 20 μM of cisplatin for 48 h. Each experiment is repeated three times. * P
    Figure Legend Snippet: TP53TG1 was associated with cisplatin sensitivity in NSCLC cells. a A549 and A549/DDP cells were exposed to different concentrations of cisplatin (1, 10, 20, 40, 80, 160 μM) for 48 h, followed by the determination of cell viability and the calculation of IC50 of cisplatin by MTT assay. b A549/DDP cells were transfected with pcDNA-TP53TG1 and A549 cells were introduced with two individual TP53TG1 siRNAs (si-TP53TG1#1 and si-TP53TG1#2), followed by the detection of TP53TG1 expression by qRT-PCR assay. c pcDNA-TP53TG1-transfected A549/DDP cells were treated with various concentrations of cisplatin for 48 h, and IC50 of cisplatin and cell proliferation capacity were measured by MTT. d si-TP53TG1#1- or si-TP53TG1#2-transfected A549 cells were treated with different doses of cisplatin for 48 h, and IC50 of cisplatin and cell proliferation capacity were monitored by MTT. e Cell apoptosis was evaluated by flow cytometry in pcDNA-TP53TG1-transfected A549/DDP cells after exposed to 60 μM of cisplatin for 48 h. f The apoptotic rate was analyzed by flow cytometry in si-TP53TG1#1- or si-TP53TG1#2-transfected A549 cells after treated with 20 μM of cisplatin for 48 h. Each experiment is repeated three times. * P

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

    TP53TG1-induced cisplatin sensitivity of NSCLC cells was decreased following miR-18a upregulation. A549/DDP cells were transfected with pcDNA-TP53TG1 alone or together with miR-18a mimics, followed by qRT-PCR assay of miR-18a expression ( a ), MTT analysis of IC50 of cisplatin ( c ) and flow cytometry analysis of apoptotic rate ( e ). A549 cells were introduced with si-TP53TG1#1 alone or together with anti-miR-18a, followed by measurement of miR-18a expression by qRT-PCR ( b ), determination of IC50 of cisplatin by MTT ( d ), detection of apoptotic rate by flow cytometry ( f ). Each experiment is repeated three times. * P
    Figure Legend Snippet: TP53TG1-induced cisplatin sensitivity of NSCLC cells was decreased following miR-18a upregulation. A549/DDP cells were transfected with pcDNA-TP53TG1 alone or together with miR-18a mimics, followed by qRT-PCR assay of miR-18a expression ( a ), MTT analysis of IC50 of cisplatin ( c ) and flow cytometry analysis of apoptotic rate ( e ). A549 cells were introduced with si-TP53TG1#1 alone or together with anti-miR-18a, followed by measurement of miR-18a expression by qRT-PCR ( b ), determination of IC50 of cisplatin by MTT ( d ), detection of apoptotic rate by flow cytometry ( f ). Each experiment is repeated three times. * P

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

    TP53TG1 inhibited miR-18a expression in NSCLC cells. a Sequence alignment of miR-18a with the putative binding sites within the wild-type regions of TP53TG1. b Subcellular fractionation assay was performed to identify the subcellular location of TP53TG1 with GAPDH and U6 as internal references. c , d The luciferase activity was detected in A549 cells transfected with TP53TG1-WT or TP53TG1-MUT and miR-con, miR-18a mimics, anti-miR-con or anti-miR-18a. e Biotin-labeled TP53TG1 RNA was obtained and added to cell lysates with Streptavidin agarose beads, followed by the detection of miR-18a enrichment by RNA pull-down assay. f RIP assay was performed to evaluate the endogenous binding between TP53TG1 and miR-18a in A549 cells using specific antibody against Ago2, followed by detection of RNA levels by qRT-PCR. g qRT-PCR assay of miR-18a expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1 for 48 h. h qRT-PCR assay of miR-18a expression in 40 pairs of NSCLC samples. i qRT-PCR assay of miR-18a expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. j The correlation between TP53TG1 and miR-18a expression was detected in NSCLC samples. All experiments are repeated three times. * P
    Figure Legend Snippet: TP53TG1 inhibited miR-18a expression in NSCLC cells. a Sequence alignment of miR-18a with the putative binding sites within the wild-type regions of TP53TG1. b Subcellular fractionation assay was performed to identify the subcellular location of TP53TG1 with GAPDH and U6 as internal references. c , d The luciferase activity was detected in A549 cells transfected with TP53TG1-WT or TP53TG1-MUT and miR-con, miR-18a mimics, anti-miR-con or anti-miR-18a. e Biotin-labeled TP53TG1 RNA was obtained and added to cell lysates with Streptavidin agarose beads, followed by the detection of miR-18a enrichment by RNA pull-down assay. f RIP assay was performed to evaluate the endogenous binding between TP53TG1 and miR-18a in A549 cells using specific antibody against Ago2, followed by detection of RNA levels by qRT-PCR. g qRT-PCR assay of miR-18a expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1 for 48 h. h qRT-PCR assay of miR-18a expression in 40 pairs of NSCLC samples. i qRT-PCR assay of miR-18a expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. j The correlation between TP53TG1 and miR-18a expression was detected in NSCLC samples. All experiments are repeated three times. * P

    Techniques Used: Expressing, Sequencing, Binding Assay, Fractionation, Luciferase, Activity Assay, Transfection, Labeling, Pull Down Assay, Quantitative RT-PCR

    6) Product Images from "Survivin-targeting miR-542-3p overcomes HER3 signaling-induced chemoresistance and enhances the antitumor activity of paclitaxel against HER2-overexpressing breast cancer"

    Article Title: Survivin-targeting miR-542-3p overcomes HER3 signaling-induced chemoresistance and enhances the antitumor activity of paclitaxel against HER2-overexpressing breast cancer

    Journal: Cancer letters

    doi: 10.1016/j.canlet.2018.01.065

    Ectopic expression of HER3 or specific knockdown of HER3 does not affect Survivin mRNA levels and its protein stability in HER2-positive breast cancer cells SKBR3 cells and the expression vector transfectants (Neo.1, B3.pool, B3.1, and B3.2) or SKBR3 and MDA-MB-453 (MDA-453) cells transfected with a specific shRNA against human HER3 were subjected to total RNA extraction. A , First-strand cDNA was synthesized using a reverse transcription kit. The partial coding sequence of Survivin or β-actin was amplified with specific primers. The PCR products were separated on a 1.8% agarose gel containing ethidium bromide and visualized under UV light. B , The mRNA levels of Survivin were measured by qRT-PCR. Bars , S.D. Data represent the results of three independent experiments. C , SKBR3 cells or the HER3 -transfectants were treated with cycloheximide (CHX) for 0, 4, 8, or 16 h. Total cell lysates of each cell line collected at indicated time points were examined by western blot analyses of Survivin. D , The intensities of Survivin signals were determined by densitometry analyses and normalized to control (0 h), which was set as 100%.
    Figure Legend Snippet: Ectopic expression of HER3 or specific knockdown of HER3 does not affect Survivin mRNA levels and its protein stability in HER2-positive breast cancer cells SKBR3 cells and the expression vector transfectants (Neo.1, B3.pool, B3.1, and B3.2) or SKBR3 and MDA-MB-453 (MDA-453) cells transfected with a specific shRNA against human HER3 were subjected to total RNA extraction. A , First-strand cDNA was synthesized using a reverse transcription kit. The partial coding sequence of Survivin or β-actin was amplified with specific primers. The PCR products were separated on a 1.8% agarose gel containing ethidium bromide and visualized under UV light. B , The mRNA levels of Survivin were measured by qRT-PCR. Bars , S.D. Data represent the results of three independent experiments. C , SKBR3 cells or the HER3 -transfectants were treated with cycloheximide (CHX) for 0, 4, 8, or 16 h. Total cell lysates of each cell line collected at indicated time points were examined by western blot analyses of Survivin. D , The intensities of Survivin signals were determined by densitometry analyses and normalized to control (0 h), which was set as 100%.

    Techniques Used: Expressing, Plasmid Preparation, Multiple Displacement Amplification, Transfection, shRNA, RNA Extraction, Synthesized, Sequencing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR, Western Blot

    Modulation of the HER3/Akt signaling specifically influences expression of two Survivin -targeting miRNAs, miR-203 and miR-542-3p Cells were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-203, miR-542-3p, Let-7c and miR-29b were measured by qRT-PCR using TaqMan miRNA assays. All results were normalized according to the internal control RNU6B. Bars , S.D. Data represent the results of three independent experiments. A B, Ectopic expression of HER3 (A) or transient transfection with an expression vector containing human HER3 cDNA (B) in SKBR3 cells significantly reduced the expression levels of miR-203 and miR-542-3p, but not Let-7c and miR-29b. C, Specific knockdown of HER3 expression in SKBR3.B3.1 and SKBR3.B3.2 sublines significantly enhanced expression of miR-203 and miR-542-3p, but not Let-7c and miR-29b. D, A specific Akt inhibitor significantly enhanced expression of miR-203 and miR-542-3p, but not Let-7c and miR-29b, in SKBR3.B3.1 and SKBR3.B3.2 cells.
    Figure Legend Snippet: Modulation of the HER3/Akt signaling specifically influences expression of two Survivin -targeting miRNAs, miR-203 and miR-542-3p Cells were collected and subjected to total RNA extraction, inclusive of the small RNA fraction. The expression levels of miR-203, miR-542-3p, Let-7c and miR-29b were measured by qRT-PCR using TaqMan miRNA assays. All results were normalized according to the internal control RNU6B. Bars , S.D. Data represent the results of three independent experiments. A B, Ectopic expression of HER3 (A) or transient transfection with an expression vector containing human HER3 cDNA (B) in SKBR3 cells significantly reduced the expression levels of miR-203 and miR-542-3p, but not Let-7c and miR-29b. C, Specific knockdown of HER3 expression in SKBR3.B3.1 and SKBR3.B3.2 sublines significantly enhanced expression of miR-203 and miR-542-3p, but not Let-7c and miR-29b. D, A specific Akt inhibitor significantly enhanced expression of miR-203 and miR-542-3p, but not Let-7c and miR-29b, in SKBR3.B3.1 and SKBR3.B3.2 cells.

    Techniques Used: Expressing, RNA Extraction, Quantitative RT-PCR, Transfection, Plasmid Preparation

    Blockade of HER3 with monoclonal antibody MM-121 downregulates Survivin via induction of miR-203 and miR-542-3p in HER2-positive breast cancer cells A , SKBR3.B3.1, SKBR3.B3.2, and MDA-MB-453 cells treated with MM-121 (10 μg/mL) for 24 or 48 h were collected and subjected to total RNA extraction. The expression levels of miR-203 and miR-542-3p were measured by qRT-PCR using TaqMan miRNA assays. RNU6B was used as an internal control. Bars , S.D. Data show the representative of three independent experiments. B C , SKBR3.B3.1 or SKBR3.B3.2 cells were transfected with either negative control miRNA inhibitor (Control) or the specific inhibitor(s) of miR-203 (miR-203) and/or miR-542-3p (miR-542-3p). After 24 h, the cells were then untreated or treated with MM-121 (10 μg/mL) for another 24 h. Cells were collected and subjected to western blot analyses with specific antibody against Survivin, Mcl-1, Bcl-xL, or β-actin. The densitometry analyses of Survivin signals are shown underneath, and the arbitrary numbers indicate the intensities of each cell line relative to controls, defined as 1.
    Figure Legend Snippet: Blockade of HER3 with monoclonal antibody MM-121 downregulates Survivin via induction of miR-203 and miR-542-3p in HER2-positive breast cancer cells A , SKBR3.B3.1, SKBR3.B3.2, and MDA-MB-453 cells treated with MM-121 (10 μg/mL) for 24 or 48 h were collected and subjected to total RNA extraction. The expression levels of miR-203 and miR-542-3p were measured by qRT-PCR using TaqMan miRNA assays. RNU6B was used as an internal control. Bars , S.D. Data show the representative of three independent experiments. B C , SKBR3.B3.1 or SKBR3.B3.2 cells were transfected with either negative control miRNA inhibitor (Control) or the specific inhibitor(s) of miR-203 (miR-203) and/or miR-542-3p (miR-542-3p). After 24 h, the cells were then untreated or treated with MM-121 (10 μg/mL) for another 24 h. Cells were collected and subjected to western blot analyses with specific antibody against Survivin, Mcl-1, Bcl-xL, or β-actin. The densitometry analyses of Survivin signals are shown underneath, and the arbitrary numbers indicate the intensities of each cell line relative to controls, defined as 1.

    Techniques Used: Multiple Displacement Amplification, RNA Extraction, Expressing, Quantitative RT-PCR, Transfection, Negative Control, Western Blot

    7) Product Images from "Glypican-3 induces a mesenchymal to epithelial transition in human breast cancer cells"

    Article Title: Glypican-3 induces a mesenchymal to epithelial transition in human breast cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11107

    Effect of GPC3 on the expression of the mesenchymal markers N-Cadherin and vimentin A, B. WB and qRT-PCR techniques were used to analyze N-Cadherin (A) and vimentin (B) expression. For WB, β-Actin was used as an internal control and numbers on the left indicate molecular mass (kDa). For qRT-PCR, GAPDH was employed as control, and values are expressed as mean ± SD. The data are representative of three independent experiments (**p
    Figure Legend Snippet: Effect of GPC3 on the expression of the mesenchymal markers N-Cadherin and vimentin A, B. WB and qRT-PCR techniques were used to analyze N-Cadherin (A) and vimentin (B) expression. For WB, β-Actin was used as an internal control and numbers on the left indicate molecular mass (kDa). For qRT-PCR, GAPDH was employed as control, and values are expressed as mean ± SD. The data are representative of three independent experiments (**p

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

    Effect of GPC3 on the expression of the epithelial marker E-Cadherin A. E-Cadherin expression was evaluated in MCF-7-sh scramble, MCF-7-sh3 and MCF-7-sh3 C2 as well as MDA-MB231-vector, MDA-MB231-GPC3×1 and MDA-MB231-GPC3×2 by IF (Magnification x1000. Scale bars 8 μm), WB and qRT-PCR. β-Actin was used as a WB internal control, while GAPDH was employed as a qRT-PCR control (**p
    Figure Legend Snippet: Effect of GPC3 on the expression of the epithelial marker E-Cadherin A. E-Cadherin expression was evaluated in MCF-7-sh scramble, MCF-7-sh3 and MCF-7-sh3 C2 as well as MDA-MB231-vector, MDA-MB231-GPC3×1 and MDA-MB231-GPC3×2 by IF (Magnification x1000. Scale bars 8 μm), WB and qRT-PCR. β-Actin was used as a WB internal control, while GAPDH was employed as a qRT-PCR control (**p

    Techniques Used: Expressing, Marker, Multiple Displacement Amplification, Plasmid Preparation, Western Blot, Quantitative RT-PCR

    GPC3 expression in breast cancer cell lines A, B, C. Left panel : GPC3 mRNA expression levels were identified by qRT-PCR analysis of MDA-MB231, Hs578T, ZR-75-1 and MCF-7 (A), MCF-7-scramble, MCF-7-sh1, MCF-7-sh2, MCF-7-sh3 and MCF-7-sh3 C2 (B), MDA-MB231-vector, MDA-MB231-GPC3×1 and MDA-MB231-GPC3×2 (C) cells. GAPDH was used as control. Values are expressed as mean ± SD. The data are representative of three independent experiments. (*p
    Figure Legend Snippet: GPC3 expression in breast cancer cell lines A, B, C. Left panel : GPC3 mRNA expression levels were identified by qRT-PCR analysis of MDA-MB231, Hs578T, ZR-75-1 and MCF-7 (A), MCF-7-scramble, MCF-7-sh1, MCF-7-sh2, MCF-7-sh3 and MCF-7-sh3 C2 (B), MDA-MB231-vector, MDA-MB231-GPC3×1 and MDA-MB231-GPC3×2 (C) cells. GAPDH was used as control. Values are expressed as mean ± SD. The data are representative of three independent experiments. (*p

    Techniques Used: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification, Plasmid Preparation

    Effect of GPC3 on the expression of the transcriptional repressor ZEB1 A. ZEB1 mRNA levels were analyzed by qRT-PCR ( upper panel ) and ZEB1 protein expression was determined by WB ( lower panel ), in MCF-7 and MDA-MB231cell sublines. In qRT-PCR reactions, GAPDH was used as an internal control. Values are expressed as mean ± SD (*p
    Figure Legend Snippet: Effect of GPC3 on the expression of the transcriptional repressor ZEB1 A. ZEB1 mRNA levels were analyzed by qRT-PCR ( upper panel ) and ZEB1 protein expression was determined by WB ( lower panel ), in MCF-7 and MDA-MB231cell sublines. In qRT-PCR reactions, GAPDH was used as an internal control. Values are expressed as mean ± SD (*p

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Multiple Displacement Amplification

    8) Product Images from "Biosynthetic CircRNA_001160 induced by PTBP1 regulates the permeability of BTB via the CircRNA_001160/miR-195-5p/ETV1 axis"

    Article Title: Biosynthetic CircRNA_001160 induced by PTBP1 regulates the permeability of BTB via the CircRNA_001160/miR-195-5p/ETV1 axis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-2191-z

    Knockdown of ETV1 increased the BTB permeability and inhibited the expression levels of tight junction-related proteins at the transcriptional level. a Relative ETV1 expression in AECs and GECs determined by qRT-PCR. Data represent mean ± SD ( n = 5, each). * P
    Figure Legend Snippet: Knockdown of ETV1 increased the BTB permeability and inhibited the expression levels of tight junction-related proteins at the transcriptional level. a Relative ETV1 expression in AECs and GECs determined by qRT-PCR. Data represent mean ± SD ( n = 5, each). * P

    Techniques Used: Permeability, Expressing, Quantitative RT-PCR

    Knockdown of PTBP1 increased BTB permeability in vitro and reduced the expression levels of tight junction-related proteins. a Relative PTBP1 expression in AECs and GECs determined by qRT-PCR. Data represent mean ± SD ( n = 5, each). * P
    Figure Legend Snippet: Knockdown of PTBP1 increased BTB permeability in vitro and reduced the expression levels of tight junction-related proteins. a Relative PTBP1 expression in AECs and GECs determined by qRT-PCR. Data represent mean ± SD ( n = 5, each). * P

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

    Overexpression of miR-195-5p increased in vitro BTB permeability and reduced the expression levels of tight junction-related proteins. a The expression and location of miR-195-5p in AECs and GECs were detected by FISH. (green, miR-195-5p; blue, DAPI nuclear staining). Scale bar represents 20 μm. b Relative miR-195-5p expression in AECs and GECs was detected by qRT-PCR. Data represent mean ± SD ( n = 5, each group). * P
    Figure Legend Snippet: Overexpression of miR-195-5p increased in vitro BTB permeability and reduced the expression levels of tight junction-related proteins. a The expression and location of miR-195-5p in AECs and GECs were detected by FISH. (green, miR-195-5p; blue, DAPI nuclear staining). Scale bar represents 20 μm. b Relative miR-195-5p expression in AECs and GECs was detected by qRT-PCR. Data represent mean ± SD ( n = 5, each group). * P

    Techniques Used: Over Expression, In Vitro, Permeability, Expressing, Fluorescence In Situ Hybridization, Staining, Quantitative RT-PCR

    PTBP1 regulated the BTB permeability via promoting the expression of circRNA_001160 in GECs. a CircRNA microarray analysis of total RNAs isolated from sh-NC and sh-PTBP1 cells. Red indicates high relative expression and green indicates low relative expression. b Relative expression levels of circRNA_0008035, circRNA_0034642, circRNA_001160, and circRNA_001569 were detected by qRT-PCR. Data represent mean ± SD ( n = 5, each group). * P
    Figure Legend Snippet: PTBP1 regulated the BTB permeability via promoting the expression of circRNA_001160 in GECs. a CircRNA microarray analysis of total RNAs isolated from sh-NC and sh-PTBP1 cells. Red indicates high relative expression and green indicates low relative expression. b Relative expression levels of circRNA_0008035, circRNA_0034642, circRNA_001160, and circRNA_001569 were detected by qRT-PCR. Data represent mean ± SD ( n = 5, each group). * P

    Techniques Used: Permeability, Expressing, Microarray, Isolation, Quantitative RT-PCR

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

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

    Journal: Basic Research in Cardiology

    doi: 10.1007/s00395-011-0181-0

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

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

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

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

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

    Techniques Used: Staining, Quantitative RT-PCR

    10) Product Images from "MiR-148b suppresses cell proliferation and invasion in hepatocellular carcinoma by targeting WNT1/β-catenin pathway"

    Article Title: MiR-148b suppresses cell proliferation and invasion in hepatocellular carcinoma by targeting WNT1/β-catenin pathway

    Journal: Scientific Reports

    doi: 10.1038/srep08087

    WNT1 inversely correlated with miR-148b levels in HCC tissues. (A) WNT1 was detected in 40 HCC tissues and matched normal liver tissues by qRT-PCR, and the WNT1 abundance was normalized to GAPDH. (B) The relationship between WNT1 and miR-148b expression was explored by Spearman's correlation analysis in HCC tissues. *P
    Figure Legend Snippet: WNT1 inversely correlated with miR-148b levels in HCC tissues. (A) WNT1 was detected in 40 HCC tissues and matched normal liver tissues by qRT-PCR, and the WNT1 abundance was normalized to GAPDH. (B) The relationship between WNT1 and miR-148b expression was explored by Spearman's correlation analysis in HCC tissues. *P

    Techniques Used: Quantitative RT-PCR, Expressing

    WNT1 was the direct target of miR-148b in HCC cells. (A. Top) Human WNT1 3′UTR fragment containing wild-type or mutated miR-148b–binding sequence. (WT: wild type; MUT: mutant type). (A. Bottom) MiR-148b and its putative binding site in the 3′UTR of WNT1. (B) Luciferase reporter assays, with co-transfection of wild-type or mutant 3′UTR (100 ng) and miR-148b or miR-NC (50 nM) in HepG2 cells as indicated. Firefly luciferase activity was normalized by Renilla luciferase activity. After transfection with miR-148b or anti-miR-148b (50 nM) for 48 h, qRT-PCR (C) and western blot (D) were used for monitoring WNT1 expression in HepG2 cells. *P
    Figure Legend Snippet: WNT1 was the direct target of miR-148b in HCC cells. (A. Top) Human WNT1 3′UTR fragment containing wild-type or mutated miR-148b–binding sequence. (WT: wild type; MUT: mutant type). (A. Bottom) MiR-148b and its putative binding site in the 3′UTR of WNT1. (B) Luciferase reporter assays, with co-transfection of wild-type or mutant 3′UTR (100 ng) and miR-148b or miR-NC (50 nM) in HepG2 cells as indicated. Firefly luciferase activity was normalized by Renilla luciferase activity. After transfection with miR-148b or anti-miR-148b (50 nM) for 48 h, qRT-PCR (C) and western blot (D) were used for monitoring WNT1 expression in HepG2 cells. *P

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

    Analysis of miR-148b expression in human HCC tissues and survivals of HCC patients. (A) The relative expression levels of miR-148b in human HCC tissues (n = 40) and matched normal liver tissues (n = 40) were detected by qRT-PCR. U6 was used as the control for RNA loading, and miR-148b abundance was normalized to U6 RNA. (B) Kaplan- Meier curves of overall survivals of 40 HCC patients, according to miR-148b expression scored as low expression (below the median value, n = 20) and high expression level (above the median value, n = 20). MiR-148b downregulation correlated significantly with shorter overall survivals. P-value was shown with log-rank test. **P
    Figure Legend Snippet: Analysis of miR-148b expression in human HCC tissues and survivals of HCC patients. (A) The relative expression levels of miR-148b in human HCC tissues (n = 40) and matched normal liver tissues (n = 40) were detected by qRT-PCR. U6 was used as the control for RNA loading, and miR-148b abundance was normalized to U6 RNA. (B) Kaplan- Meier curves of overall survivals of 40 HCC patients, according to miR-148b expression scored as low expression (below the median value, n = 20) and high expression level (above the median value, n = 20). MiR-148b downregulation correlated significantly with shorter overall survivals. P-value was shown with log-rank test. **P

    Techniques Used: Expressing, Quantitative RT-PCR

    11) Product Images from "Evaluation of Schistosome Promoter Expression for Transgenesis and Genetic Analysis"

    Article Title: Evaluation of Schistosome Promoter Expression for Transgenesis and Genetic Analysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0098302

    qRT-PCR quantitation of the mCherry transcript in schistosomes transfected with vectors expressing a reporter gene at day 2 and day 7. The viral promoter CMV and the endogenous schistosome promoter SmActin1 were assayed for their ability to express an mCherry reporter at different time frames, without maintaining the schistosomula under transfection conditions.
    Figure Legend Snippet: qRT-PCR quantitation of the mCherry transcript in schistosomes transfected with vectors expressing a reporter gene at day 2 and day 7. The viral promoter CMV and the endogenous schistosome promoter SmActin1 were assayed for their ability to express an mCherry reporter at different time frames, without maintaining the schistosomula under transfection conditions.

    Techniques Used: Quantitative RT-PCR, Quantitation Assay, Transfection, Expressing

    qRT-PCR quantitation of SmCaspase7 transcript in schistosomula expressing plasmid-based SmCaspase7 gene regulated by different promoters. SmCaspase7 was cloned into plasmids and its expression was controlled by the SmActin, CMV, or SmHsp70 promoter. Non-transfected, wild-type schistosomula were used as a negative control. Transcript levels were assessed by quantitative RT-PCR.
    Figure Legend Snippet: qRT-PCR quantitation of SmCaspase7 transcript in schistosomula expressing plasmid-based SmCaspase7 gene regulated by different promoters. SmCaspase7 was cloned into plasmids and its expression was controlled by the SmActin, CMV, or SmHsp70 promoter. Non-transfected, wild-type schistosomula were used as a negative control. Transcript levels were assessed by quantitative RT-PCR.

    Techniques Used: Quantitative RT-PCR, Quantitation Assay, Expressing, Plasmid Preparation, Clone Assay, Transfection, Negative Control

    Schematic of expression plasmids used for transfection experiments. (A) Six promoters, two viral (CMV and SV40) and four endogenous (SmHsp70, SmActin1, Sm23, and SmCalcineurinA), were used to regulate expression of an mCherry reporter gene. These promoters were separately subcloned into vector pCI-Neo to make plasmids pEJ1175, pEJ1500, pEJ1501, pEJ1502, pEJ1503, pEJ1504, respectively. Forward arrow (a) and reverse arrow (b) represent forward oligonucleotide (a) and reverse oligonucleotide (b), used to quantify mCherry transcript levels by qRT-PCR. (B) The schistosome Actin1, CyclinB, Caspase3, and Caspase7 genes are separately regulated by the plasmid-based SmActin1 promoter. Forward and reverse arrows represent DNA oligonucleotides used for qRT-PCR analysis of each transcript ( Table S2 ). (C) Transcript levels of the schistosome Caspase 7 gene were regulated by plasmid-based SmActin1, SmHsp70, or CMV promoters. DNA oligos (c) and (d) were used for qRT-PCR analysis to measure SmCaspase7 transcript levels directed by each promoter.
    Figure Legend Snippet: Schematic of expression plasmids used for transfection experiments. (A) Six promoters, two viral (CMV and SV40) and four endogenous (SmHsp70, SmActin1, Sm23, and SmCalcineurinA), were used to regulate expression of an mCherry reporter gene. These promoters were separately subcloned into vector pCI-Neo to make plasmids pEJ1175, pEJ1500, pEJ1501, pEJ1502, pEJ1503, pEJ1504, respectively. Forward arrow (a) and reverse arrow (b) represent forward oligonucleotide (a) and reverse oligonucleotide (b), used to quantify mCherry transcript levels by qRT-PCR. (B) The schistosome Actin1, CyclinB, Caspase3, and Caspase7 genes are separately regulated by the plasmid-based SmActin1 promoter. Forward and reverse arrows represent DNA oligonucleotides used for qRT-PCR analysis of each transcript ( Table S2 ). (C) Transcript levels of the schistosome Caspase 7 gene were regulated by plasmid-based SmActin1, SmHsp70, or CMV promoters. DNA oligos (c) and (d) were used for qRT-PCR analysis to measure SmCaspase7 transcript levels directed by each promoter.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR

    qRT-PCR quantitation of the mCherry transcript in schistosomes transfected with vectors expressing this reporter gene regulated by different promoters. Two viral promoters and four endogenous schistosome promoters were assayed for their ability to express an mCherry reporter. Schistosome samples treated with PEI only or DNA only were used as negative controls. All samples were normalized against the CMV samples.
    Figure Legend Snippet: qRT-PCR quantitation of the mCherry transcript in schistosomes transfected with vectors expressing this reporter gene regulated by different promoters. Two viral promoters and four endogenous schistosome promoters were assayed for their ability to express an mCherry reporter. Schistosome samples treated with PEI only or DNA only were used as negative controls. All samples were normalized against the CMV samples.

    Techniques Used: Quantitative RT-PCR, Quantitation Assay, Transfection, Expressing

    12) Product Images from "A Site-Specific, Single-Copy Transgenesis Strategy to Identify 5? Regulatory Sequences of the Mouse Testis-Determining Gene Sry"

    Article Title: A Site-Specific, Single-Copy Transgenesis Strategy to Identify 5? Regulatory Sequences of the Mouse Testis-Determining Gene Sry

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094813

    Temporal analysis of gonadal Sry expression. qRT-PCR (2 − Δ Ct , normalized against Sdha ) was performed for gonad-mesonephros pairs from precisely staged embryos between 10.5 dpc (∼8 ts) and 12.5 dpc (∼30 ts), and for gonad pairs at 13.5 dpc. Each circle represents a single sample. (A) Wild type XY (white) and XX (black) gonads. Dashed line shows curve of fit generated by non-linear regression for XY wild type gonads (5–30 ts, 6 th order polynomial function, least squares fit, R 2 = 0.7012). (B) XX Sry#49 (white), XY Sry#49 (grey), and XX Sry-L741 (black) gonads. XY Sry#49 gonads are included at 13.5 dpc only, as endogenous Sry expression would mask transgenic Sry expression at the earlier time points. Dashed line shows curve of fit generated by non-linear regression for XX Sry#49 gonads (5–30 ts, 6 th order polynomial function, least squares fit, R 2 = 0.2081).
    Figure Legend Snippet: Temporal analysis of gonadal Sry expression. qRT-PCR (2 − Δ Ct , normalized against Sdha ) was performed for gonad-mesonephros pairs from precisely staged embryos between 10.5 dpc (∼8 ts) and 12.5 dpc (∼30 ts), and for gonad pairs at 13.5 dpc. Each circle represents a single sample. (A) Wild type XY (white) and XX (black) gonads. Dashed line shows curve of fit generated by non-linear regression for XY wild type gonads (5–30 ts, 6 th order polynomial function, least squares fit, R 2 = 0.7012). (B) XX Sry#49 (white), XY Sry#49 (grey), and XX Sry-L741 (black) gonads. XY Sry#49 gonads are included at 13.5 dpc only, as endogenous Sry expression would mask transgenic Sry expression at the earlier time points. Dashed line shows curve of fit generated by non-linear regression for XX Sry#49 gonads (5–30 ts, 6 th order polynomial function, least squares fit, R 2 = 0.2081).

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

    Transgene constructs and relative transcription of Sry in the five transgenic mouse lines. Left panel shows the structure of the five transgene constructs integrated into the Col1a1 locus, featuring sequential deletions of the Sry 5′ region. Length of sequence 5′ to the transcriptional start site (TSS) is indicated beneath the constructs (base-pairs). Grey box, Sry coding region. PolyA, polyadenylation signal. Black arrows and italics indicate primer sites used for genotyping. Grey arrows indicate inverted repeat sequences flanking the Sry coding region. Right hand panel shows corresponding qRT-PCR analysis of Sry mRNA levels in samples (gonad and mesonephros pairs) collected at 11.5 dpc (2 − Δ Ct , normalized against Sdha ). Error bars are 95% confidence intervals. Numbers beside bars indicate sample size (individual gonad-mesonephros pairs).
    Figure Legend Snippet: Transgene constructs and relative transcription of Sry in the five transgenic mouse lines. Left panel shows the structure of the five transgene constructs integrated into the Col1a1 locus, featuring sequential deletions of the Sry 5′ region. Length of sequence 5′ to the transcriptional start site (TSS) is indicated beneath the constructs (base-pairs). Grey box, Sry coding region. PolyA, polyadenylation signal. Black arrows and italics indicate primer sites used for genotyping. Grey arrows indicate inverted repeat sequences flanking the Sry coding region. Right hand panel shows corresponding qRT-PCR analysis of Sry mRNA levels in samples (gonad and mesonephros pairs) collected at 11.5 dpc (2 − Δ Ct , normalized against Sdha ). Error bars are 95% confidence intervals. Numbers beside bars indicate sample size (individual gonad-mesonephros pairs).

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

    Sry transgene expression in gonadal versus non-gonadal tissue. Analyses were performed by qRT-PCR (2 − Δ Ct , normalized against Sdha ). (A) Expression of Sry mRNA in XX Sry#49 (black) and XY Sry#49 (white) tissues at 13.5 dpc. (B) Expression of Sry mRNA in XX Sry-L741 (black) and XY wild type (white) tissues at 11.5 dpc. Error bars are 95% confidence intervals. Numbers above bars indicate sample size.
    Figure Legend Snippet: Sry transgene expression in gonadal versus non-gonadal tissue. Analyses were performed by qRT-PCR (2 − Δ Ct , normalized against Sdha ). (A) Expression of Sry mRNA in XX Sry#49 (black) and XY Sry#49 (white) tissues at 13.5 dpc. (B) Expression of Sry mRNA in XX Sry-L741 (black) and XY wild type (white) tissues at 11.5 dpc. Error bars are 95% confidence intervals. Numbers above bars indicate sample size.

    Techniques Used: Expressing, Quantitative RT-PCR

    qRT-PCR analysis of Sry and Sox9 expression at 11.5 dpc. (A) Total Sry mRNA expression for wild type and transgenic XX (black) and XY (white) embryos. (B) Sox9 mRNA expression for the same embryos. Relative expression is measured as 2 − Δ Ct values, normalized against Sdha . Numbers above bars indicate sample size (individual gonad-mesonephros pairs). Error bars are 95% confidence intervals. * P
    Figure Legend Snippet: qRT-PCR analysis of Sry and Sox9 expression at 11.5 dpc. (A) Total Sry mRNA expression for wild type and transgenic XX (black) and XY (white) embryos. (B) Sox9 mRNA expression for the same embryos. Relative expression is measured as 2 − Δ Ct values, normalized against Sdha . Numbers above bars indicate sample size (individual gonad-mesonephros pairs). Error bars are 95% confidence intervals. * P

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

    13) Product Images from "Role of the venus kinase receptor in the female reproductive physiology of the desert locust, Schistocerca gregaria"

    Article Title: Role of the venus kinase receptor in the female reproductive physiology of the desert locust, Schistocerca gregaria

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-11434-3

    Effect of RNAi-mediated knockdown of SgVKR on ecdysteroid biosynthesis in 12-day-old adult female S . gregaria . ( A ) Overview of the ecdysteroid biosynthesis pathway in larval D. melanogaster . ( B ) Relative transcript levels of the Halloween genes, SgSpo, SgPhm, SgDib, SgSad and SgShd , involved in ecdysteroid biosynthesis were measured in the gonads from control and ds VKR -treated 12-day-old female locusts, using qRT-PCR. The data represent box plots (min to max) of five independent pools of three locusts, run in duplicate and normalized to g lyceraldehyde-3-phosphate dehydrogenase ( GAPDH) and α- tubulin1A transcript levels. ( C ) Ecdysteroid titers in the hemolymph and ecdysteroid levels (free and total) in the gonads of 12-day-old control and ds VKR -treated female locusts. Ecdysteroid titers, expressed in pg 20E equivalents per µL hemolymph, and ecdysteroid levels, expressed in ng 20E equivalents per ovary, were measured with an EIA. The data represent mean ± S.E.M. of individual animals (N = 16), as well as the individual values (grey dots). Locusts were injected with 200 ng of dsRNA against SgVKR or GFP (control) one day, five days and nine days after molting to the adult stage. Significant differences (p
    Figure Legend Snippet: Effect of RNAi-mediated knockdown of SgVKR on ecdysteroid biosynthesis in 12-day-old adult female S . gregaria . ( A ) Overview of the ecdysteroid biosynthesis pathway in larval D. melanogaster . ( B ) Relative transcript levels of the Halloween genes, SgSpo, SgPhm, SgDib, SgSad and SgShd , involved in ecdysteroid biosynthesis were measured in the gonads from control and ds VKR -treated 12-day-old female locusts, using qRT-PCR. The data represent box plots (min to max) of five independent pools of three locusts, run in duplicate and normalized to g lyceraldehyde-3-phosphate dehydrogenase ( GAPDH) and α- tubulin1A transcript levels. ( C ) Ecdysteroid titers in the hemolymph and ecdysteroid levels (free and total) in the gonads of 12-day-old control and ds VKR -treated female locusts. Ecdysteroid titers, expressed in pg 20E equivalents per µL hemolymph, and ecdysteroid levels, expressed in ng 20E equivalents per ovary, were measured with an EIA. The data represent mean ± S.E.M. of individual animals (N = 16), as well as the individual values (grey dots). Locusts were injected with 200 ng of dsRNA against SgVKR or GFP (control) one day, five days and nine days after molting to the adult stage. Significant differences (p

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

    Effect of RNAi-mediated knockdown of SgVKR on transcripts coding for neuroparsins and insulin-related peptide in 12-day-old adult female S. gregaria . Relative ( A ) SgNP3 , ( B ) SgNP4 and ( C ) SgIRP transcript levels were measured in the fat body from control and ds VKR -treated 12-day-old female locusts, using qRT-PCR. Locusts were injected with 200 ng of dsRNA against SgVKR or GFP (control) one, five and nine days after molting to the adult stage. The data represent box plots (min to max) of five independent pools of three locusts, run in duplicate and normalized to GAPDH and ribosomal protein 49 ( RP49 ) transcript levels. Significant differences (p
    Figure Legend Snippet: Effect of RNAi-mediated knockdown of SgVKR on transcripts coding for neuroparsins and insulin-related peptide in 12-day-old adult female S. gregaria . Relative ( A ) SgNP3 , ( B ) SgNP4 and ( C ) SgIRP transcript levels were measured in the fat body from control and ds VKR -treated 12-day-old female locusts, using qRT-PCR. Locusts were injected with 200 ng of dsRNA against SgVKR or GFP (control) one, five and nine days after molting to the adult stage. The data represent box plots (min to max) of five independent pools of three locusts, run in duplicate and normalized to GAPDH and ribosomal protein 49 ( RP49 ) transcript levels. Significant differences (p

    Techniques Used: Quantitative RT-PCR, Injection

    Tissue and temporal distribution of SgVKR . ( A ) Tissue distribution of SgVKR in immature (white) and mature (black) adult locusts. Relative transcript levels of SgVKR were measured in different adult tissues using qRT-PCR. All tissues, except the male reproductive system (Gon M), were dissected from immature, 3-day-old female locusts or mature female locusts with an oocyte size between 3 and 6 mm. The male reproductive system was dissected from immature, 3-day-old and mature, 12-day-old male locusts. The data represent mean ± S.E.M. of three independent pools of five animals, run in duplicate and normalized to α- tubulin1A, CG13220 and ubiquitin transcript levels. Abbreviations X-axis: Br + OL: brain + optic lobes; Fb: fat body; Mal: Malpighian tubules; PG: prothoracic glands; CA + CC: corpora allata + corpora cardiaca; Gon M: male reproductive system (testes + accessory glands); Gon F: female gonads (ovaries); Gut: for-, mid- and hindgut; VNC: ventral nerve cord. ( B ) Temporal distribution profile of SgVKR in the female gonads during the first reproductive cycle. Using qRT-PCR, relative transcript levels of SgVKR were measured every other day in the female gonads, starting on the day of molting to the adult stage (day 0). Data represent mean ± S.E.M. of three independent pools of six animals, run in duplicate and normalized to CG13220 and ubiquitin transcript levels. The ecdysteroid titre (red line), expressed in nM, throughout the first reproductive cycle was measured with an EIA. The data represent mean ± S.E.M. of 6-18 hemolymph samples taken from different animals.
    Figure Legend Snippet: Tissue and temporal distribution of SgVKR . ( A ) Tissue distribution of SgVKR in immature (white) and mature (black) adult locusts. Relative transcript levels of SgVKR were measured in different adult tissues using qRT-PCR. All tissues, except the male reproductive system (Gon M), were dissected from immature, 3-day-old female locusts or mature female locusts with an oocyte size between 3 and 6 mm. The male reproductive system was dissected from immature, 3-day-old and mature, 12-day-old male locusts. The data represent mean ± S.E.M. of three independent pools of five animals, run in duplicate and normalized to α- tubulin1A, CG13220 and ubiquitin transcript levels. Abbreviations X-axis: Br + OL: brain + optic lobes; Fb: fat body; Mal: Malpighian tubules; PG: prothoracic glands; CA + CC: corpora allata + corpora cardiaca; Gon M: male reproductive system (testes + accessory glands); Gon F: female gonads (ovaries); Gut: for-, mid- and hindgut; VNC: ventral nerve cord. ( B ) Temporal distribution profile of SgVKR in the female gonads during the first reproductive cycle. Using qRT-PCR, relative transcript levels of SgVKR were measured every other day in the female gonads, starting on the day of molting to the adult stage (day 0). Data represent mean ± S.E.M. of three independent pools of six animals, run in duplicate and normalized to CG13220 and ubiquitin transcript levels. The ecdysteroid titre (red line), expressed in nM, throughout the first reproductive cycle was measured with an EIA. The data represent mean ± S.E.M. of 6-18 hemolymph samples taken from different animals.

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

    14) Product Images from "Gene Expression and Yeast Two-Hybrid Studies of 1R-MYB Transcription Factor Mediating Drought Stress Response in Chickpea (Cicer arietinum L.)"

    Article Title: Gene Expression and Yeast Two-Hybrid Studies of 1R-MYB Transcription Factor Mediating Drought Stress Response in Chickpea (Cicer arietinum L.)

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2015.01117

    A hypothetical model proposing a possible role for the 1R-MYB TF in co-regulating drought tolerance in chickpea . The Y2H analysis involving the candidate TF, 1R-MYB suggests GSGT2, CIPK25, and ABR17-like proteins as possible co-regulators for drought tolerance in chickpea root. These proteins have been known to be involved in various signal transduction and stress responses to drought from previous reports and has been depicted in the figure. The color intensity of the arrows indicate the strength of interactions observed through biochemical and qRT-PCR analyses in the present study.
    Figure Legend Snippet: A hypothetical model proposing a possible role for the 1R-MYB TF in co-regulating drought tolerance in chickpea . The Y2H analysis involving the candidate TF, 1R-MYB suggests GSGT2, CIPK25, and ABR17-like proteins as possible co-regulators for drought tolerance in chickpea root. These proteins have been known to be involved in various signal transduction and stress responses to drought from previous reports and has been depicted in the figure. The color intensity of the arrows indicate the strength of interactions observed through biochemical and qRT-PCR analyses in the present study.

    Techniques Used: Transduction, Quantitative RT-PCR

    The relative expression ratio of 8 candidate TF genes analyzed using qRT-PCR under drought stress . The relative expression ratio of each gene was calculated relative to its expression in control sample. GAPDH was used as an internal control to normalize the data. The error bars representing standard deviation were calculated based on three biological and two technical replicates.
    Figure Legend Snippet: The relative expression ratio of 8 candidate TF genes analyzed using qRT-PCR under drought stress . The relative expression ratio of each gene was calculated relative to its expression in control sample. GAPDH was used as an internal control to normalize the data. The error bars representing standard deviation were calculated based on three biological and two technical replicates.

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

    Confirmation of PPI strength using qRT-PCR . The qRT-PCR analysis quantified the expression of two reporter genes HIS3 and ADE2 for determining the strength of PPI for the selected yeast colonies (PPI-1, PPI-4, and PPI-5). These genes are under the control of Gal4-responsive promoters. The interaction between the bait and prey proteins allow the Gal4-responsive HIS3 and ADE2 genes to biosynthesis histidine and adenine for cell growth.
    Figure Legend Snippet: Confirmation of PPI strength using qRT-PCR . The qRT-PCR analysis quantified the expression of two reporter genes HIS3 and ADE2 for determining the strength of PPI for the selected yeast colonies (PPI-1, PPI-4, and PPI-5). These genes are under the control of Gal4-responsive promoters. The interaction between the bait and prey proteins allow the Gal4-responsive HIS3 and ADE2 genes to biosynthesis histidine and adenine for cell growth.

    Techniques Used: Quantitative RT-PCR, Expressing

    15) Product Images from "Effect of Radiation Dose-Rate on Hematopoietic Cell Engraftment in Adult Zebrafish"

    Article Title: Effect of Radiation Dose-Rate on Hematopoietic Cell Engraftment in Adult Zebrafish

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073745

    Dose-Rate effect does not require cxcr4b in transplanted cells. A) WT zebrafish were irradiated with 20 Gy at either dose rate and transplanted with WKM from Tg( bactin :EGFP) WKM that was either heterozygous or homozygous for the odysseus ( cxcr4b ) mutation. 9 dpt, kidneys were harvested and analyzed by flow cytometry. Data shows pooled results from two biologically independent trials, n = 13–15 fish per group. Heterozygous: p = 0.0420, Homozygous mutant: p = 0.0141, (unpaired t-test). B) 1 day after radiation, samples enriched for renal tubules were collected from WT zebrafish and analyzed by qRT-PCR for sdf-1a expression. No significant differences were seen between dose-rate groups. Pooled results from two biological experiments, each with four fish per group, are shown. RQ: Relative Quantitation.
    Figure Legend Snippet: Dose-Rate effect does not require cxcr4b in transplanted cells. A) WT zebrafish were irradiated with 20 Gy at either dose rate and transplanted with WKM from Tg( bactin :EGFP) WKM that was either heterozygous or homozygous for the odysseus ( cxcr4b ) mutation. 9 dpt, kidneys were harvested and analyzed by flow cytometry. Data shows pooled results from two biologically independent trials, n = 13–15 fish per group. Heterozygous: p = 0.0420, Homozygous mutant: p = 0.0141, (unpaired t-test). B) 1 day after radiation, samples enriched for renal tubules were collected from WT zebrafish and analyzed by qRT-PCR for sdf-1a expression. No significant differences were seen between dose-rate groups. Pooled results from two biological experiments, each with four fish per group, are shown. RQ: Relative Quantitation.

    Techniques Used: Irradiation, Mutagenesis, Flow Cytometry, Cytometry, Fluorescence In Situ Hybridization, Quantitative RT-PCR, Expressing, Quantitation Assay

    qRT-PCR of hematopoietic and non-hematopoietic kidney after radiation. 3/min (HDR) or a lower dose rate of 25 cGy/min (LDR), WT zebrafish kidneys were pooled and filtered to obtain samples enriched for either hematopoietic whole kidney marrow (WKM) (A), or renal tubules (B). 0 Gy control samples were harvested concurrently at each time point and set as biological references. Each biological trial was composed of kidneys from four fish per time point, and data summarizes pooled results from 2–3 independent biological trials per time point, per condition. Although LDR hematopoietic cells showed significantly higher p53 expression 2 dpr than HDR hematopoietic cells (.001
    Figure Legend Snippet: qRT-PCR of hematopoietic and non-hematopoietic kidney after radiation. 3/min (HDR) or a lower dose rate of 25 cGy/min (LDR), WT zebrafish kidneys were pooled and filtered to obtain samples enriched for either hematopoietic whole kidney marrow (WKM) (A), or renal tubules (B). 0 Gy control samples were harvested concurrently at each time point and set as biological references. Each biological trial was composed of kidneys from four fish per time point, and data summarizes pooled results from 2–3 independent biological trials per time point, per condition. Although LDR hematopoietic cells showed significantly higher p53 expression 2 dpr than HDR hematopoietic cells (.001

    Techniques Used: Quantitative RT-PCR, Fluorescence In Situ Hybridization, Expressing

    Myelosuppression after 20Gy at 25 cGy/min vs 800cGy/min in non-transplanted fish. A, Flow cytometry analysis of hematopoietic cell recovery in the kidney on days 2, 6, and 20–21 following radiation at either 25cGy/min (LDR) or 800cGy/min (HDR), showing initial myelosuppression followed by hematopoietic recovery. Plot shows the means and SDs of pooled data from two independent biological trials, for a total of at least 7–10 fish per data point. 100,000 events were collected per sample. No significant differences were noted between dose-rate groups. B, qRT-PCR of hematopoietic cells 2 days post radiation. Data shows pooled results from two independent biological trials; each trial composed of 4 fish per group. No statistically significant differences between dose-rate groups were noted. RQ: Relative Quantitation.
    Figure Legend Snippet: Myelosuppression after 20Gy at 25 cGy/min vs 800cGy/min in non-transplanted fish. A, Flow cytometry analysis of hematopoietic cell recovery in the kidney on days 2, 6, and 20–21 following radiation at either 25cGy/min (LDR) or 800cGy/min (HDR), showing initial myelosuppression followed by hematopoietic recovery. Plot shows the means and SDs of pooled data from two independent biological trials, for a total of at least 7–10 fish per data point. 100,000 events were collected per sample. No significant differences were noted between dose-rate groups. B, qRT-PCR of hematopoietic cells 2 days post radiation. Data shows pooled results from two independent biological trials; each trial composed of 4 fish per group. No statistically significant differences between dose-rate groups were noted. RQ: Relative Quantitation.

    Techniques Used: Fluorescence In Situ Hybridization, Flow Cytometry, Cytometry, Quantitative RT-PCR, Quantitation Assay

    16) Product Images from "SP1 and STAT3 Functionally Synergize to Induce the RhoU Small GTPase and a Subclass of Non-canonical WNT Responsive Genes Correlating with Poor Prognosis in Breast Cancer"

    Article Title: SP1 and STAT3 Functionally Synergize to Induce the RhoU Small GTPase and a Subclass of Non-canonical WNT Responsive Genes Correlating with Poor Prognosis in Breast Cancer

    Journal: Cancers

    doi: 10.3390/cancers11010101

    JNK-mediated SP1 recruitment is responsible for RhoU promoter induction by WNT ligands. ( A ) RhoU mRNA levels measured by qRT-PCR in MEF cells stimulated for 24 h with the indicated WNT ligands as described in the legend to Figure 1 , in the presence or absence of the SP1 inhibitor Mithramycin A (Mit. A). Mean ± SEM of the relative expression levels normalized to the 18S rRNA; n = 3. ( B ) The indicated RhoU promoter constructs were transfected in MEF cells as described in the legend to Figure 1 C, followed by WNT1 stimulation in the presence or absence of Mit. A. Data are mean ± SEM of the luciferase induction fold, relative to unstimulated condition. n = 3. ( C , D ) SP1 binding to the RhoU promoter measured by ChIP assay in MEF cells stimulated for 24 h with the indicated WNTs, in the presence or absence of Mit. A ( C ) or of the JNK inhibitor JNKi ( D ). Immunoprecipitated chromatin was analyzed by qRT-PCR, and data (mean ± SEM) obtained upon normalization to total input (T.I.) and IgG. n = 3. ( E ) Western blot showing WNT-dependent increase of phosphorylated SP1 (upper band). MEF cells were stimulated for 24 h with the indicated WNTs, in the presence or absence of Mit. A or JNKi, and analyzed with anti-SP1 antibodies recognizing both the phosphorylated and unphosphorylated forms. Representative of one of four independent experiments. ( F ) Ratio between phosphorylated SP1 and total SP1 signals, expressed as fold induction relative to the unstimulated condition. Mean ± SEM of four independent experiments. ** p
    Figure Legend Snippet: JNK-mediated SP1 recruitment is responsible for RhoU promoter induction by WNT ligands. ( A ) RhoU mRNA levels measured by qRT-PCR in MEF cells stimulated for 24 h with the indicated WNT ligands as described in the legend to Figure 1 , in the presence or absence of the SP1 inhibitor Mithramycin A (Mit. A). Mean ± SEM of the relative expression levels normalized to the 18S rRNA; n = 3. ( B ) The indicated RhoU promoter constructs were transfected in MEF cells as described in the legend to Figure 1 C, followed by WNT1 stimulation in the presence or absence of Mit. A. Data are mean ± SEM of the luciferase induction fold, relative to unstimulated condition. n = 3. ( C , D ) SP1 binding to the RhoU promoter measured by ChIP assay in MEF cells stimulated for 24 h with the indicated WNTs, in the presence or absence of Mit. A ( C ) or of the JNK inhibitor JNKi ( D ). Immunoprecipitated chromatin was analyzed by qRT-PCR, and data (mean ± SEM) obtained upon normalization to total input (T.I.) and IgG. n = 3. ( E ) Western blot showing WNT-dependent increase of phosphorylated SP1 (upper band). MEF cells were stimulated for 24 h with the indicated WNTs, in the presence or absence of Mit. A or JNKi, and analyzed with anti-SP1 antibodies recognizing both the phosphorylated and unphosphorylated forms. Representative of one of four independent experiments. ( F ) Ratio between phosphorylated SP1 and total SP1 signals, expressed as fold induction relative to the unstimulated condition. Mean ± SEM of four independent experiments. ** p

    Techniques Used: Quantitative RT-PCR, Expressing, Construct, Transfection, Luciferase, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Western Blot

    Both RHOU expression and JNK and SP1 activities are required for migration of MDA-MB-231 cells. ( A ) qRT-PCR analysis of RHOU, Ror1, Wnt5a and Wnt5b mRNAs in MDA-MB-231 and MCF-7 cells. ( B ) RHOU mRNA downregulation in MDA-MB-231 cells treated with the JNK or SP1 inhibitors (JNKi, Mit. A). Data are mean ± SEM of the relative expression levels, normalized to 18S rRNA. ( C – F ) Wound healing assays. MDA-MB-231 cells were infected with lentiviral vectors expressing two distinct RHOU shRNAs or a scrambled control (sh1, sh2, scr) ( C , D ), or treated with the JNK or SP1 inhibitors (JNKi, Mit.A) ( E , F ), and migration was assessed by in vitro wound healing assays. The phase contrast pictures (C ,E ) are representative of three independent experiments (Field of View: 895 × 671 µm). Migrating distance ( D , F ) was calculated as described in the Materials and Methods, and shown as mean ± SEM. n = 3. * p
    Figure Legend Snippet: Both RHOU expression and JNK and SP1 activities are required for migration of MDA-MB-231 cells. ( A ) qRT-PCR analysis of RHOU, Ror1, Wnt5a and Wnt5b mRNAs in MDA-MB-231 and MCF-7 cells. ( B ) RHOU mRNA downregulation in MDA-MB-231 cells treated with the JNK or SP1 inhibitors (JNKi, Mit. A). Data are mean ± SEM of the relative expression levels, normalized to 18S rRNA. ( C – F ) Wound healing assays. MDA-MB-231 cells were infected with lentiviral vectors expressing two distinct RHOU shRNAs or a scrambled control (sh1, sh2, scr) ( C , D ), or treated with the JNK or SP1 inhibitors (JNKi, Mit.A) ( E , F ), and migration was assessed by in vitro wound healing assays. The phase contrast pictures (C ,E ) are representative of three independent experiments (Field of View: 895 × 671 µm). Migrating distance ( D , F ) was calculated as described in the Materials and Methods, and shown as mean ± SEM. n = 3. * p

    Techniques Used: Expressing, Migration, Multiple Displacement Amplification, Quantitative RT-PCR, Infection, In Vitro

    RhoU (Ras Homolog Family Member U) is a common Wingless-type MMTV Integration site (WNT) transcriptional target through the non-canonical c-JUN N-terminal kinase (JNK)-dependent pathway. ( A – C ) RhoU mRNA was measured by qRT-PCR in MEF cells co-cultured for 24 h with HEK-293 cells expressing or not the indicated WNTs ( A ), in the presence or absence of the JNK-inhibitor SP600125 (JNKi, B ), or upon knockdown of JNK 1 or JNK 2 by means of 2 independent shRNAs/each ( C ). Data are mean ± SEM of the relative expression levels, normalized to the 18S rRNA. n = 3. ( D ) RhoU promoter activity in mouse embryonic fibroblasts (MEF) cells transiently co-transfected with the indicated reporter constructs and a Secreted Embryonic Alkaline Phosphatase (SEAP)-expressing vector for normalization. TOPflash (TOP) and FOPflash (FOP) were used as positive and negative controls, respectively, for the β-catenin-dependent pathway. Transfected cells were stimulated or not with WNT ligands as described above. Data are mean ± SEM of the luciferase induction fold, relative to unstimulated condition. All WNT ligands significantly induced the activity of the −1765, −1300 and −756 RhoU promoter constructs ( p
    Figure Legend Snippet: RhoU (Ras Homolog Family Member U) is a common Wingless-type MMTV Integration site (WNT) transcriptional target through the non-canonical c-JUN N-terminal kinase (JNK)-dependent pathway. ( A – C ) RhoU mRNA was measured by qRT-PCR in MEF cells co-cultured for 24 h with HEK-293 cells expressing or not the indicated WNTs ( A ), in the presence or absence of the JNK-inhibitor SP600125 (JNKi, B ), or upon knockdown of JNK 1 or JNK 2 by means of 2 independent shRNAs/each ( C ). Data are mean ± SEM of the relative expression levels, normalized to the 18S rRNA. n = 3. ( D ) RhoU promoter activity in mouse embryonic fibroblasts (MEF) cells transiently co-transfected with the indicated reporter constructs and a Secreted Embryonic Alkaline Phosphatase (SEAP)-expressing vector for normalization. TOPflash (TOP) and FOPflash (FOP) were used as positive and negative controls, respectively, for the β-catenin-dependent pathway. Transfected cells were stimulated or not with WNT ligands as described above. Data are mean ± SEM of the luciferase induction fold, relative to unstimulated condition. All WNT ligands significantly induced the activity of the −1765, −1300 and −756 RhoU promoter constructs ( p

    Techniques Used: Quantitative RT-PCR, Cell Culture, Expressing, Activity Assay, Transfection, Construct, Plasmid Preparation, Luciferase

    17) Product Images from "Control of chrysanthemum flowering through integration with an aging pathway"

    Article Title: Control of chrysanthemum flowering through integration with an aging pathway

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00812-0

    Expression of CmSPL genes ( a , b , c ) and cmo-MIR156 ( d , e ) in CmNF-YB8 -RNAi chrysanthemum plants. qRT-PCR was performed to evaluate the expression of each gene. UBIQUITIN was used as the control gene for CmSPL3 , CmSPL5 , CmSPL9 , and primary cmo-MIR156 expression. U6 was used as the control gene for cmo-miR156. Three independent experiments were performed and error bars indicate standard deviation. Asterisks indicate significant differences according to a Student’s t test (* P
    Figure Legend Snippet: Expression of CmSPL genes ( a , b , c ) and cmo-MIR156 ( d , e ) in CmNF-YB8 -RNAi chrysanthemum plants. qRT-PCR was performed to evaluate the expression of each gene. UBIQUITIN was used as the control gene for CmSPL3 , CmSPL5 , CmSPL9 , and primary cmo-MIR156 expression. U6 was used as the control gene for cmo-miR156. Three independent experiments were performed and error bars indicate standard deviation. Asterisks indicate significant differences according to a Student’s t test (* P

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

    Transcript abundance of chrysanthemum CmNF-YB8 . a Transcript abundance of CmNF-YB8 in different organs. Plants at 5 days after transplanting and growth under long day (LD) conditions were used. Apical buds (AB), leaves (L), stems (St), and roots (R) were harvested. b Transcript abundance of CmNF-YB8 in apical buds and leaves of differently aged chrysanthemum. Plants were grown under LD conditions. Samples were harvested every 10 days from 5 days after propagation. c Transcript abundance of CmNF-YB8 in chrysanthemum grown under different day lengths conditions, including LD and SD, under a low-temperature (4 °C) regime, or following treatment with gibberellic acid (GA 4/7 ). Leaves were harvested 30 days after each treatment. qRT-PCR was performed to evaluate expression of CmNF-YB8 , using UBIQUITIN as the control. Three independent experiments were performed and error bars indicate standard deviation. Significant differences were determined using Duncan’s multiple range test ( P
    Figure Legend Snippet: Transcript abundance of chrysanthemum CmNF-YB8 . a Transcript abundance of CmNF-YB8 in different organs. Plants at 5 days after transplanting and growth under long day (LD) conditions were used. Apical buds (AB), leaves (L), stems (St), and roots (R) were harvested. b Transcript abundance of CmNF-YB8 in apical buds and leaves of differently aged chrysanthemum. Plants were grown under LD conditions. Samples were harvested every 10 days from 5 days after propagation. c Transcript abundance of CmNF-YB8 in chrysanthemum grown under different day lengths conditions, including LD and SD, under a low-temperature (4 °C) regime, or following treatment with gibberellic acid (GA 4/7 ). Leaves were harvested 30 days after each treatment. qRT-PCR was performed to evaluate expression of CmNF-YB8 , using UBIQUITIN as the control. Three independent experiments were performed and error bars indicate standard deviation. Significant differences were determined using Duncan’s multiple range test ( P

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

    The juvenile vegetative phase of CmNF-YB8 -RNAi chrysanthemum plants. a Transcript abundance of CmNF-YB8 in wild type (WT) and CmNF-YB8 -RNAi plants determined by qRT-PCR. RNAi-4, -13, and -22 correspond to three independent CmNF-YB8 -RNAi lines. b Morphology of leaves of seven-leaf-old WT and CmNF-YB8 -RNAi chrysanthemum plants. Juvenile leaves were defined as small, with no, or minimal, marginal serration. c Percentage of juvenile leaves among the first five leaves in WT and CmNF-YB8 -RNAi chrysanthemum plants. Three independent experiments were performed and error bars indicate standard deviation. Asterisks indicate significant differences according to a Student’s t test (** P
    Figure Legend Snippet: The juvenile vegetative phase of CmNF-YB8 -RNAi chrysanthemum plants. a Transcript abundance of CmNF-YB8 in wild type (WT) and CmNF-YB8 -RNAi plants determined by qRT-PCR. RNAi-4, -13, and -22 correspond to three independent CmNF-YB8 -RNAi lines. b Morphology of leaves of seven-leaf-old WT and CmNF-YB8 -RNAi chrysanthemum plants. Juvenile leaves were defined as small, with no, or minimal, marginal serration. c Percentage of juvenile leaves among the first five leaves in WT and CmNF-YB8 -RNAi chrysanthemum plants. Three independent experiments were performed and error bars indicate standard deviation. Asterisks indicate significant differences according to a Student’s t test (** P

    Techniques Used: Quantitative RT-PCR, Standard Deviation

    18) Product Images from "The ecdysis triggering hormone system is essential for successful moulting of a major hemimetabolous pest insect, Schistocerca gregaria"

    Article Title: The ecdysis triggering hormone system is essential for successful moulting of a major hemimetabolous pest insect, Schistocerca gregaria

    Journal: Scientific Reports

    doi: 10.1038/srep46502

    Temporal expression profile of SchgrETHR and SchgrETHpre as well as hemolymph ecdysteroid titres in fourth and fifth instar S. gregaria . Using qRT-PCR, the relative transcript levels of SchgrETHR and SchgrETHpre (bars) were measured every day in the heads during fourth instar development and every other day in the trachea during fifth instar development. SchgrETHR transcript levels were also measured every other day in the epidermis and brain during fifth instar development. The data during fourth instar development represent mean ± S.E.M. of six independent pools of three animals, run in duplicate and normalized to RP49 and EF1α transcript levels. The data during fifth instar development represent mean ± S.E.M. of three independent pools of five animals, run in duplicate. The data in the epidermis were normalized to RP49 and EF1α, the data in the brain were normalized to GAPDH and CG13220 , and the data in the trachea were normalized to α-tubulin1A, β-actin and EF1α transcript levels. Ecdysteroid titres (red line), expressed in pg 20E equivalents per μl hemolymph, throughout the fourth and fifth instar stage were measured with an EIA. The data represent mean ± S.E.M. of five pools of three hemolymph samples taken from different animals.
    Figure Legend Snippet: Temporal expression profile of SchgrETHR and SchgrETHpre as well as hemolymph ecdysteroid titres in fourth and fifth instar S. gregaria . Using qRT-PCR, the relative transcript levels of SchgrETHR and SchgrETHpre (bars) were measured every day in the heads during fourth instar development and every other day in the trachea during fifth instar development. SchgrETHR transcript levels were also measured every other day in the epidermis and brain during fifth instar development. The data during fourth instar development represent mean ± S.E.M. of six independent pools of three animals, run in duplicate and normalized to RP49 and EF1α transcript levels. The data during fifth instar development represent mean ± S.E.M. of three independent pools of five animals, run in duplicate. The data in the epidermis were normalized to RP49 and EF1α, the data in the brain were normalized to GAPDH and CG13220 , and the data in the trachea were normalized to α-tubulin1A, β-actin and EF1α transcript levels. Ecdysteroid titres (red line), expressed in pg 20E equivalents per μl hemolymph, throughout the fourth and fifth instar stage were measured with an EIA. The data represent mean ± S.E.M. of five pools of three hemolymph samples taken from different animals.

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

    Tissue distribution profile of SchgrETHR and SchgrETHpre in fifth instar S. gregaria. Relative transcript levels of SchgrETHR and SchgrETHpre were measured in different fifth instar tissues using qRT-PCR. Tissues were dissected from 8-day-old male fifth instar locusts, except for the female gonads. The data represent mean ± S.E.M. of three independent pools of five animals, run in duplicate and normalized to RP49 and EF1α transcript levels. Abbreviations X-axis: Br + OL: brain + optic lobes; Epi: epidermis; Fb: fat body; Mal: Malpighian tubules; PG: prothoracic glands; CA + CC: corpora allata + corpora cardiaca; Gon M: male gonads; Gon F: female gonads.
    Figure Legend Snippet: Tissue distribution profile of SchgrETHR and SchgrETHpre in fifth instar S. gregaria. Relative transcript levels of SchgrETHR and SchgrETHpre were measured in different fifth instar tissues using qRT-PCR. Tissues were dissected from 8-day-old male fifth instar locusts, except for the female gonads. The data represent mean ± S.E.M. of three independent pools of five animals, run in duplicate and normalized to RP49 and EF1α transcript levels. Abbreviations X-axis: Br + OL: brain + optic lobes; Epi: epidermis; Fb: fat body; Mal: Malpighian tubules; PG: prothoracic glands; CA + CC: corpora allata + corpora cardiaca; Gon M: male gonads; Gon F: female gonads.

    Techniques Used: Quantitative RT-PCR

    Effect of RNAi-mediated knockdown of the ecdysone receptor complex, SchgrEcR / SchgrRXR , on the transcript levels of SchgrETHR and SchgrETHpre in 6-day-old fifth instar male S. gregaria . Relative transcript levels were measured in the epidermis from control locusts and locusts injected with SchgrEcR and SchgrRXR dsRNA using qRT-PCR. Newly emerged fifth instar locusts were injected with 200 ng of SchgrEcR and SchgrRXR dsRNA. A boost injection was given three days later. The data represent box plots (min to max) of six independent pools of three animals, run in duplicate and normalized to β-actin and EF1α transcript levels. Significant differences (p
    Figure Legend Snippet: Effect of RNAi-mediated knockdown of the ecdysone receptor complex, SchgrEcR / SchgrRXR , on the transcript levels of SchgrETHR and SchgrETHpre in 6-day-old fifth instar male S. gregaria . Relative transcript levels were measured in the epidermis from control locusts and locusts injected with SchgrEcR and SchgrRXR dsRNA using qRT-PCR. Newly emerged fifth instar locusts were injected with 200 ng of SchgrEcR and SchgrRXR dsRNA. A boost injection was given three days later. The data represent box plots (min to max) of six independent pools of three animals, run in duplicate and normalized to β-actin and EF1α transcript levels. Significant differences (p

    Techniques Used: Injection, Quantitative RT-PCR

    19) Product Images from "Identification of early indicators of altered metabolism in normal development using a rodent model system"

    Article Title: Identification of early indicators of altered metabolism in normal development using a rodent model system

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.031815

    Expression of insulin, glucagon and the Pi3k/Akt pathway genes differs between ABW and LBW pups. (A) Distribution of insulin-secreting β cells (green) and glucagon-secreting α cells (red) in the islets of the pancreas of 1-day-old ABW and LBW pups (scale bar: 50 µm). (B) Percentage of insulin-secreting β cells (green) and glucagon-secreting α cells (red) in 1-day-old ABW and LBW pups. (C) qRT-PCR showing fold changes in expression of mRNAs of insulin and glucagon in pancreata of 1-day-old ABW and LBW pups. 18S was used to normalize the expression. (D) Differences in the level of insulin and glucagon expression, determined by western blot analysis, between 1-day-old ABW and LBW pups. Gapdh was used as a loading control. (E) Glucose tolerance test at 0, 1 h and 2 h after 6 h fasting and administration of 2 mg/kg body weight of glucose in 1-day-old ABW and LBW pups. (F) Serum insulin concentration at 0, 1 h and 2 h after 6 h fasting and administration of 2 mg/kg body weight of glucose determined by ELISA in 1-day-old ABW and LBW pups. (G) Western blot analysis of the components of the Pi3k/Akt pathway (Akt, Irs1, Glut4) showing a reduction in the phosphorylated levels of Akt and Irs1, and the membrane fraction of Glut 4 in LBW pups. Gapdh was used as a loading control. * P
    Figure Legend Snippet: Expression of insulin, glucagon and the Pi3k/Akt pathway genes differs between ABW and LBW pups. (A) Distribution of insulin-secreting β cells (green) and glucagon-secreting α cells (red) in the islets of the pancreas of 1-day-old ABW and LBW pups (scale bar: 50 µm). (B) Percentage of insulin-secreting β cells (green) and glucagon-secreting α cells (red) in 1-day-old ABW and LBW pups. (C) qRT-PCR showing fold changes in expression of mRNAs of insulin and glucagon in pancreata of 1-day-old ABW and LBW pups. 18S was used to normalize the expression. (D) Differences in the level of insulin and glucagon expression, determined by western blot analysis, between 1-day-old ABW and LBW pups. Gapdh was used as a loading control. (E) Glucose tolerance test at 0, 1 h and 2 h after 6 h fasting and administration of 2 mg/kg body weight of glucose in 1-day-old ABW and LBW pups. (F) Serum insulin concentration at 0, 1 h and 2 h after 6 h fasting and administration of 2 mg/kg body weight of glucose determined by ELISA in 1-day-old ABW and LBW pups. (G) Western blot analysis of the components of the Pi3k/Akt pathway (Akt, Irs1, Glut4) showing a reduction in the phosphorylated levels of Akt and Irs1, and the membrane fraction of Glut 4 in LBW pups. Gapdh was used as a loading control. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Expression of DNA methyltransferases in the pancreas and skeletal muscle, and methylation of the InsII promoter in pancreata, differ between ABW and LBW pups. (A) qRT-PCR analysis of the expression of DNA methyltransferases Dnmt1 , Dmnt3a and Dnmt3b in the pancreas of ABW and LBW pups at day 1. (B) Western blot analysis of the expression of DNA methyltransferases reveal an increase in the expression of Dnmt1, but a decrease in Dnmt3a and Dnmt3b expression, in the pancreas of LBW pups. (C) The rat InsII promoter depicting the position of CPGs analyzed with the Sequenom mass array system. (D) Percentage methylation of CpGs analyzed by the Sequenom microarray system in ABW and LBW pups. (E) qRT-PCR analyses showing differences in the expression of DNA methyltransferases Dnmt1 , Dnmt3a and Dnmt3b in skeletal muscle of ABW and LBW pups. Expression of Dnmt1 is increased, expression of Dnmt3a is decreased, and the expression of Dnmt3b remains unaltered in the two groups. (F) Western blot analyses of the expression of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b reveal an increase in Dnmt1 but a decrease in Dnmt3a, without any significant change in the expression of Dnmt3b, in LBW pups. * P
    Figure Legend Snippet: Expression of DNA methyltransferases in the pancreas and skeletal muscle, and methylation of the InsII promoter in pancreata, differ between ABW and LBW pups. (A) qRT-PCR analysis of the expression of DNA methyltransferases Dnmt1 , Dmnt3a and Dnmt3b in the pancreas of ABW and LBW pups at day 1. (B) Western blot analysis of the expression of DNA methyltransferases reveal an increase in the expression of Dnmt1, but a decrease in Dnmt3a and Dnmt3b expression, in the pancreas of LBW pups. (C) The rat InsII promoter depicting the position of CPGs analyzed with the Sequenom mass array system. (D) Percentage methylation of CpGs analyzed by the Sequenom microarray system in ABW and LBW pups. (E) qRT-PCR analyses showing differences in the expression of DNA methyltransferases Dnmt1 , Dnmt3a and Dnmt3b in skeletal muscle of ABW and LBW pups. Expression of Dnmt1 is increased, expression of Dnmt3a is decreased, and the expression of Dnmt3b remains unaltered in the two groups. (F) Western blot analyses of the expression of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b reveal an increase in Dnmt1 but a decrease in Dnmt3a, without any significant change in the expression of Dnmt3b, in LBW pups. * P

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

    Expression of the Pparg gene and its targets are different in ABW and LBW pups. (A) qRT-PCR reveals a reduction in the expression of Rxra and AdipoQ in LBW compared to ABW pups at day 1. (B) Western blot analysis showing the reduction in the expression of Rxrα, Pparγ 1 and 2 and AdipoQ in LBW pups. (C) The rat adiponectin gene promoter showing the position of PPRE elements tested in this study (+1 is the transcription start site). (D,E) ChIP analysis of the occupancy of Pparγ in the adiponectin promoter of ABW (D) and LBW (E) pups. * P
    Figure Legend Snippet: Expression of the Pparg gene and its targets are different in ABW and LBW pups. (A) qRT-PCR reveals a reduction in the expression of Rxra and AdipoQ in LBW compared to ABW pups at day 1. (B) Western blot analysis showing the reduction in the expression of Rxrα, Pparγ 1 and 2 and AdipoQ in LBW pups. (C) The rat adiponectin gene promoter showing the position of PPRE elements tested in this study (+1 is the transcription start site). (D,E) ChIP analysis of the occupancy of Pparγ in the adiponectin promoter of ABW (D) and LBW (E) pups. * P

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

    20) Product Images from "lncRNA differentiation antagonizing nonprotein coding RNA overexpression accelerates progression and indicates poor prognosis in pancreatic ductal adenocarcinoma"

    Article Title: lncRNA differentiation antagonizing nonprotein coding RNA overexpression accelerates progression and indicates poor prognosis in pancreatic ductal adenocarcinoma

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S167065

    DANCR overexpression implicates cancer progression in PDAC. Notes: ( A ) Expression of DANCR in five PDAC cell lines (BxPC3, Panc28, AsPC1, MiaPaCa2 and Panc1) and human pancreatic ductal epithelial cell line, HPDE, was measured by qRT-PCR assay. ( B ) The expression level of DANCR in PDAC tissues relative to paired tumor-adjacent tissues was detected by qRT-PCR assay and calculated. ( C ) The expression level of DANCR in PDAC tissues with vascular invasion evaluated by qRT-PCR assay was compared with PDAC tissues without vascular invasion. ( D ) The expression level of DANCR in PDAC tissues with T-stage T1 and T2 detected by qRT-PCR assay was compared with PDAC tissues with T-stage T3 and T4. ( E ) The expression level of DANCR in PDAC tissues with lymph node metastasis detected by qRT-PCR assay was compared with PDAC tissues without lymph node metastasis. ( F ) The expression level of DANCR in PDAC tissues with TNM early stages detected by qRT-PCR assay was compared with PDAC tissues with TNM advanced stages. * P
    Figure Legend Snippet: DANCR overexpression implicates cancer progression in PDAC. Notes: ( A ) Expression of DANCR in five PDAC cell lines (BxPC3, Panc28, AsPC1, MiaPaCa2 and Panc1) and human pancreatic ductal epithelial cell line, HPDE, was measured by qRT-PCR assay. ( B ) The expression level of DANCR in PDAC tissues relative to paired tumor-adjacent tissues was detected by qRT-PCR assay and calculated. ( C ) The expression level of DANCR in PDAC tissues with vascular invasion evaluated by qRT-PCR assay was compared with PDAC tissues without vascular invasion. ( D ) The expression level of DANCR in PDAC tissues with T-stage T1 and T2 detected by qRT-PCR assay was compared with PDAC tissues with T-stage T3 and T4. ( E ) The expression level of DANCR in PDAC tissues with lymph node metastasis detected by qRT-PCR assay was compared with PDAC tissues without lymph node metastasis. ( F ) The expression level of DANCR in PDAC tissues with TNM early stages detected by qRT-PCR assay was compared with PDAC tissues with TNM advanced stages. * P

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

    DANCR functions through upregulating AXL via microRNA-33a-5p inhibition. Notes: ( A ) Expression of miR-33a-5p and miR-634 was measured by qRT-PCR in BxPC3 cells with DANCR overexpression. ( B ) Expression of AXL and RAB1A was detected by qRT-PCR in BxPC3 cells with DANCR overexpression. ( C ) Expression of miR-33a-5p and miR-634 was measured by qRT-PCR in Panc1 cells with DANCR deficiency. ( D ) Expression of AXL and RAB1A was detected by qRT-PCR in Panc1 cells with DANCR deficiency. * P
    Figure Legend Snippet: DANCR functions through upregulating AXL via microRNA-33a-5p inhibition. Notes: ( A ) Expression of miR-33a-5p and miR-634 was measured by qRT-PCR in BxPC3 cells with DANCR overexpression. ( B ) Expression of AXL and RAB1A was detected by qRT-PCR in BxPC3 cells with DANCR overexpression. ( C ) Expression of miR-33a-5p and miR-634 was measured by qRT-PCR in Panc1 cells with DANCR deficiency. ( D ) Expression of AXL and RAB1A was detected by qRT-PCR in Panc1 cells with DANCR deficiency. * P

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

    DANCR accelerates metastasis of PDAC cells. Notes: ( A ) The relative expression level of DANCR in Panc1 cells after DANCR interference was determined by qRT-PCR assay. ( B ) The relative expression level of DANCR in BxPC3 cells after DANCR overexpression was confirmed by qRT-PCR assay. ( C ) The migration and invasion abilities of Panc1 cells with DANCR silencing were analyzed with Transwell assay and Matrigel assay, respectively (right panel). Typical images are shown in the left panel. ( D ) The migration and invasion abilities of BxPC3 cells with DANCR overexpression were revealed with Transwell assay and Matrigel assay, respectively (right panel). Typical images are shown in the left panel. * P
    Figure Legend Snippet: DANCR accelerates metastasis of PDAC cells. Notes: ( A ) The relative expression level of DANCR in Panc1 cells after DANCR interference was determined by qRT-PCR assay. ( B ) The relative expression level of DANCR in BxPC3 cells after DANCR overexpression was confirmed by qRT-PCR assay. ( C ) The migration and invasion abilities of Panc1 cells with DANCR silencing were analyzed with Transwell assay and Matrigel assay, respectively (right panel). Typical images are shown in the left panel. ( D ) The migration and invasion abilities of BxPC3 cells with DANCR overexpression were revealed with Transwell assay and Matrigel assay, respectively (right panel). Typical images are shown in the left panel. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Over Expression, Migration, Transwell Assay, Matrigel Assay

    21) Product Images from "Identification of early indicators of altered metabolism in normal development using a rodent model system"

    Article Title: Identification of early indicators of altered metabolism in normal development using a rodent model system

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.031815

    Expression of insulin, glucagon and the Pi3k/Akt pathway genes differs between ABW and LBW pups. (A) Distribution of insulin-secreting β cells (green) and glucagon-secreting α cells (red) in the islets of the pancreas of 1-day-old ABW and LBW pups (scale bar: 50 µm). (B) Percentage of insulin-secreting β cells (green) and glucagon-secreting α cells (red) in 1-day-old ABW and LBW pups. (C) qRT-PCR showing fold changes in expression of mRNAs of insulin and glucagon in pancreata of 1-day-old ABW and LBW pups. 18S was used to normalize the expression. (D) Differences in the level of insulin and glucagon expression, determined by western blot analysis, between 1-day-old ABW and LBW pups. Gapdh was used as a loading control. (E) Glucose tolerance test at 0, 1 h and 2 h after 6 h fasting and administration of 2 mg/kg body weight of glucose in 1-day-old ABW and LBW pups. (F) Serum insulin concentration at 0, 1 h and 2 h after 6 h fasting and administration of 2 mg/kg body weight of glucose determined by ELISA in 1-day-old ABW and LBW pups. (G) Western blot analysis of the components of the Pi3k/Akt pathway (Akt, Irs1, Glut4) showing a reduction in the phosphorylated levels of Akt and Irs1, and the membrane fraction of Glut 4 in LBW pups. Gapdh was used as a loading control. * P
    Figure Legend Snippet: Expression of insulin, glucagon and the Pi3k/Akt pathway genes differs between ABW and LBW pups. (A) Distribution of insulin-secreting β cells (green) and glucagon-secreting α cells (red) in the islets of the pancreas of 1-day-old ABW and LBW pups (scale bar: 50 µm). (B) Percentage of insulin-secreting β cells (green) and glucagon-secreting α cells (red) in 1-day-old ABW and LBW pups. (C) qRT-PCR showing fold changes in expression of mRNAs of insulin and glucagon in pancreata of 1-day-old ABW and LBW pups. 18S was used to normalize the expression. (D) Differences in the level of insulin and glucagon expression, determined by western blot analysis, between 1-day-old ABW and LBW pups. Gapdh was used as a loading control. (E) Glucose tolerance test at 0, 1 h and 2 h after 6 h fasting and administration of 2 mg/kg body weight of glucose in 1-day-old ABW and LBW pups. (F) Serum insulin concentration at 0, 1 h and 2 h after 6 h fasting and administration of 2 mg/kg body weight of glucose determined by ELISA in 1-day-old ABW and LBW pups. (G) Western blot analysis of the components of the Pi3k/Akt pathway (Akt, Irs1, Glut4) showing a reduction in the phosphorylated levels of Akt and Irs1, and the membrane fraction of Glut 4 in LBW pups. Gapdh was used as a loading control. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Expression of DNA methyltransferases in the pancreas and skeletal muscle, and methylation of the InsII promoter in pancreata, differ between ABW and LBW pups. (A) qRT-PCR analysis of the expression of DNA methyltransferases Dnmt1 , Dmnt3a and Dnmt3b in the pancreas of ABW and LBW pups at day 1. (B) Western blot analysis of the expression of DNA methyltransferases reveal an increase in the expression of Dnmt1, but a decrease in Dnmt3a and Dnmt3b expression, in the pancreas of LBW pups. (C) The rat InsII promoter depicting the position of CPGs analyzed with the Sequenom mass array system. (D) Percentage methylation of CpGs analyzed by the Sequenom microarray system in ABW and LBW pups. (E) qRT-PCR analyses showing differences in the expression of DNA methyltransferases Dnmt1 , Dnmt3a and Dnmt3b in skeletal muscle of ABW and LBW pups. Expression of Dnmt1 is increased, expression of Dnmt3a is decreased, and the expression of Dnmt3b remains unaltered in the two groups. (F) Western blot analyses of the expression of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b reveal an increase in Dnmt1 but a decrease in Dnmt3a, without any significant change in the expression of Dnmt3b, in LBW pups. * P
    Figure Legend Snippet: Expression of DNA methyltransferases in the pancreas and skeletal muscle, and methylation of the InsII promoter in pancreata, differ between ABW and LBW pups. (A) qRT-PCR analysis of the expression of DNA methyltransferases Dnmt1 , Dmnt3a and Dnmt3b in the pancreas of ABW and LBW pups at day 1. (B) Western blot analysis of the expression of DNA methyltransferases reveal an increase in the expression of Dnmt1, but a decrease in Dnmt3a and Dnmt3b expression, in the pancreas of LBW pups. (C) The rat InsII promoter depicting the position of CPGs analyzed with the Sequenom mass array system. (D) Percentage methylation of CpGs analyzed by the Sequenom microarray system in ABW and LBW pups. (E) qRT-PCR analyses showing differences in the expression of DNA methyltransferases Dnmt1 , Dnmt3a and Dnmt3b in skeletal muscle of ABW and LBW pups. Expression of Dnmt1 is increased, expression of Dnmt3a is decreased, and the expression of Dnmt3b remains unaltered in the two groups. (F) Western blot analyses of the expression of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b reveal an increase in Dnmt1 but a decrease in Dnmt3a, without any significant change in the expression of Dnmt3b, in LBW pups. * P

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

    Expression of the Pparg gene and its targets are different in ABW and LBW pups. (A) qRT-PCR reveals a reduction in the expression of Rxra and AdipoQ in LBW compared to ABW pups at day 1. (B) Western blot analysis showing the reduction in the expression of Rxrα, Pparγ 1 and 2 and AdipoQ in LBW pups. (C) The rat adiponectin gene promoter showing the position of PPRE elements tested in this study (+1 is the transcription start site). (D,E) ChIP analysis of the occupancy of Pparγ in the adiponectin promoter of ABW (D) and LBW (E) pups. * P
    Figure Legend Snippet: Expression of the Pparg gene and its targets are different in ABW and LBW pups. (A) qRT-PCR reveals a reduction in the expression of Rxra and AdipoQ in LBW compared to ABW pups at day 1. (B) Western blot analysis showing the reduction in the expression of Rxrα, Pparγ 1 and 2 and AdipoQ in LBW pups. (C) The rat adiponectin gene promoter showing the position of PPRE elements tested in this study (+1 is the transcription start site). (D,E) ChIP analysis of the occupancy of Pparγ in the adiponectin promoter of ABW (D) and LBW (E) pups. * P

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

    22) Product Images from "Control of chrysanthemum flowering through integration with an aging pathway"

    Article Title: Control of chrysanthemum flowering through integration with an aging pathway

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00812-0

    Expression of CmSPL genes ( a , b , c ) and cmo-MIR156 ( d , e ) in CmNF-YB8 -RNAi chrysanthemum plants. qRT-PCR was performed to evaluate the expression of each gene. UBIQUITIN was used as the control gene for CmSPL3 , CmSPL5 , CmSPL9 , and primary cmo-MIR156 expression. U6 was used as the control gene for cmo-miR156. Three independent experiments were performed and error bars indicate standard deviation. Asterisks indicate significant differences according to a Student’s t test (* P
    Figure Legend Snippet: Expression of CmSPL genes ( a , b , c ) and cmo-MIR156 ( d , e ) in CmNF-YB8 -RNAi chrysanthemum plants. qRT-PCR was performed to evaluate the expression of each gene. UBIQUITIN was used as the control gene for CmSPL3 , CmSPL5 , CmSPL9 , and primary cmo-MIR156 expression. U6 was used as the control gene for cmo-miR156. Three independent experiments were performed and error bars indicate standard deviation. Asterisks indicate significant differences according to a Student’s t test (* P

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

    Transcript abundance of chrysanthemum CmNF-YB8 . a Transcript abundance of CmNF-YB8 in different organs. Plants at 5 days after transplanting and growth under long day (LD) conditions were used. Apical buds (AB), leaves (L), stems (St), and roots (R) were harvested. b Transcript abundance of CmNF-YB8 in apical buds and leaves of differently aged chrysanthemum. Plants were grown under LD conditions. Samples were harvested every 10 days from 5 days after propagation. c Transcript abundance of CmNF-YB8 in chrysanthemum grown under different day lengths conditions, including LD and SD, under a low-temperature (4 °C) regime, or following treatment with gibberellic acid (GA 4/7 ). Leaves were harvested 30 days after each treatment. qRT-PCR was performed to evaluate expression of CmNF-YB8 , using UBIQUITIN as the control. Three independent experiments were performed and error bars indicate standard deviation. Significant differences were determined using Duncan’s multiple range test ( P
    Figure Legend Snippet: Transcript abundance of chrysanthemum CmNF-YB8 . a Transcript abundance of CmNF-YB8 in different organs. Plants at 5 days after transplanting and growth under long day (LD) conditions were used. Apical buds (AB), leaves (L), stems (St), and roots (R) were harvested. b Transcript abundance of CmNF-YB8 in apical buds and leaves of differently aged chrysanthemum. Plants were grown under LD conditions. Samples were harvested every 10 days from 5 days after propagation. c Transcript abundance of CmNF-YB8 in chrysanthemum grown under different day lengths conditions, including LD and SD, under a low-temperature (4 °C) regime, or following treatment with gibberellic acid (GA 4/7 ). Leaves were harvested 30 days after each treatment. qRT-PCR was performed to evaluate expression of CmNF-YB8 , using UBIQUITIN as the control. Three independent experiments were performed and error bars indicate standard deviation. Significant differences were determined using Duncan’s multiple range test ( P

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

    The juvenile vegetative phase of CmNF-YB8 -RNAi chrysanthemum plants. a Transcript abundance of CmNF-YB8 in wild type (WT) and CmNF-YB8 -RNAi plants determined by qRT-PCR. RNAi-4, -13, and -22 correspond to three independent CmNF-YB8 -RNAi lines. b Morphology of leaves of seven-leaf-old WT and CmNF-YB8 -RNAi chrysanthemum plants. Juvenile leaves were defined as small, with no, or minimal, marginal serration. c Percentage of juvenile leaves among the first five leaves in WT and CmNF-YB8 -RNAi chrysanthemum plants. Three independent experiments were performed and error bars indicate standard deviation. Asterisks indicate significant differences according to a Student’s t test (** P
    Figure Legend Snippet: The juvenile vegetative phase of CmNF-YB8 -RNAi chrysanthemum plants. a Transcript abundance of CmNF-YB8 in wild type (WT) and CmNF-YB8 -RNAi plants determined by qRT-PCR. RNAi-4, -13, and -22 correspond to three independent CmNF-YB8 -RNAi lines. b Morphology of leaves of seven-leaf-old WT and CmNF-YB8 -RNAi chrysanthemum plants. Juvenile leaves were defined as small, with no, or minimal, marginal serration. c Percentage of juvenile leaves among the first five leaves in WT and CmNF-YB8 -RNAi chrysanthemum plants. Three independent experiments were performed and error bars indicate standard deviation. Asterisks indicate significant differences according to a Student’s t test (** P

    Techniques Used: Quantitative RT-PCR, Standard Deviation

    23) Product Images from "RNA-Seq Reveals Spliceosome and Proteasome Genes as Most Consistent Transcripts in Human Cancer Cells"

    Article Title: RNA-Seq Reveals Spliceosome and Proteasome Genes as Most Consistent Transcripts in Human Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072884

    Correlation between RPKM and delta Ct of CD33 calculated with different control genes. dCt represents the difference between the Ct value of CD33 and that of the indicated control gene, for a given leukemic sample, measured by qRT-PCR. RPKM is plotted on a log-2 scale and represents the Reads Per Kilobase of transcript per Million mapped reads obtained for each leukemic sample by RNA-seq. ρ represents the Spearman correlation coefficient between the RPKM and the dCt obtained with the indicated control gene.
    Figure Legend Snippet: Correlation between RPKM and delta Ct of CD33 calculated with different control genes. dCt represents the difference between the Ct value of CD33 and that of the indicated control gene, for a given leukemic sample, measured by qRT-PCR. RPKM is plotted on a log-2 scale and represents the Reads Per Kilobase of transcript per Million mapped reads obtained for each leukemic sample by RNA-seq. ρ represents the Spearman correlation coefficient between the RPKM and the dCt obtained with the indicated control gene.

    Techniques Used: Quantitative RT-PCR, RNA Sequencing Assay

    Comparison of EIF4H gene expression values calculated with GAPDH or HNRNPL . RQ represents relative quantification of EIF4H determined by qRT-PCR, calculated using the ddCt method with either GAPDH or HNRNPL as the control gene, relative to the CD34+ cord blood (CB) sample. The X axis indicates the leukemic sample ID. CV (expressed as a percentage) indicates the coefficient of variation and equals the standard deviation divided by the mean RQ of CD33 calculated using the indicated control gene. MFC (mean fold change) represents the maximum divided by minimum RQ value.
    Figure Legend Snippet: Comparison of EIF4H gene expression values calculated with GAPDH or HNRNPL . RQ represents relative quantification of EIF4H determined by qRT-PCR, calculated using the ddCt method with either GAPDH or HNRNPL as the control gene, relative to the CD34+ cord blood (CB) sample. The X axis indicates the leukemic sample ID. CV (expressed as a percentage) indicates the coefficient of variation and equals the standard deviation divided by the mean RQ of CD33 calculated using the indicated control gene. MFC (mean fold change) represents the maximum divided by minimum RQ value.

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

    Average expression consistency of control genes in qRT-PCR. Average expression consistency (M) was calculated with the GeNorm algorithm [18] based on qRT-PCR for the indicated control gene on a panel of 14 leukemia samples and one cord blood sample. Lower M values relate to genes which proved to have more consistent expression levels across the samples used.
    Figure Legend Snippet: Average expression consistency of control genes in qRT-PCR. Average expression consistency (M) was calculated with the GeNorm algorithm [18] based on qRT-PCR for the indicated control gene on a panel of 14 leukemia samples and one cord blood sample. Lower M values relate to genes which proved to have more consistent expression levels across the samples used.

    Techniques Used: Expressing, Quantitative RT-PCR

    24) Product Images from "PIWIL1/piRNA-DQ593109 Regulates the Permeability of the Blood-Tumor Barrier via the MEG3/miR-330-5p/RUNX3 Axis"

    Article Title: PIWIL1/piRNA-DQ593109 Regulates the Permeability of the Blood-Tumor Barrier via the MEG3/miR-330-5p/RUNX3 Axis

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2017.12.020

    miR-330-5p Was a Mediator of MEG3-Regulated BTB Permeability (A) Effect of MEG3 overexpression on miR-330-5p levels in GECs by qRT-PCR. Data represent mean ± SD (n = 5, each). * p
    Figure Legend Snippet: miR-330-5p Was a Mediator of MEG3-Regulated BTB Permeability (A) Effect of MEG3 overexpression on miR-330-5p levels in GECs by qRT-PCR. Data represent mean ± SD (n = 5, each). * p

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

    Downregulation of piR-DQ593109 Increased BTB Permeability as well as Inhibited the Expression Levels of ZO-1, Occludin, and Claudin-5 (A) piRNA microarray analysis of total RNAs isolated from EC and GEC cells. Red indicates high relative expression and green indicates low relative expression. (B) Relative expression levels of piR-DQ569979, piR-DQ585094, piR-DQ-582921, and piR-DQ593109 determined by qRT-PCR. Data represent mean ± SD (n = 5, each). * p
    Figure Legend Snippet: Downregulation of piR-DQ593109 Increased BTB Permeability as well as Inhibited the Expression Levels of ZO-1, Occludin, and Claudin-5 (A) piRNA microarray analysis of total RNAs isolated from EC and GEC cells. Red indicates high relative expression and green indicates low relative expression. (B) Relative expression levels of piR-DQ569979, piR-DQ585094, piR-DQ-582921, and piR-DQ593109 determined by qRT-PCR. Data represent mean ± SD (n = 5, each). * p

    Techniques Used: Permeability, Expressing, Microarray, Isolation, Quantitative RT-PCR

    MEG3 Was Involved in PIWIL1/piR-DQ593109 Complex-Regulated BTB Permeability (A) Relative MEG3 expression levels determined by qRT-PCR in ECs and GECs. Data represent mean ± SD (n = 5, each). * p
    Figure Legend Snippet: MEG3 Was Involved in PIWIL1/piR-DQ593109 Complex-Regulated BTB Permeability (A) Relative MEG3 expression levels determined by qRT-PCR in ECs and GECs. Data represent mean ± SD (n = 5, each). * p

    Techniques Used: Permeability, Expressing, Quantitative RT-PCR

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

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

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

    doi: 10.1073/pnas.1519197113

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

    Techniques Used: Mouse Assay, Quantitative RT-PCR

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

    Techniques Used: Migration, Quantitative RT-PCR

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

    Techniques Used: Migration, Quantitative RT-PCR

    26) Product Images from "Host-to-Host Transmission of Streptococcus pneumoniae Is Driven by Its Inflammatory Toxin, Pneumolysin"

    Article Title: Host-to-Host Transmission of Streptococcus pneumoniae Is Driven by Its Inflammatory Toxin, Pneumolysin

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2016.12.005

    Pneumococcal Shedding Requires URT Inflammation (A–C) Pups colonized at age 4 days with T4S were treated IN twice daily with dexamethasone (Dex) or PBS-vehicle control. Daily shedding quantified in secretions from ages 5 to 9 days from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup on a single day (A). Colonization density in URT lavages obtained from Dex- or PBS-treated pups at age 9 days with the median value shown (B). Gene expression for the Dex-treated relative to PBS-treated pups at age 9 days measured by qRT-PCR for the chemokine/cytokine shown (C). Values are ±SEM (n = 5–7). (D–F) WT or congenic pafr −/− pups were colonized at age 4 days with T4S. Daily shedding quantified in secretions from ages 5 to 9 days from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup (D). Colonization density in URT lavages obtained at age 9 days with the median value shown (E). Gene expression in colonized pafr −/− pups at age 9 days relative to PBS (mock)-inoculated pups measured by qRT-PCR for the chemokine/cytokine shown (F). Values are ±SEM (n = 7–9). Dashed line represents the 300 CFU threshold level described in the Results. **p
    Figure Legend Snippet: Pneumococcal Shedding Requires URT Inflammation (A–C) Pups colonized at age 4 days with T4S were treated IN twice daily with dexamethasone (Dex) or PBS-vehicle control. Daily shedding quantified in secretions from ages 5 to 9 days from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup on a single day (A). Colonization density in URT lavages obtained from Dex- or PBS-treated pups at age 9 days with the median value shown (B). Gene expression for the Dex-treated relative to PBS-treated pups at age 9 days measured by qRT-PCR for the chemokine/cytokine shown (C). Values are ±SEM (n = 5–7). (D–F) WT or congenic pafr −/− pups were colonized at age 4 days with T4S. Daily shedding quantified in secretions from ages 5 to 9 days from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup (D). Colonization density in URT lavages obtained at age 9 days with the median value shown (E). Gene expression in colonized pafr −/− pups at age 9 days relative to PBS (mock)-inoculated pups measured by qRT-PCR for the chemokine/cytokine shown (F). Values are ±SEM (n = 7–9). Dashed line represents the 300 CFU threshold level described in the Results. **p

    Techniques Used: Expressing, Quantitative RT-PCR

    Recognition of Pneumococci and Pneumococcal Products Increases URT Inflammation and Promotes Shedding (A–D) WT or congenic tlr2 −/− pups were colonized at age 4 days with T4S. Daily shedding quantified in secretions from ages 5 to 9 days from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup on a single day (A). Dashed line represents the 300 CFU threshold level described in the Results. Colonization density in URT lavages obtained at age 9 days with the median value shown (B). Numbers of neutrophils as determined by flow cytometry (CD45 + , CD11b + , and Ly6G + events) in URT lavages obtained from tlr2 −/− or PBS (mock)-inoculated pups at age 7 days (C). Values are ±SEM (n = 8–11). Gene expression in colonized tlr2 −/− pups relative to PBS (mock)-inoculated pups at age 7 days measured by qRT-PCR for the chemokine/cytokine shown (D). Values are ±SEM (n = 8). (E–H) WT pups were colonized with the ply − strain at age 4 days. From ages 4 to 8 days, pups were given a daily IN dose (100 ng/pup) of purified pneumolysin (Ply), a non-pore-forming toxoid with the W433F point mutation (PdB), or PBS-vehicle control. Daily shedding quantified in secretions from ages 5 to 9 days from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup (E). Colonization density in URT lavages obtained at age 9 days with the median value shown (F). Numbers of neutrophils as determined by flow cytometry (CD45 + , CD11b + , and Ly6G + events) in URT lavages at age 9 days (G). Values are ±SEM (n = 8). Gene expression relative to PBS (mock)-inoculated pups measured by qRT-PCR for the chemokine/cytokine shown at age 9 days (H). Values are ±SEM (n = 10–18). *p
    Figure Legend Snippet: Recognition of Pneumococci and Pneumococcal Products Increases URT Inflammation and Promotes Shedding (A–D) WT or congenic tlr2 −/− pups were colonized at age 4 days with T4S. Daily shedding quantified in secretions from ages 5 to 9 days from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup on a single day (A). Dashed line represents the 300 CFU threshold level described in the Results. Colonization density in URT lavages obtained at age 9 days with the median value shown (B). Numbers of neutrophils as determined by flow cytometry (CD45 + , CD11b + , and Ly6G + events) in URT lavages obtained from tlr2 −/− or PBS (mock)-inoculated pups at age 7 days (C). Values are ±SEM (n = 8–11). Gene expression in colonized tlr2 −/− pups relative to PBS (mock)-inoculated pups at age 7 days measured by qRT-PCR for the chemokine/cytokine shown (D). Values are ±SEM (n = 8). (E–H) WT pups were colonized with the ply − strain at age 4 days. From ages 4 to 8 days, pups were given a daily IN dose (100 ng/pup) of purified pneumolysin (Ply), a non-pore-forming toxoid with the W433F point mutation (PdB), or PBS-vehicle control. Daily shedding quantified in secretions from ages 5 to 9 days from nasal secretions with median values indicated and each symbol representing the CFU observed from a single pup (E). Colonization density in URT lavages obtained at age 9 days with the median value shown (F). Numbers of neutrophils as determined by flow cytometry (CD45 + , CD11b + , and Ly6G + events) in URT lavages at age 9 days (G). Values are ±SEM (n = 8). Gene expression relative to PBS (mock)-inoculated pups measured by qRT-PCR for the chemokine/cytokine shown at age 9 days (H). Values are ±SEM (n = 10–18). *p

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR, Purification, Mutagenesis

    Pneumococcal Shedding Correlates with Upper Respiratory Tract Inflammation (A) 4-day-old pups were intranasally (IN) inoculated with ~2,000 CFUs S. pneumoniae strain T4S. (B) Daily shedding was collected and quantified from nasal secretions on the days shown with median values indicated and each symbol representing the CFU observed from a single pup. Statistical analysis compares shedding on ages 6 and 9 days. Dashed line represents the 300 CFU threshold level described in the Results. (C) Colonization density for T4S in cultures of upper respiratory tract (URT) lavages obtained from pups at the age indicated with the median value shown. (D) Numbers of neutrophils as determined by flow cytometry (CD45 + , CD11b + , and Ly6G + events) in URT lavages obtained at the age indicated in colonized pups. Values are ±SEM (n = 5–11). (E) The URT was lavaged with RLT RNA lysis buffer to isolate RNA from the epithelium to make cDNA from T4S colonized pups. Gene expression relative to PBS (mock)-inoculated mice was measured by qRT-PCR for the chemokine/cytokine shown. Values are ±SEM (n = 5–8). (F) IL-1β measured by ELISA in URT lavages obtained at age 7 days for T4S or PBS (mock)-colonized pups. Values are ±SEM (n = 6–11). *p
    Figure Legend Snippet: Pneumococcal Shedding Correlates with Upper Respiratory Tract Inflammation (A) 4-day-old pups were intranasally (IN) inoculated with ~2,000 CFUs S. pneumoniae strain T4S. (B) Daily shedding was collected and quantified from nasal secretions on the days shown with median values indicated and each symbol representing the CFU observed from a single pup. Statistical analysis compares shedding on ages 6 and 9 days. Dashed line represents the 300 CFU threshold level described in the Results. (C) Colonization density for T4S in cultures of upper respiratory tract (URT) lavages obtained from pups at the age indicated with the median value shown. (D) Numbers of neutrophils as determined by flow cytometry (CD45 + , CD11b + , and Ly6G + events) in URT lavages obtained at the age indicated in colonized pups. Values are ±SEM (n = 5–11). (E) The URT was lavaged with RLT RNA lysis buffer to isolate RNA from the epithelium to make cDNA from T4S colonized pups. Gene expression relative to PBS (mock)-inoculated mice was measured by qRT-PCR for the chemokine/cytokine shown. Values are ±SEM (n = 5–8). (F) IL-1β measured by ELISA in URT lavages obtained at age 7 days for T4S or PBS (mock)-colonized pups. Values are ±SEM (n = 6–11). *p

    Techniques Used: Flow Cytometry, Cytometry, Lysis, Expressing, Mouse Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    27) Product Images from "Down-regulation of cholinergic signaling in the habenula induces anhedonia-like behavior"

    Article Title: Down-regulation of cholinergic signaling in the habenula induces anhedonia-like behavior

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01088-6

    Effect of chronic restraint stress on the expression of cholinergic system genes in the rat habenula. ( a ) Schematic diagram of a generalized cholinergic synapse. The choline transporter protein (CHT) delivers choline into the cytoplasm, where choline acetyltransferase (CHAT) catalyze the transfer of an acetyl group from the coenzyme, acetyl Co-A, to choline, generating acetylcholine, and the vesicular acetylcholine transporter (VACHT) packs acetylcholine into the vesicle. Presynaptic nicotinic acetylcholine receptors (nAChR; CHRNA3, CHRNB3 and CHRNB4) modulate the release of neurotransmitter. ( b – h ) qRT-PCR was used to quantify mRNA expression levels of six cholinergic system genes in the habenula of rats exposed to CRS. The mRNA expression levels of CHAT, VACHT, CHT, CHRNA3, CHRNB3, and CHRNB4 were significantly reduced by CRS. Values for each individual gene were normalized to the mean of the reference gene GAPDH. CAMK2B was not changed by CRS. Data represent mean ± SEM. NS, n = 4; CRS, n = 4; * P
    Figure Legend Snippet: Effect of chronic restraint stress on the expression of cholinergic system genes in the rat habenula. ( a ) Schematic diagram of a generalized cholinergic synapse. The choline transporter protein (CHT) delivers choline into the cytoplasm, where choline acetyltransferase (CHAT) catalyze the transfer of an acetyl group from the coenzyme, acetyl Co-A, to choline, generating acetylcholine, and the vesicular acetylcholine transporter (VACHT) packs acetylcholine into the vesicle. Presynaptic nicotinic acetylcholine receptors (nAChR; CHRNA3, CHRNB3 and CHRNB4) modulate the release of neurotransmitter. ( b – h ) qRT-PCR was used to quantify mRNA expression levels of six cholinergic system genes in the habenula of rats exposed to CRS. The mRNA expression levels of CHAT, VACHT, CHT, CHRNA3, CHRNB3, and CHRNB4 were significantly reduced by CRS. Values for each individual gene were normalized to the mean of the reference gene GAPDH. CAMK2B was not changed by CRS. Data represent mean ± SEM. NS, n = 4; CRS, n = 4; * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Expression of cholinergic system genes in habenula tissue obtained from persons with MDD who died by suicide. ( a ) The human brain was separated into two hemispheres by slicing through the center of the brain stem and cerebellum. Each hemisphere was sliced coronally into 18–20 slabs (slab 1, frontal lobe; slab 18, occipital lobe) and these slabs were frozen. The habenula is nearly adjacent to the pineal gland and is contained in the 11th of 18 slabs. To isolate habenula samples, a carving tool with a round tip was used to make long grooves one at a time. ( c – i ) The mRNA expression levels of CHAT, VACHT, CHT, CHRNA3, CHRNB3, and CHRNB4 were evaluated using TaqMan probe-based qRT-PCR. All cholinergic system genes examined were down-regulated in suicide, but not the control CAMK2B gene ( b ), an excitatory neuronal marker. GAPDH and β-Actin were used as reference genes to normalize qRT-PCR data. Data represent mean ± SEM (CON, Control, n = 11; MDD, n = 12 subjects; * P
    Figure Legend Snippet: Expression of cholinergic system genes in habenula tissue obtained from persons with MDD who died by suicide. ( a ) The human brain was separated into two hemispheres by slicing through the center of the brain stem and cerebellum. Each hemisphere was sliced coronally into 18–20 slabs (slab 1, frontal lobe; slab 18, occipital lobe) and these slabs were frozen. The habenula is nearly adjacent to the pineal gland and is contained in the 11th of 18 slabs. To isolate habenula samples, a carving tool with a round tip was used to make long grooves one at a time. ( c – i ) The mRNA expression levels of CHAT, VACHT, CHT, CHRNA3, CHRNB3, and CHRNB4 were evaluated using TaqMan probe-based qRT-PCR. All cholinergic system genes examined were down-regulated in suicide, but not the control CAMK2B gene ( b ), an excitatory neuronal marker. GAPDH and β-Actin were used as reference genes to normalize qRT-PCR data. Data represent mean ± SEM (CON, Control, n = 11; MDD, n = 12 subjects; * P

    Techniques Used: Expressing, Quantitative RT-PCR, Marker

    28) Product Images from "Alpha or beta human chorionic gonadotropin knockdown decrease BeWo cell fusion by down-regulating PKA and CREB activation"

    Article Title: Alpha or beta human chorionic gonadotropin knockdown decrease BeWo cell fusion by down-regulating PKA and CREB activation

    Journal: Scientific Reports

    doi: 10.1038/srep11210

    Effect of α- and β-hCG silencing on the expression of syncytin-1 and syndecan-1. Control, α- and β-hCG silenced cells were treated with forskolin (25 μM) for 24, 48 and 72 h. Subsequently, total RNA and cell lysates were prepared and profiles of syncytin-1 and syndecan-1 were determined by qRT-PCR and Western blotting. Panels ( a ) and ( b ) show qRT-PCR data comparing transcript levels of syncytin-1 and syndecan-1 respectively, in control, α- and β-hCG silenced cells after 24, 48 and 72 h of foskolin treatment. Each bar represents relative ΔCt values after normalization with the 18S rRNA, expressed as mean ± s.e.m. of three independent experiments performed in triplicates. Panel ( c ) represents comparative expression profiles of syncytin-1 and syndecan-1 between control and α- and β-hCG silenced cells on forskolin treatment with β-actin used as an internal control. The cropped blots were run under the same experimental conditions and the full length blots can be viewed in Supplementary Fig. S12 online . Values are expressed as mean ± s.e.m. of band intensity of three independent experiments. Representative blots for the same are appended with the graphs. # p
    Figure Legend Snippet: Effect of α- and β-hCG silencing on the expression of syncytin-1 and syndecan-1. Control, α- and β-hCG silenced cells were treated with forskolin (25 μM) for 24, 48 and 72 h. Subsequently, total RNA and cell lysates were prepared and profiles of syncytin-1 and syndecan-1 were determined by qRT-PCR and Western blotting. Panels ( a ) and ( b ) show qRT-PCR data comparing transcript levels of syncytin-1 and syndecan-1 respectively, in control, α- and β-hCG silenced cells after 24, 48 and 72 h of foskolin treatment. Each bar represents relative ΔCt values after normalization with the 18S rRNA, expressed as mean ± s.e.m. of three independent experiments performed in triplicates. Panel ( c ) represents comparative expression profiles of syncytin-1 and syndecan-1 between control and α- and β-hCG silenced cells on forskolin treatment with β-actin used as an internal control. The cropped blots were run under the same experimental conditions and the full length blots can be viewed in Supplementary Fig. S12 online . Values are expressed as mean ± s.e.m. of band intensity of three independent experiments. Representative blots for the same are appended with the graphs. # p

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

    Expression profiles of β-catenin and transcript for Wnt 10b in α- and β-hCG silenced BeWo cells treated with forskolin. Control and α- and β-hCG silenced BeWo cells were treated with forskolin (25 μM) for 24, 48 and 72 h; cell lysates for Western blotting and total RNA for qRT-PCR were processed as described in Methods . Panel (a) shows densitometeric plots of β-catenin with β-actin used as an internal control. Data is represented as mean ± s.e.m. of at least three experiments. Representative blots for the same are also shown. Panel (b) represents Wnt 10b transcript profile at varying time points in the control and α- and β-hCG silenced BeWo cells. Each bar represents relative ΔCt values after normalization with the 18S rRNA, expressed as mean ± s.e.m. of three independent experiments performed in triplicates. *p
    Figure Legend Snippet: Expression profiles of β-catenin and transcript for Wnt 10b in α- and β-hCG silenced BeWo cells treated with forskolin. Control and α- and β-hCG silenced BeWo cells were treated with forskolin (25 μM) for 24, 48 and 72 h; cell lysates for Western blotting and total RNA for qRT-PCR were processed as described in Methods . Panel (a) shows densitometeric plots of β-catenin with β-actin used as an internal control. Data is represented as mean ± s.e.m. of at least three experiments. Representative blots for the same are also shown. Panel (b) represents Wnt 10b transcript profile at varying time points in the control and α- and β-hCG silenced BeWo cells. Each bar represents relative ΔCt values after normalization with the 18S rRNA, expressed as mean ± s.e.m. of three independent experiments performed in triplicates. *p

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

    Effect of α- and β-hCG silencing on forskolin mediated syncytialization of BeWo cells. BeWo cells knockdown for α- and β-hCG were made using lentiviral shRNA as described in Methods . Efficacy of silencing of α- and β-hCG transcript was confirmed by qRT-PCR using specific primers and secreted hCG by ELISA. The effect of α- and β-hCG silencing on fusion was studied at 48 and 72 h of forskolin (25 μM) treatment by desmoplakin I + II staining. Panels ( a ) and ( b ) show qRT-PCR data comparing transcript levels of α- and β-hCG respectively, in control, α- and β-hCG silenced cells on foskolin treatment. Each bar represents relative ΔCt values after normalization with the 18 S rRNA, expressed as mean ± s.e.m. of three independent experiments performed in triplicates. Panel ( c ) compares the fold change in fusion on treatment with forskolin (25 μM) in control and α- and β-hCG silenced BeWo cells when compared with their respective untreated controls at 48 and 72 h. Values are shown as mean ± s.e.m. of three independent experiments. Panel ( d ) shows hCG secreted by control, α- and β-hCG silenced cells in response to forskolin treatment and represented as mean ± s.e.m. of three independent experiments performed in duplicates. # p
    Figure Legend Snippet: Effect of α- and β-hCG silencing on forskolin mediated syncytialization of BeWo cells. BeWo cells knockdown for α- and β-hCG were made using lentiviral shRNA as described in Methods . Efficacy of silencing of α- and β-hCG transcript was confirmed by qRT-PCR using specific primers and secreted hCG by ELISA. The effect of α- and β-hCG silencing on fusion was studied at 48 and 72 h of forskolin (25 μM) treatment by desmoplakin I + II staining. Panels ( a ) and ( b ) show qRT-PCR data comparing transcript levels of α- and β-hCG respectively, in control, α- and β-hCG silenced cells on foskolin treatment. Each bar represents relative ΔCt values after normalization with the 18 S rRNA, expressed as mean ± s.e.m. of three independent experiments performed in triplicates. Panel ( c ) compares the fold change in fusion on treatment with forskolin (25 μM) in control and α- and β-hCG silenced BeWo cells when compared with their respective untreated controls at 48 and 72 h. Values are shown as mean ± s.e.m. of three independent experiments. Panel ( d ) shows hCG secreted by control, α- and β-hCG silenced cells in response to forskolin treatment and represented as mean ± s.e.m. of three independent experiments performed in duplicates. # p

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

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

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

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14854

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

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

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

    Techniques Used: Expressing, Quantitative RT-PCR

    30) Product Images from "Long Non-Coding RNA HOTAIR Promotes Cell Migration and Invasion via Down-Regulation of RNA Binding Motif Protein 38 in Hepatocellular Carcinoma Cells"

    Article Title: Long Non-Coding RNA HOTAIR Promotes Cell Migration and Invasion via Down-Regulation of RNA Binding Motif Protein 38 in Hepatocellular Carcinoma Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms15034060

    Effects of QKI, CD82, and RBM38 knockdown on cell proliferation. ( A ) effective silencing of QKI, CD82, and RBM38 expression in Bel-7402 cells after 48 h siRNA treatment according to qRT-PCR analysis; and ( B ) Viable cell numbers were detected by CCK-8 assay after transferred to 96-wells plates for 24, 48, and 72 h.
    Figure Legend Snippet: Effects of QKI, CD82, and RBM38 knockdown on cell proliferation. ( A ) effective silencing of QKI, CD82, and RBM38 expression in Bel-7402 cells after 48 h siRNA treatment according to qRT-PCR analysis; and ( B ) Viable cell numbers were detected by CCK-8 assay after transferred to 96-wells plates for 24, 48, and 72 h.

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

    Upregulation of QKI, CD82, and RBM38 were validated after knockdown of HOTAIR . ( A ) Bel-7402 cells; ( B ) HepG2 cells were transiently transfected with HOTAIR siRNA for 48 h to detect QKI, CD82, and RBM38 expression by qRT-PCR compared with the siGFP group ( * indicated p
    Figure Legend Snippet: Upregulation of QKI, CD82, and RBM38 were validated after knockdown of HOTAIR . ( A ) Bel-7402 cells; ( B ) HepG2 cells were transiently transfected with HOTAIR siRNA for 48 h to detect QKI, CD82, and RBM38 expression by qRT-PCR compared with the siGFP group ( * indicated p

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR

    The expression levels of RBM38 were downregulated in HCC resection specimens. The expression levels of QKI ( A ); CD82 ( B ) and RBM38 ( C ) in HCC and paired adjacent noncancerous tissues were measured by qRT-PCR and normalized to GAPDH. The significant differences between samples were analyzed using the Student’s t test. (log 2, ** p
    Figure Legend Snippet: The expression levels of RBM38 were downregulated in HCC resection specimens. The expression levels of QKI ( A ); CD82 ( B ) and RBM38 ( C ) in HCC and paired adjacent noncancerous tissues were measured by qRT-PCR and normalized to GAPDH. The significant differences between samples were analyzed using the Student’s t test. (log 2, ** p

    Techniques Used: Expressing, Quantitative RT-PCR

    31) Product Images from "Inactivation of SACE_3446, a TetR family transcriptional regulator, stimulates erythromycin production in Saccharopolyspora erythraea"

    Article Title: Inactivation of SACE_3446, a TetR family transcriptional regulator, stimulates erythromycin production in Saccharopolyspora erythraea

    Journal: Synthetic and Systems Biotechnology

    doi: 10.1016/j.synbio.2016.01.004

    Effects of SACE_3446/SACE_3986 deletion and SACE_7301 overexpression on erythromycin production and gene transcription in the industrial strain WB. (a) Erythromycin A yield of WB and its derivatives cultured in flasks for 6 days. The mean values of at least three replicates are shown, with the standard deviation indicated by error bars. (b) qRT-PCR analysis of eryAI and ermE in WB, WBΔ 3446 Δ 3986 , and WBΔ 3446 Δ 3986 /3 × 7301 cultured for 4 days. (c) Time course of erythromycin A production of WB, WBΔ 3446 and WBΔ 3446 Δ 3986 in a 5-L fermentor. One of the representative datasets is shown.
    Figure Legend Snippet: Effects of SACE_3446/SACE_3986 deletion and SACE_7301 overexpression on erythromycin production and gene transcription in the industrial strain WB. (a) Erythromycin A yield of WB and its derivatives cultured in flasks for 6 days. The mean values of at least three replicates are shown, with the standard deviation indicated by error bars. (b) qRT-PCR analysis of eryAI and ermE in WB, WBΔ 3446 Δ 3986 , and WBΔ 3446 Δ 3986 /3 × 7301 cultured for 4 days. (c) Time course of erythromycin A production of WB, WBΔ 3446 and WBΔ 3446 Δ 3986 in a 5-L fermentor. One of the representative datasets is shown.

    Techniques Used: Over Expression, Western Blot, Cell Culture, Standard Deviation, Quantitative RT-PCR

    SACE_7301 indirectly regulated the transcriptions of SACE_3446 and SACE_3986 . Gene transcription was compared between A226 and deletion mutants of three TetR family genes ( SACE_3446 , SACE_3986 , and SACE_7301 ) cultured for 4 days via qRT-PCR assay. (a) Effects of SACE_3446 disruption on the transcriptions of SACE_3986 and SACE_7301 . (b) Effects of SACE_3986 disruption on the transcriptions of SACE_3446 and SACE_7301 . (c) Effects of SACE_7301 disruption on the transcriptions of SACE_3446 and SACE_3986 . The mean values of three independent experiments are shown, with the standard deviation indicated by error bars.
    Figure Legend Snippet: SACE_7301 indirectly regulated the transcriptions of SACE_3446 and SACE_3986 . Gene transcription was compared between A226 and deletion mutants of three TetR family genes ( SACE_3446 , SACE_3986 , and SACE_7301 ) cultured for 4 days via qRT-PCR assay. (a) Effects of SACE_3446 disruption on the transcriptions of SACE_3986 and SACE_7301 . (b) Effects of SACE_3986 disruption on the transcriptions of SACE_3446 and SACE_7301 . (c) Effects of SACE_7301 disruption on the transcriptions of SACE_3446 and SACE_3986 . The mean values of three independent experiments are shown, with the standard deviation indicated by error bars.

    Techniques Used: Cell Culture, Quantitative RT-PCR, Standard Deviation

    Effects of SACE_3446 disruption on the transcription of eryAI , ermE , and SACE_3447 . qRT-PCR was used to quantify the amounts of transcripts produced by A226 and Δ SACE_3446 cultured for 2 days (a) and 4 days (b). The mean values of three independent experiments are shown, with the standard deviation indicated by error bars.
    Figure Legend Snippet: Effects of SACE_3446 disruption on the transcription of eryAI , ermE , and SACE_3447 . qRT-PCR was used to quantify the amounts of transcripts produced by A226 and Δ SACE_3446 cultured for 2 days (a) and 4 days (b). The mean values of three independent experiments are shown, with the standard deviation indicated by error bars.

    Techniques Used: Quantitative RT-PCR, Produced, Cell Culture, Standard Deviation

    32) Product Images from "The Helicase Aquarius/EMB-4 Is Required to Overcome Intronic Barriers to Allow Nuclear RNAi Pathways to Heritably Silence Transcription"

    Article Title: The Helicase Aquarius/EMB-4 Is Required to Overcome Intronic Barriers to Allow Nuclear RNAi Pathways to Heritably Silence Transcription

    Journal: Developmental Cell

    doi: 10.1016/j.devcel.2017.07.002

    Aquarius/EMB-4 Is Required for the piRNA-Mediated Silencing of a Sensor Transgene (A) piRNA sensor transgene and its expression pattern in C. elegans germline. (B) Fluorescent microscope images of wild-type and mutant animal germlines with an integrated single-copy piRNA sensor transgene (germline boundaries are marked by white dotted lines). (C) qRT-PCR analysis of GFP expression in animals described in Figure 3 B (two biological replicates with at least two technical replicates). See also Figure S3 .
    Figure Legend Snippet: Aquarius/EMB-4 Is Required for the piRNA-Mediated Silencing of a Sensor Transgene (A) piRNA sensor transgene and its expression pattern in C. elegans germline. (B) Fluorescent microscope images of wild-type and mutant animal germlines with an integrated single-copy piRNA sensor transgene (germline boundaries are marked by white dotted lines). (C) qRT-PCR analysis of GFP expression in animals described in Figure 3 B (two biological replicates with at least two technical replicates). See also Figure S3 .

    Techniques Used: Expressing, Microscopy, Mutagenesis, Quantitative RT-PCR

    SILAC Proteomics Identifies Aquarius/EMB-4 as an Interactor of the Nuclear Argonaute HRDE-1 (A) SILAC labeling and IP scheme for wild-type (heavy labeled) and hrde-1 ( tm1200 ) mutants (light labeled) using anti-HRDE-1 antibodies. (B) Known protein interactions identified by the STRING database for mammalian orthologs of nuclear HRDE-1 interactors. Gray lines indicate known protein-protein interactions, red circles highlight known RNA-processing factors, blue circles highlight RNA pol II subunits, and gray circles highlight nuclear pore complex subunits. (C) Aquarius and two exon-junction complex proteins eIF4A3 and ALY are detected in HRDE-1 IPs (second column, mean log 2 fold enrichment heavy/light and number of replicates detected) and in D. melanogaster PIWI IPs (third column, number of IPs detected/total number of IPs). The effect on TE desilencing in D. melanogaster upon RNAi knockdown (fourth column, number of TEs desilenced out of four tested). (D) EMB-4 domain structure based on the mammalian homolog Aquarius and the position of emb-4 mutations used in this study. (E) Validation of protein-protein interactions between HRDE-1 and EMB-4 using anti-HRDE-1 antibodies to immunoprecipitate HRDE-1 complexes in CRISPR-tagged OLLAS-EMB-4 strain with or without RNase treatment (immunoglobulin G [IgG] negative control). (F) qRT-PCR analysis of emb-4 mRNA expression at different developmental stages (embryo to gravid adult), in animals lacking sperm (female germline fem-1 ( hc17 )), in animals lacking oocytes (male germline fog-2 ( q71 )), and in animals lacking a germline (soma only glp-4 ( bn2 )). fem-1 ( hc17 ), fog-2 ( q71 ), and glp-4 ( bn2 ) are temperature-sensitive mutants and were grown at 25°C alongside the wild-type gravid adult control. Error bars represent SD. (G) Western blot analysis of EMB-4 protein levels using anti-EMB-4 antibodies for the same conditions as in Figure 2 A. (H) Localization of EMB-4 protein in the germline of adult animals. See also Figures S1 and S2 .
    Figure Legend Snippet: SILAC Proteomics Identifies Aquarius/EMB-4 as an Interactor of the Nuclear Argonaute HRDE-1 (A) SILAC labeling and IP scheme for wild-type (heavy labeled) and hrde-1 ( tm1200 ) mutants (light labeled) using anti-HRDE-1 antibodies. (B) Known protein interactions identified by the STRING database for mammalian orthologs of nuclear HRDE-1 interactors. Gray lines indicate known protein-protein interactions, red circles highlight known RNA-processing factors, blue circles highlight RNA pol II subunits, and gray circles highlight nuclear pore complex subunits. (C) Aquarius and two exon-junction complex proteins eIF4A3 and ALY are detected in HRDE-1 IPs (second column, mean log 2 fold enrichment heavy/light and number of replicates detected) and in D. melanogaster PIWI IPs (third column, number of IPs detected/total number of IPs). The effect on TE desilencing in D. melanogaster upon RNAi knockdown (fourth column, number of TEs desilenced out of four tested). (D) EMB-4 domain structure based on the mammalian homolog Aquarius and the position of emb-4 mutations used in this study. (E) Validation of protein-protein interactions between HRDE-1 and EMB-4 using anti-HRDE-1 antibodies to immunoprecipitate HRDE-1 complexes in CRISPR-tagged OLLAS-EMB-4 strain with or without RNase treatment (immunoglobulin G [IgG] negative control). (F) qRT-PCR analysis of emb-4 mRNA expression at different developmental stages (embryo to gravid adult), in animals lacking sperm (female germline fem-1 ( hc17 )), in animals lacking oocytes (male germline fog-2 ( q71 )), and in animals lacking a germline (soma only glp-4 ( bn2 )). fem-1 ( hc17 ), fog-2 ( q71 ), and glp-4 ( bn2 ) are temperature-sensitive mutants and were grown at 25°C alongside the wild-type gravid adult control. Error bars represent SD. (G) Western blot analysis of EMB-4 protein levels using anti-EMB-4 antibodies for the same conditions as in Figure 2 A. (H) Localization of EMB-4 protein in the germline of adult animals. See also Figures S1 and S2 .

    Techniques Used: Labeling, CRISPR, Negative Control, Quantitative RT-PCR, Expressing, Western Blot

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

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

    Journal: Nature

    doi: 10.1038/nature12447

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

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

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

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

    34) Product Images from "Myb proteins regulate expression of histone variant H2A.Z during thymocyte development"

    Article Title: Myb proteins regulate expression of histone variant H2A.Z during thymocyte development

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2007.02697.x

    Elevated histone variant H2A.Z mRNA in vMyb4 T-cell populations in vivo . (a) Flow cytometry of thymocytes (top and centre panels) and splenocytes (bottom panels) from vMyb4 transgenic mice and wild-type littermate controls (WT), stained with anti-CD4 and anti-CD8 antibodies. (b) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of H2A.Z mRNA expression using RNA from sorted T lymphocytes. Data are presented as means from triplicate qRT-PCR reactions performed from each of three independent sorts. Error bars represent standard error. DP, double positive; HPRT, hypoxanthine-guanine phosphoribosyltransferase.
    Figure Legend Snippet: Elevated histone variant H2A.Z mRNA in vMyb4 T-cell populations in vivo . (a) Flow cytometry of thymocytes (top and centre panels) and splenocytes (bottom panels) from vMyb4 transgenic mice and wild-type littermate controls (WT), stained with anti-CD4 and anti-CD8 antibodies. (b) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of H2A.Z mRNA expression using RNA from sorted T lymphocytes. Data are presented as means from triplicate qRT-PCR reactions performed from each of three independent sorts. Error bars represent standard error. DP, double positive; HPRT, hypoxanthine-guanine phosphoribosyltransferase.

    Techniques Used: Variant Assay, In Vivo, Flow Cytometry, Cytometry, Transgenic Assay, Mouse Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    Deletion of c-myb at the double negative 4 (DN4) stage reduces histone variant H2A.Z expression in thymic double positive (DP) subsets. (a) Fluorescence-activated cell sorting (FACS) profiles of thymocytes stained with antibodies against CD4 ( y -axis) and CD8 ( x -axis) from Myb F/F mice in the absence or presence of the CD4Cre transgene. (b) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of H2A.Z mRNA expression using RNA isolated from sorted T lymphocytes of Myb F/F CD4Cre mice. Data are presented as means from triplicate qRT-PCR reactions performed from each of three independent sorts. Error bars represent standard error. HPRT, hypoxanthine guanine phosphoribosyltransferase.
    Figure Legend Snippet: Deletion of c-myb at the double negative 4 (DN4) stage reduces histone variant H2A.Z expression in thymic double positive (DP) subsets. (a) Fluorescence-activated cell sorting (FACS) profiles of thymocytes stained with antibodies against CD4 ( y -axis) and CD8 ( x -axis) from Myb F/F mice in the absence or presence of the CD4Cre transgene. (b) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of H2A.Z mRNA expression using RNA isolated from sorted T lymphocytes of Myb F/F CD4Cre mice. Data are presented as means from triplicate qRT-PCR reactions performed from each of three independent sorts. Error bars represent standard error. HPRT, hypoxanthine guanine phosphoribosyltransferase.

    Techniques Used: Variant Assay, Expressing, Fluorescence, FACS, Staining, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Isolation

    Deletion of c-myb at the double negative 2 (DN2) stage reduces histone variant H2A.Z expression in DN3 thymocytes. (a) Fluorescence-activated cell sorting (FACS) profiles of thymocytes. Upper panel: DN thymocytes stained with antibodies against CD44 ( y -axis) and CD25 ( x -axis) from Myb F/F mice in the absence or presence of the CD2Cre transgene. A recombination activating gene (RAG)-2 –/– profile is also shown. DN2 (grey boxes) and DN3 (black boxes) populations used for mRNA preparation are indicated. Lower panel: FACS profiles of total thymocytes stained with antibodies against CD4 ( y -axis) and CD8 ( x -axis) from Myb F/F mice in the absence or presence of the CD2Cre transgene. Percentages of cells in each quadrant are shown. (b) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of H2A.Z mRNA expression using RNA isolated from sorted DN2 and DN3 cells of the indicated genotypes. Data are presented as means from triplicate qRT-PCR reactions. Error bars represent standard deviation.
    Figure Legend Snippet: Deletion of c-myb at the double negative 2 (DN2) stage reduces histone variant H2A.Z expression in DN3 thymocytes. (a) Fluorescence-activated cell sorting (FACS) profiles of thymocytes. Upper panel: DN thymocytes stained with antibodies against CD44 ( y -axis) and CD25 ( x -axis) from Myb F/F mice in the absence or presence of the CD2Cre transgene. A recombination activating gene (RAG)-2 –/– profile is also shown. DN2 (grey boxes) and DN3 (black boxes) populations used for mRNA preparation are indicated. Lower panel: FACS profiles of total thymocytes stained with antibodies against CD4 ( y -axis) and CD8 ( x -axis) from Myb F/F mice in the absence or presence of the CD2Cre transgene. Percentages of cells in each quadrant are shown. (b) Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis of H2A.Z mRNA expression using RNA isolated from sorted DN2 and DN3 cells of the indicated genotypes. Data are presented as means from triplicate qRT-PCR reactions. Error bars represent standard deviation.

    Techniques Used: Variant Assay, Expressing, Fluorescence, FACS, Staining, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Standard Deviation

    35) Product Images from "Decreased AGO2 and DCR1 in PBMCs from War Veterans with PTSD leads to diminished miRNA resulting in elevated inflammation"

    Article Title: Decreased AGO2 and DCR1 in PBMCs from War Veterans with PTSD leads to diminished miRNA resulting in elevated inflammation

    Journal: Translational Psychiatry

    doi: 10.1038/tp.2017.185

    STAT3 expression is lowered in the PBMCs of post-traumatic stress disorder (PTSD) patients. ( a , b ) Network showing predicted transcription factors for AGO2 and DCR1. ( c ) Transcript level of the three reported STAT3 variants in the PBMCs of PTSD patients as analyzed by RNA-seq (the values above the bars indicate log 2 fold-change). ( d ) Relative abundance of STAT3 transcripts in the PBMCs of PTSD patients after analysis by qRT-PCR with 22 control and 18 PTSD samples. Here, 18S rRNA was used as an internal control. ( e ) Relative abundance of AGO2, DCR1 and STAT3 transcripts 72 h post-knockdown of STAT3. ( f ) STAT3 was knocked down using siRNA in THP-1 cells and miRNAs were quantified after 72 h. The figure shows RE of miRNAs after knockdown of STAT3. (RE: relative enrichment). PBMC, peripheral blood mononuclear cell.
    Figure Legend Snippet: STAT3 expression is lowered in the PBMCs of post-traumatic stress disorder (PTSD) patients. ( a , b ) Network showing predicted transcription factors for AGO2 and DCR1. ( c ) Transcript level of the three reported STAT3 variants in the PBMCs of PTSD patients as analyzed by RNA-seq (the values above the bars indicate log 2 fold-change). ( d ) Relative abundance of STAT3 transcripts in the PBMCs of PTSD patients after analysis by qRT-PCR with 22 control and 18 PTSD samples. Here, 18S rRNA was used as an internal control. ( e ) Relative abundance of AGO2, DCR1 and STAT3 transcripts 72 h post-knockdown of STAT3. ( f ) STAT3 was knocked down using siRNA in THP-1 cells and miRNAs were quantified after 72 h. The figure shows RE of miRNAs after knockdown of STAT3. (RE: relative enrichment). PBMC, peripheral blood mononuclear cell.

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

    miRNAs downregulated in post-traumatic stress disorder (PTSD) target several pro-inflammatory genes. ( a ) Ingenuity Pathway Analysis (IPA) was performed with the dysregulated miRNAs in PTSD. The genes were included in the network and the miRNAs were connected by the IPA algorithm. The network shows direct interaction (predicted and proven) between miRNAs and target genes as indicated by solid arrows. ( b ) Principal component analysis (PCA) plot showing the relatedness of the samples. ( c ) IPA-generated network showing the interaction between miRNAs and targets after analyzing with the miRNAs obtained in the replicate samples. The red and blue colored target genes in the network correspond to up- and downregulated, respectively, validated for its expression level and reported either in the current manuscript or in our previous reports with studies on the same samples. For the miRNAs, the red color indicates upregulated and green indicates downregulated as per microarray analysis. ( d ) To validate the transcript levels of miRNA-targeted genes, qRT-PCR was performed to detect transcript of eight genes as shown in the graph (genes were selected based on the miRNA–gene interaction networks). The P- values of the genes under the curly brackets were
    Figure Legend Snippet: miRNAs downregulated in post-traumatic stress disorder (PTSD) target several pro-inflammatory genes. ( a ) Ingenuity Pathway Analysis (IPA) was performed with the dysregulated miRNAs in PTSD. The genes were included in the network and the miRNAs were connected by the IPA algorithm. The network shows direct interaction (predicted and proven) between miRNAs and target genes as indicated by solid arrows. ( b ) Principal component analysis (PCA) plot showing the relatedness of the samples. ( c ) IPA-generated network showing the interaction between miRNAs and targets after analyzing with the miRNAs obtained in the replicate samples. The red and blue colored target genes in the network correspond to up- and downregulated, respectively, validated for its expression level and reported either in the current manuscript or in our previous reports with studies on the same samples. For the miRNAs, the red color indicates upregulated and green indicates downregulated as per microarray analysis. ( d ) To validate the transcript levels of miRNA-targeted genes, qRT-PCR was performed to detect transcript of eight genes as shown in the graph (genes were selected based on the miRNA–gene interaction networks). The P- values of the genes under the curly brackets were

    Techniques Used: Indirect Immunoperoxidase Assay, Generated, Expressing, Microarray, Quantitative RT-PCR

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

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

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-676

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

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

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

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

    37) Product Images from "Androgen Receptor Pathway-Independent Prostate Cancer Is Sustained through FGF Signaling"

    Article Title: Androgen Receptor Pathway-Independent Prostate Cancer Is Sustained through FGF Signaling

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2017.09.003

    FGF Pathway and MAPK Activity in Cell Line and PDX Models of DNPC (A) Quantitation of the indicated transcripts by qRT-PCR in parental PacMet-UT1 cells and two independent PacMet-UT1 clones propagated after CRISPR/Cas9-mediated AR deletion. (B) Western immunoblot of AR protein in the indicated cell lines. (C) Quantitation of the indicated transcripts by qRT-PCR in the indicated cell lines. ***p
    Figure Legend Snippet: FGF Pathway and MAPK Activity in Cell Line and PDX Models of DNPC (A) Quantitation of the indicated transcripts by qRT-PCR in parental PacMet-UT1 cells and two independent PacMet-UT1 clones propagated after CRISPR/Cas9-mediated AR deletion. (B) Western immunoblot of AR protein in the indicated cell lines. (C) Quantitation of the indicated transcripts by qRT-PCR in the indicated cell lines. ***p

    Techniques Used: Activity Assay, Quantitation Assay, Quantitative RT-PCR, Clone Assay, CRISPR, Western Blot

    Characterization of a Model of AR Program-Independent Prostate Cancer (A) LNCaP cells with a doxycycline (Dox)-inducible shRNA targeting the AR (shAR) and an androgen-driven thymidine kinase gene (pATK) were starved of androgens (ADT) and treated with Dox to induce the AR-directed shRNA, then treated with ganciclovir to eliminate cells with AR-driven thymidine kinase expression. Scale bars, 10 µm. (B) qRT-PCR analysis of AR and PSA expression in LNCaP shAR/pATK and LNCaP APIPC with 1 nM R1881 or 1 µg/mL Dox treatment. Significance was determined by Student’s t test and data are presented as mean ± SEM (n = 4 replicates per data point); **p
    Figure Legend Snippet: Characterization of a Model of AR Program-Independent Prostate Cancer (A) LNCaP cells with a doxycycline (Dox)-inducible shRNA targeting the AR (shAR) and an androgen-driven thymidine kinase gene (pATK) were starved of androgens (ADT) and treated with Dox to induce the AR-directed shRNA, then treated with ganciclovir to eliminate cells with AR-driven thymidine kinase expression. Scale bars, 10 µm. (B) qRT-PCR analysis of AR and PSA expression in LNCaP shAR/pATK and LNCaP APIPC with 1 nM R1881 or 1 µg/mL Dox treatment. Significance was determined by Student’s t test and data are presented as mean ± SEM (n = 4 replicates per data point); **p

    Techniques Used: shRNA, Expressing, Quantitative RT-PCR

    38) Product Images from "High IFN- γ and low SLPI mark severe asthma in mice and humans"

    Article Title: High IFN- γ and low SLPI mark severe asthma in mice and humans

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI80911

    Inverse correlation between IFN-γ and SLPI expression in human SA. ( A ) SLPI mRNA expression analyzed by qRT-PCR in epithelial brushings of MMA and SA subjects. n = 17 and n = 20 for MMA and SA, respectively. ** P ≤ 0.01, Student’s unpaired t test. ( B ) Correlation analysis between BAL cell IFNG and airway epithelial cell–expressed SLPI mRNA in MMA (top) and SA (bottom) subjects. Spearman’s rank correlation test was used to calculate the correlation coefficient ( r s ) using GraphPad Prism software. Regression line for the SA cohorts is shown in the right panel. n = 9 and n = 13 for MMA and SA, respectively. ( C ) Primary airway (bronchial) epithelial cells from nonasthmatic human subjects were stimulated with rhIFN-γ ± anti–IFN-γ or left untreated for 8 hours. SLPI mRNA expression was analyzed by qRT-PCR, and fold change over untreated was calculated using HPRT as internal reference control. n = 5. *** P ≤ 0.001, 1-way ANOVA with Tukey’s post-hoc test. ( D ) Detection of hSLPI protein in sera of mice that received hSLPI expression plasmid by tail vein injection. * P ≤ 0.05, 1-way ANOVA with Tukey’s post-hoc test. ( E ) AHR measurement in mice subjected to the SA model with or without hSLPI introduced in an expression plasmid via hydrodynamic tail vein injection. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, 1-way ANOVA with Tukey’s post-hoc test. ( F ) Cytokine concentrations in whole lungs of mice treated as in E . ** P ≤ 0.01, 1-way ANOVA with Tukey’s post-hoc test. For D – F , data shown represent mean ± SEM and representative of 2 independent experiments.
    Figure Legend Snippet: Inverse correlation between IFN-γ and SLPI expression in human SA. ( A ) SLPI mRNA expression analyzed by qRT-PCR in epithelial brushings of MMA and SA subjects. n = 17 and n = 20 for MMA and SA, respectively. ** P ≤ 0.01, Student’s unpaired t test. ( B ) Correlation analysis between BAL cell IFNG and airway epithelial cell–expressed SLPI mRNA in MMA (top) and SA (bottom) subjects. Spearman’s rank correlation test was used to calculate the correlation coefficient ( r s ) using GraphPad Prism software. Regression line for the SA cohorts is shown in the right panel. n = 9 and n = 13 for MMA and SA, respectively. ( C ) Primary airway (bronchial) epithelial cells from nonasthmatic human subjects were stimulated with rhIFN-γ ± anti–IFN-γ or left untreated for 8 hours. SLPI mRNA expression was analyzed by qRT-PCR, and fold change over untreated was calculated using HPRT as internal reference control. n = 5. *** P ≤ 0.001, 1-way ANOVA with Tukey’s post-hoc test. ( D ) Detection of hSLPI protein in sera of mice that received hSLPI expression plasmid by tail vein injection. * P ≤ 0.05, 1-way ANOVA with Tukey’s post-hoc test. ( E ) AHR measurement in mice subjected to the SA model with or without hSLPI introduced in an expression plasmid via hydrodynamic tail vein injection. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, 1-way ANOVA with Tukey’s post-hoc test. ( F ) Cytokine concentrations in whole lungs of mice treated as in E . ** P ≤ 0.01, 1-way ANOVA with Tukey’s post-hoc test. For D – F , data shown represent mean ± SEM and representative of 2 independent experiments.

    Techniques Used: Expressing, Quantitative RT-PCR, Software, Mouse Assay, Plasmid Preparation, Injection

    Comparison of AHR-linked IFN-γ–regulated genes in WT and Ifng –/– mice subjected to the SA model. qRT-PCR analysis of the indicated genes in WT and Ifng –/– mice subjected to the SA model. ** P ≤ 0.01, Student’s unpaired t test. Data shown are mean ± SEM and representative of 3 independent experiments.
    Figure Legend Snippet: Comparison of AHR-linked IFN-γ–regulated genes in WT and Ifng –/– mice subjected to the SA model. qRT-PCR analysis of the indicated genes in WT and Ifng –/– mice subjected to the SA model. ** P ≤ 0.01, Student’s unpaired t test. Data shown are mean ± SEM and representative of 3 independent experiments.

    Techniques Used: Mouse Assay, Quantitative RT-PCR

    39) Product Images from "Circ-DLG1 promotes the proliferation of esophageal squamous cell carcinoma"

    Article Title: Circ-DLG1 promotes the proliferation of esophageal squamous cell carcinoma

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S175826

    The expression of circ-DLG1 in ESCC cell lines. Notes: ( A ) The expression of circ-DLG1 in K140, K70, K150, K180, and T10 cells was higher than that in HET-1 significantly. ( B ) Knockdown of circP-DLG1 was confirmed via qRT-PCR, demonstrating the effective knockdown in ESCC cells. ( C ) Knockdown of circ-DLG1 inhibited T10 cell proliferation significantly. ( D ) Knockdown of circ-DLG1 inhibited K180 cell proliferation significantly. ( E ) Cloning formation experiment showed that knockdown of circ-DLG1 inhibited K180 and T10 cell proliferation significantly. ( F ) Cell clone formation ratio after knockdown of circ-DLG1 was lower than that of the control group in K180 and T10 cells. ** P
    Figure Legend Snippet: The expression of circ-DLG1 in ESCC cell lines. Notes: ( A ) The expression of circ-DLG1 in K140, K70, K150, K180, and T10 cells was higher than that in HET-1 significantly. ( B ) Knockdown of circP-DLG1 was confirmed via qRT-PCR, demonstrating the effective knockdown in ESCC cells. ( C ) Knockdown of circ-DLG1 inhibited T10 cell proliferation significantly. ( D ) Knockdown of circ-DLG1 inhibited K180 cell proliferation significantly. ( E ) Cloning formation experiment showed that knockdown of circ-DLG1 inhibited K180 and T10 cell proliferation significantly. ( F ) Cell clone formation ratio after knockdown of circ-DLG1 was lower than that of the control group in K180 and T10 cells. ** P

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

    ( A ) Schematics showed that circ-DLG1 was derived from exons 13 to 18 of DLG1. ( B ) qRT-PCR for the abundance of circ-DLG1 and DLG1 mRNA in ESCC cells treated with RNase R. ( C ) qRT-PCR for the abundance of circ-DLG1 and DLG1 mRNA in ESCC cells treated with actinomycin. Abbreviations: ESCC, esophageal squamous cell carcinoma; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.
    Figure Legend Snippet: ( A ) Schematics showed that circ-DLG1 was derived from exons 13 to 18 of DLG1. ( B ) qRT-PCR for the abundance of circ-DLG1 and DLG1 mRNA in ESCC cells treated with RNase R. ( C ) qRT-PCR for the abundance of circ-DLG1 and DLG1 mRNA in ESCC cells treated with actinomycin. Abbreviations: ESCC, esophageal squamous cell carcinoma; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

    Techniques Used: Derivative Assay, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    40) Product Images from "Melanoma dormancy in a mouse model is linked to GILZ/FOXO3A-dependent quiescence of disseminated stem-like cells"

    Article Title: Melanoma dormancy in a mouse model is linked to GILZ/FOXO3A-dependent quiescence of disseminated stem-like cells

    Journal: Scientific Reports

    doi: 10.1038/srep30405

    Down-regulation of GILZ expression induced quiescence of dormant DMC-derived B16F1-GFP-D cells in the G0 phase. ( a ) Cell cycle analysis of dormant DMC-derived B16F1-GFP-D (lower panel) and maternal B16F1-GFP-M (upper panel) cells transfected with control (left) or GILZ-specific (right) siRNA revealed that the dormant DMC-derived cells possessed the novel, GILZ-dependent ability to control the G0-to-G1 transition in addition to the known GILZ-dependent ability to control the G1-to-S transition of the cell cycle. A fraction of cells in the G0 phase of the cell cycle are shown in red. ( b ) Sphere-forming units (SFUs) formed by B16F1-GFP-D (grey) or B16F1-GFP-M (white) cells transfected with control and GILZ-specific siRNA. ( c ) qRT-PCR assay of Tsc22d3 expression in spheres obtained from B16F1-GFP-M and B16F1-GFP-D cells transfected with either control or GILZ-specific siRNA and cultured for 7 days. The values indicate the level of GILZ-encoding mRNA relative to the control (B16-F1GFP-M adherent cells) (ΔΔCt); siCTR (control siRNA), siGILZ (GILZ-specific siRNA). The results represent 3 independent experiments. *p
    Figure Legend Snippet: Down-regulation of GILZ expression induced quiescence of dormant DMC-derived B16F1-GFP-D cells in the G0 phase. ( a ) Cell cycle analysis of dormant DMC-derived B16F1-GFP-D (lower panel) and maternal B16F1-GFP-M (upper panel) cells transfected with control (left) or GILZ-specific (right) siRNA revealed that the dormant DMC-derived cells possessed the novel, GILZ-dependent ability to control the G0-to-G1 transition in addition to the known GILZ-dependent ability to control the G1-to-S transition of the cell cycle. A fraction of cells in the G0 phase of the cell cycle are shown in red. ( b ) Sphere-forming units (SFUs) formed by B16F1-GFP-D (grey) or B16F1-GFP-M (white) cells transfected with control and GILZ-specific siRNA. ( c ) qRT-PCR assay of Tsc22d3 expression in spheres obtained from B16F1-GFP-M and B16F1-GFP-D cells transfected with either control or GILZ-specific siRNA and cultured for 7 days. The values indicate the level of GILZ-encoding mRNA relative to the control (B16-F1GFP-M adherent cells) (ΔΔCt); siCTR (control siRNA), siGILZ (GILZ-specific siRNA). The results represent 3 independent experiments. *p

    Techniques Used: Expressing, Derivative Assay, Cell Cycle Assay, Transfection, Quantitative RT-PCR, Cell Culture

    Down-regulation of GILZ induced cellular quiescence of human melanoma in vitro and in vivo . Human melanoma HBL-H2B-GFP cells were treated with tetracycline to induce the cell division-tracking H2B-GFP protein, a marker of quiescence, and were subsequently transfected transiently with control (siCTR) or GILZ-specific (siGILZ) siRNA. ( a ) qRT-PCR analysis of GILZ expression in FACS-sorted melanoma GFP low fast-cycling cells and GFP high quiescent cells. *p
    Figure Legend Snippet: Down-regulation of GILZ induced cellular quiescence of human melanoma in vitro and in vivo . Human melanoma HBL-H2B-GFP cells were treated with tetracycline to induce the cell division-tracking H2B-GFP protein, a marker of quiescence, and were subsequently transfected transiently with control (siCTR) or GILZ-specific (siGILZ) siRNA. ( a ) qRT-PCR analysis of GILZ expression in FACS-sorted melanoma GFP low fast-cycling cells and GFP high quiescent cells. *p

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

    41) Product Images from "Role of the Macrophage Migration Inhibitory Factor (MIF) in the survival of first trimester human placenta under induced stress conditions"

    Article Title: Role of the Macrophage Migration Inhibitory Factor (MIF) in the survival of first trimester human placenta under induced stress conditions

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-29797-6

    MIF and CD74 in placental explant cultures after exposure to hypoxia/re-oxygenation (H/R) and FCCP. ( A ) MIF concentration in culture supernatants quantified by ELISA assay and expressed as pg/mg (means ± SEM) of total explant tissue proteins. MIF ( B ) and CD74 ( C ) mRNA expression level assessed by qRT-PCR analysis. Results were normalized to those of 18 S and expressed as fold increase relative to control cultures. *p
    Figure Legend Snippet: MIF and CD74 in placental explant cultures after exposure to hypoxia/re-oxygenation (H/R) and FCCP. ( A ) MIF concentration in culture supernatants quantified by ELISA assay and expressed as pg/mg (means ± SEM) of total explant tissue proteins. MIF ( B ) and CD74 ( C ) mRNA expression level assessed by qRT-PCR analysis. Results were normalized to those of 18 S and expressed as fold increase relative to control cultures. *p

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

    CD74 expression in first trimester placental tissues and interaction with MIF. CD74 mRNA expression assessed by qRT-PCR. ( A ) CD74 mRNA levels were normalized to those of 18S and expressed as fold increase relative to 8 weeks placental tissue selected as calibrator sample. Representative western blot ( B ) and densitometric analysis ( C ) in placental tissues at different weeks of gestation (n = 5 for each week). ( D ) Representative immunohistochemical analysis of CD74 in placenta at 9 weeks of gestation. Slides were counterstained with Mayer’s haematoxylin. Reddish staining represents positive immunoreactivity for CD74. Arrow-head indicates villous trophoblasts; asterisk marks the mesenchymal cells. Ct: cytotrophoblast; Sy: syncytiotrophoblast. Bar = 25 μm. ( E ) Representative immunoprecipitation (IP) of CD74 in placental tissues from 7 to −10 weeks of gestation followed by western blot (WB) for CD74 (left panel) and MIF (right panel). IgG: isotype control; rMIF: recombinant MIF.
    Figure Legend Snippet: CD74 expression in first trimester placental tissues and interaction with MIF. CD74 mRNA expression assessed by qRT-PCR. ( A ) CD74 mRNA levels were normalized to those of 18S and expressed as fold increase relative to 8 weeks placental tissue selected as calibrator sample. Representative western blot ( B ) and densitometric analysis ( C ) in placental tissues at different weeks of gestation (n = 5 for each week). ( D ) Representative immunohistochemical analysis of CD74 in placenta at 9 weeks of gestation. Slides were counterstained with Mayer’s haematoxylin. Reddish staining represents positive immunoreactivity for CD74. Arrow-head indicates villous trophoblasts; asterisk marks the mesenchymal cells. Ct: cytotrophoblast; Sy: syncytiotrophoblast. Bar = 25 μm. ( E ) Representative immunoprecipitation (IP) of CD74 in placental tissues from 7 to −10 weeks of gestation followed by western blot (WB) for CD74 (left panel) and MIF (right panel). IgG: isotype control; rMIF: recombinant MIF.

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Staining, Immunoprecipitation, Recombinant

    42) Product Images from "Preconditioning with far-infrared irradiation enhances proliferation, cell survival, and migration of rat bone marrow-derived stem cells via CXCR4-ERK pathways"

    Article Title: Preconditioning with far-infrared irradiation enhances proliferation, cell survival, and migration of rat bone marrow-derived stem cells via CXCR4-ERK pathways

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14219-w

    Higher expression of pluripotency markers and cardiac lineage-associated markers in BMSCs FIR 50 min . ( A ) Representative agarose gel images of RT-PCR products for targeted genes in BMSCs con and BMSCs FIR 50 min at 0, 1, 4, and 8 h. ( B – E ) qRT-PCR analysis of relative targeted gene expression in BMSCs con (gray) and BMSCs FIR 50 min (black). ( F ) Western blot analysis of targeted proteins. ( G – J ) Confocal images of Sox2 + (Cyan, 100 μm), Nanog + (Pink, 100 μm), c-Kit + (green, 20 μm), and Nkx2.5 + (red, 20 μm)-expressing cells. ( K ) Overlay of c-Kit + and Nkx2.5 + -expressing cells. Data were analyzed using AVOVA followed by Tukey’s post hoc tests and displayed as mean ± SD (n = 6). * P
    Figure Legend Snippet: Higher expression of pluripotency markers and cardiac lineage-associated markers in BMSCs FIR 50 min . ( A ) Representative agarose gel images of RT-PCR products for targeted genes in BMSCs con and BMSCs FIR 50 min at 0, 1, 4, and 8 h. ( B – E ) qRT-PCR analysis of relative targeted gene expression in BMSCs con (gray) and BMSCs FIR 50 min (black). ( F ) Western blot analysis of targeted proteins. ( G – J ) Confocal images of Sox2 + (Cyan, 100 μm), Nanog + (Pink, 100 μm), c-Kit + (green, 20 μm), and Nkx2.5 + (red, 20 μm)-expressing cells. ( K ) Overlay of c-Kit + and Nkx2.5 + -expressing cells. Data were analyzed using AVOVA followed by Tukey’s post hoc tests and displayed as mean ± SD (n = 6). * P

    Techniques Used: Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

    BMSCs FIR 50 min overexpress SDF-1α and IGF-1 at the mRNA level. ( A – F ) Graphs of qRT-PCR analysis results depicting the mRNA expression of IGF-1, TNF-α, TGF-β, SDF-1α, CXCR4, and CXCR7 in BMSCs con (−) and BMSCs FIR 50 min ( + ) at the indicated times after FIR 50 min preconditioning. Data were analyzed using Student’s t-test or AVOVA followed by Tukey’s post hoc tests and displayed as mean ± SD (n = 6). * P
    Figure Legend Snippet: BMSCs FIR 50 min overexpress SDF-1α and IGF-1 at the mRNA level. ( A – F ) Graphs of qRT-PCR analysis results depicting the mRNA expression of IGF-1, TNF-α, TGF-β, SDF-1α, CXCR4, and CXCR7 in BMSCs con (−) and BMSCs FIR 50 min ( + ) at the indicated times after FIR 50 min preconditioning. Data were analyzed using Student’s t-test or AVOVA followed by Tukey’s post hoc tests and displayed as mean ± SD (n = 6). * P

    Techniques Used: Quantitative RT-PCR, Expressing

    43) Product Images from "Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues"

    Article Title: Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues

    Journal: Toxicological Sciences

    doi: 10.1093/toxsci/kfw069

    Gene expression upregulation in cardiac microtissues. Fold change of (A) S100A1 , (B) TCAP , (C) PDE3A , (D) NOS3 , (E) ADRB1 , (F) KCND3 , (G) MYH6 , and (H) MYH7 mRNA expression in foetal heart tissue, cardiac microtissues, and individual cardiac cells relative to adult heart tissue (n = 3, ± SD) was determined by qRT-PCR. * P -value
    Figure Legend Snippet: Gene expression upregulation in cardiac microtissues. Fold change of (A) S100A1 , (B) TCAP , (C) PDE3A , (D) NOS3 , (E) ADRB1 , (F) KCND3 , (G) MYH6 , and (H) MYH7 mRNA expression in foetal heart tissue, cardiac microtissues, and individual cardiac cells relative to adult heart tissue (n = 3, ± SD) was determined by qRT-PCR. * P -value

    Techniques Used: Expressing, Quantitative RT-PCR

    S100A1 regulates contraction in response to caffeine. CMEF microtissues were formed and incubated for 14 days prior to transfection with 10 nM nonsilencing siRNA (−ve control) and 10 nM S100A1 siRNA. A, RNA was extracted from microtissues and expression of GAPDH and S100A1 mRNA analysed by qRT-PCR (n = 3, ± SD). Contractile response recorded using the IonOptix cell geometry system under vehicle control (0.1% v/v DMSO) and 10 mM caffeine in (B) nonsilencing siRNA control CMEF microtissue and (C) S100A1 siRNA transfected CMEF microtissue (results from 1 experiment representative of 3 separate experiments).
    Figure Legend Snippet: S100A1 regulates contraction in response to caffeine. CMEF microtissues were formed and incubated for 14 days prior to transfection with 10 nM nonsilencing siRNA (−ve control) and 10 nM S100A1 siRNA. A, RNA was extracted from microtissues and expression of GAPDH and S100A1 mRNA analysed by qRT-PCR (n = 3, ± SD). Contractile response recorded using the IonOptix cell geometry system under vehicle control (0.1% v/v DMSO) and 10 mM caffeine in (B) nonsilencing siRNA control CMEF microtissue and (C) S100A1 siRNA transfected CMEF microtissue (results from 1 experiment representative of 3 separate experiments).

    Techniques Used: Incubation, Transfection, Expressing, Quantitative RT-PCR

    44) Product Images from "MicroRNAs 373 and 520c Are Downregulated in Prostate Cancer, Suppress CD44 Translation and Enhance Invasion of Prostate Cancer Cells in vitro"

    Article Title: MicroRNAs 373 and 520c Are Downregulated in Prostate Cancer, Suppress CD44 Translation and Enhance Invasion of Prostate Cancer Cells in vitro

    Journal:

    doi:

    A. Left y-axis: Transfection of 6 nM dose of synthetic miR-373 maximized its expression by qRT-PCR. Right y-axis: Doses up to 6 nM caused dose-dependent 35-fold overexpression of CD44 total RNA, normalized to untreated cells (using a primer and probe
    Figure Legend Snippet: A. Left y-axis: Transfection of 6 nM dose of synthetic miR-373 maximized its expression by qRT-PCR. Right y-axis: Doses up to 6 nM caused dose-dependent 35-fold overexpression of CD44 total RNA, normalized to untreated cells (using a primer and probe

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

    qRT-PCR of CD44 total (left) and variant (right) in 5 cell lines and 5 clinical prostatectomy specimens normalized to 18S RNA. Left: CD44 total, comprising the more abundant CD44s isoform, is higher in benign BPH-1 cell line than in androgen-sensitive
    Figure Legend Snippet: qRT-PCR of CD44 total (left) and variant (right) in 5 cell lines and 5 clinical prostatectomy specimens normalized to 18S RNA. Left: CD44 total, comprising the more abundant CD44s isoform, is higher in benign BPH-1 cell line than in androgen-sensitive

    Techniques Used: Quantitative RT-PCR, Variant Assay

    qRT-PCR for miR-373 by primer and probe method normalized to 18S rRNA ( A and B ). A. Among cell lines, benign BPH-1 cells have the most miR-373 , while slow-growing, androgen-dependent LNCaP cancer cells have decreased but detectable miR-373 . Androgen-independent
    Figure Legend Snippet: qRT-PCR for miR-373 by primer and probe method normalized to 18S rRNA ( A and B ). A. Among cell lines, benign BPH-1 cells have the most miR-373 , while slow-growing, androgen-dependent LNCaP cancer cells have decreased but detectable miR-373 . Androgen-independent

    Techniques Used: Quantitative RT-PCR

    45) Product Images from "The APETALA-2-Like Transcription Factor OsAP2-39 Controls Key Interactions between Abscisic Acid and Gibberellin in Rice"

    Article Title: The APETALA-2-Like Transcription Factor OsAP2-39 Controls Key Interactions between Abscisic Acid and Gibberellin in Rice

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001098

    Alteration the expression of OsAP2-39 affects the expression level of ABA and GA related genes are altered in the transgenic rice. Quantitative gene expression analysis using qRT-PCR of several genes showed variation between the wild-type and the transgenic line. (A) Knocking down the OsAP2-39 reduces the expression of EUI and OsNCED-1 in the T 0 plants. Similar results were obtained in the T 1 plants. (B) Overexpression of OsAP2-39 affects various hormone related genes. Bars of the ABA, GA and auxin-related genes are shaded with grey, dark grey and white, respectively. Actin 2 ( Os10g0510000 ) was used as an internal control. Bars represent mean ±SE (n = 3).
    Figure Legend Snippet: Alteration the expression of OsAP2-39 affects the expression level of ABA and GA related genes are altered in the transgenic rice. Quantitative gene expression analysis using qRT-PCR of several genes showed variation between the wild-type and the transgenic line. (A) Knocking down the OsAP2-39 reduces the expression of EUI and OsNCED-1 in the T 0 plants. Similar results were obtained in the T 1 plants. (B) Overexpression of OsAP2-39 affects various hormone related genes. Bars of the ABA, GA and auxin-related genes are shaded with grey, dark grey and white, respectively. Actin 2 ( Os10g0510000 ) was used as an internal control. Bars represent mean ±SE (n = 3).

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

    Exogenous application of ABA induced EUI in the wild-type rice leaves. A quantitative gene expression study using the qRT-PCR analysis of EUI after 2 hours (2 H), 6 hours (6 H) and 24 hours (24 H) of 10 µM ABA application showing that EUI is highly upregulated in rice leaves when treated with ABA after 6 hours. Actin 2 was used as an internal control. Bars represent mean ±SE (n = 3).
    Figure Legend Snippet: Exogenous application of ABA induced EUI in the wild-type rice leaves. A quantitative gene expression study using the qRT-PCR analysis of EUI after 2 hours (2 H), 6 hours (6 H) and 24 hours (24 H) of 10 µM ABA application showing that EUI is highly upregulated in rice leaves when treated with ABA after 6 hours. Actin 2 was used as an internal control. Bars represent mean ±SE (n = 3).

    Techniques Used: Expressing, Quantitative RT-PCR

    46) Product Images from "Carbon dioxide receptor genes and their expression profile in Diabrotica virgifera virgifera"

    Article Title: Carbon dioxide receptor genes and their expression profile in Diabrotica virgifera virgifera

    Journal: BMC Research Notes

    doi: 10.1186/s13104-015-1794-4

    Expression of CO 2 receptors ( a Dvv_Gr1, b Dvv_Gr3, c Dvv_Gr2) in different development stages of Diabrotica v. virgifera . For qRT-PCR, relative expression of Dvv_Gr genes in different stages was measured and normalized to an endogenous control (actin) as described in the “ Methods ” section. Values represent the means and the standard deviation of three analytical replicates on samples that contain tissue from five 3rd instar larvae. Different letters above the bars reflect significantly different expression levels (ANOVA of Tukey Test, P
    Figure Legend Snippet: Expression of CO 2 receptors ( a Dvv_Gr1, b Dvv_Gr3, c Dvv_Gr2) in different development stages of Diabrotica v. virgifera . For qRT-PCR, relative expression of Dvv_Gr genes in different stages was measured and normalized to an endogenous control (actin) as described in the “ Methods ” section. Values represent the means and the standard deviation of three analytical replicates on samples that contain tissue from five 3rd instar larvae. Different letters above the bars reflect significantly different expression levels (ANOVA of Tukey Test, P

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

    Expression of CO 2 receptors ( a Dvv_Gr1, b Dvv_Gr3, c Dvv_Gr2) in different tissues of Diabrotica v. virgifera . For qRT-PCR, relative expression of Dvv_Gr genes in different tissues was measured and normalized to an endogenous control (EF1a) as described in the “ Methods ” section. Values represent the means and the standard deviation of three analytical replicates on samples that contain tissue from five 3rd instar larvae. Different letters above the bars reflect significantly different expression levels (ANOVA of Tukey Test, P
    Figure Legend Snippet: Expression of CO 2 receptors ( a Dvv_Gr1, b Dvv_Gr3, c Dvv_Gr2) in different tissues of Diabrotica v. virgifera . For qRT-PCR, relative expression of Dvv_Gr genes in different tissues was measured and normalized to an endogenous control (EF1a) as described in the “ Methods ” section. Values represent the means and the standard deviation of three analytical replicates on samples that contain tissue from five 3rd instar larvae. Different letters above the bars reflect significantly different expression levels (ANOVA of Tukey Test, P

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

    47) Product Images from "The BAF chromatin remodelling complex is an epigenetic regulator of lineage specification in the early mouse embryo"

    Article Title: The BAF chromatin remodelling complex is an epigenetic regulator of lineage specification in the early mouse embryo

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.131961

    Upregulation of BAF155 shifts the developmental programme towards the extra-embryonic lineage. (A) Overexpression (OE) experiments of BAF155 using the HA-tagged BAF155 construct or of Ruby (control). (B) qRT-PCR on whole embryos, comparing lineage marker transcripts of control and BAF155 OE blastocysts. (C) Immunofluorescence images of control and BAF155 OE blastocysts at E4.5. (D) Total cell number was reduced in BAF155 OE blastocysts (61±6) compared with the control (90±5). (E) The total number of ICM cells was reduced in BAF155 OE blastocysts (14±2) compared with the control (21±3); the majority of ICM cells in BAF155 OE blastocysts co-express NANOG and SOX17 (9±3), unlike control blastocysts (1±2). (F) z -projections of control and BAF155 OE blastocysts: Ruby blastocyst contributes equally to the ICM and CDX2 + TE cell populations, whereas BAF155 OE blastocyst infrequently contributes to the CDX2 − ICM cells (arrows). (G) The percentage of clones injected with BAF155 contributing to the total blastocyst was lower than that injected with Ruby. (H) Clones injected with BAF155 showed a higher contribution to the CDX2 + TE lineage compared with Ruby + clones. Error bars represent s.d. * P
    Figure Legend Snippet: Upregulation of BAF155 shifts the developmental programme towards the extra-embryonic lineage. (A) Overexpression (OE) experiments of BAF155 using the HA-tagged BAF155 construct or of Ruby (control). (B) qRT-PCR on whole embryos, comparing lineage marker transcripts of control and BAF155 OE blastocysts. (C) Immunofluorescence images of control and BAF155 OE blastocysts at E4.5. (D) Total cell number was reduced in BAF155 OE blastocysts (61±6) compared with the control (90±5). (E) The total number of ICM cells was reduced in BAF155 OE blastocysts (14±2) compared with the control (21±3); the majority of ICM cells in BAF155 OE blastocysts co-express NANOG and SOX17 (9±3), unlike control blastocysts (1±2). (F) z -projections of control and BAF155 OE blastocysts: Ruby blastocyst contributes equally to the ICM and CDX2 + TE cell populations, whereas BAF155 OE blastocyst infrequently contributes to the CDX2 − ICM cells (arrows). (G) The percentage of clones injected with BAF155 contributing to the total blastocyst was lower than that injected with Ruby. (H) Clones injected with BAF155 showed a higher contribution to the CDX2 + TE lineage compared with Ruby + clones. Error bars represent s.d. * P

    Techniques Used: Over Expression, Construct, Quantitative RT-PCR, Marker, Immunofluorescence, Clone Assay, Injection

    Upregulation of BAF155 causes upregulation of BAF complex components. (A) HA-tagged human BAF155 , mouse Baf57 or Ruby mRNAs were injected into one blastomere at the 2-cell stage and analysed at the 8-cell stage. (B-B″) Clonal overexpression of BAF155 results in the upregulation of protein levels of the complex subunits (B′), whereas overexpression of Ruby (B) or BAF57 (B″) does not. (C) The protein levels of BAF57 and BRG1 upon BAF155 overexpression (OE) were upregulated by ∼3-fold. (D) qRT-PCR analysis of transcripts for key components of the BAF complex 24 h after BAF155 OE. INI1 refers to Baf47 ( Smarcb1 ). (E) HA-tagged BAF155 mRNA was injected into one blastomere at the 2-cell stage and analysed at the 4-cell stage by PLA. (F) Clonal BAF155 OE caused an increase in BAF155-BRG1 interaction in the injected clones (dashed outline). (G) Overexpression of exogenous BAF155 resulted in 2-fold upregulation of BAF155-BRG1 contact. Error bars represent s.d. * P
    Figure Legend Snippet: Upregulation of BAF155 causes upregulation of BAF complex components. (A) HA-tagged human BAF155 , mouse Baf57 or Ruby mRNAs were injected into one blastomere at the 2-cell stage and analysed at the 8-cell stage. (B-B″) Clonal overexpression of BAF155 results in the upregulation of protein levels of the complex subunits (B′), whereas overexpression of Ruby (B) or BAF57 (B″) does not. (C) The protein levels of BAF57 and BRG1 upon BAF155 overexpression (OE) were upregulated by ∼3-fold. (D) qRT-PCR analysis of transcripts for key components of the BAF complex 24 h after BAF155 OE. INI1 refers to Baf47 ( Smarcb1 ). (E) HA-tagged BAF155 mRNA was injected into one blastomere at the 2-cell stage and analysed at the 4-cell stage by PLA. (F) Clonal BAF155 OE caused an increase in BAF155-BRG1 interaction in the injected clones (dashed outline). (G) Overexpression of exogenous BAF155 resulted in 2-fold upregulation of BAF155-BRG1 contact. Error bars represent s.d. * P

    Techniques Used: Injection, Over Expression, Quantitative RT-PCR, Proximity Ligation Assay, Clone Assay

    48) Product Images from "Serum long non-coding RNAs MALAT1, AFAP1-AS1 and AL359062 as diagnostic and prognostic biomarkers for nasopharyngeal carcinoma"

    Article Title: Serum long non-coding RNAs MALAT1, AFAP1-AS1 and AL359062 as diagnostic and prognostic biomarkers for nasopharyngeal carcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17083

    High MALAT1, AFAP1-AS1 and AL359062 levels were related to EBV infection (A) qRT-PCR assay examined the differences of expression levels of circulating MALAT1, AFAP1-AS1 and AL359062 between EBV-positive NPC group and EBV-negative NPC group. (B) Expression levels of MALAT1, AFAP1-AS1 and AL359062 were detected in culture supernatant of EBV infected NP69 on Day 5, 10, and 15 of post-infection. EBV genes EBNA1 and LMP1 were used to measure the level of viral replication. (* p
    Figure Legend Snippet: High MALAT1, AFAP1-AS1 and AL359062 levels were related to EBV infection (A) qRT-PCR assay examined the differences of expression levels of circulating MALAT1, AFAP1-AS1 and AL359062 between EBV-positive NPC group and EBV-negative NPC group. (B) Expression levels of MALAT1, AFAP1-AS1 and AL359062 were detected in culture supernatant of EBV infected NP69 on Day 5, 10, and 15 of post-infection. EBV genes EBNA1 and LMP1 were used to measure the level of viral replication. (* p

    Techniques Used: Infection, Quantitative RT-PCR, Expressing

    AL359062 knockdown suppress NPC cell metastasis, invasion and proliferation in vitro (A) . siRNA1+2 efficiently suppressed AL359062 expression compared with the siNC in 5-8F and CNE2 cells by qRT-PCR. (B-C) . AL359062 knockdown inhibited NPC cell migratory and invasive abilities as measured by transwell migration assay (B) and matrigel invasion assay (C). (D) . CCK-8 assay was applied to detect the effect of AL359062 knockdown on NPC cell viability. (E-F) . Forty-eight hours after AL359062 knockdown, total RNA was extracted and mRNA expression levels of metastasis and invasion-related genes CDH1, CDH2, MMP2, 3 and 9 (E), and cell cycle-related genes CDKN1B, CCND1, CCNE1, CDK2 and 4 (F) were measured by qRT-PCR. The graph summarizes the data from three independent experiments. (* p
    Figure Legend Snippet: AL359062 knockdown suppress NPC cell metastasis, invasion and proliferation in vitro (A) . siRNA1+2 efficiently suppressed AL359062 expression compared with the siNC in 5-8F and CNE2 cells by qRT-PCR. (B-C) . AL359062 knockdown inhibited NPC cell migratory and invasive abilities as measured by transwell migration assay (B) and matrigel invasion assay (C). (D) . CCK-8 assay was applied to detect the effect of AL359062 knockdown on NPC cell viability. (E-F) . Forty-eight hours after AL359062 knockdown, total RNA was extracted and mRNA expression levels of metastasis and invasion-related genes CDH1, CDH2, MMP2, 3 and 9 (E), and cell cycle-related genes CDKN1B, CCND1, CCNE1, CDK2 and 4 (F) were measured by qRT-PCR. The graph summarizes the data from three independent experiments. (* p

    Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transwell Migration Assay, Invasion Assay, CCK-8 Assay

    49) Product Images from "Transcriptional analysis of tyrosinase gene expression during Bufo bufo development"

    Article Title: Transcriptional analysis of tyrosinase gene expression during Bufo bufo development

    Journal: Saudi Journal of Biological Sciences

    doi: 10.1016/j.sjbs.2016.10.018

    qRT-PCR expression level of tyrosinase gene and melanin pigment content at the different developmental stages of Bufo bufo . (A) Expression levels of TYR mRNA at different developmental stages of Bufo bufo measured by qRT-PCR. (B) About 50 embryos at the different developmental stage were dissolved in lysis buffer; the melanin pigment content was spectrophotometrically determined at 405 nm and the obtained values were normalized to the total protein concentration. Data represent mean ± SEM of triplicate assays. * Significant differences ( P
    Figure Legend Snippet: qRT-PCR expression level of tyrosinase gene and melanin pigment content at the different developmental stages of Bufo bufo . (A) Expression levels of TYR mRNA at different developmental stages of Bufo bufo measured by qRT-PCR. (B) About 50 embryos at the different developmental stage were dissolved in lysis buffer; the melanin pigment content was spectrophotometrically determined at 405 nm and the obtained values were normalized to the total protein concentration. Data represent mean ± SEM of triplicate assays. * Significant differences ( P

    Techniques Used: Quantitative RT-PCR, Expressing, Lysis, Protein Concentration

    Specificity of qRT-PCR amplification. (A) Melting curves of the 3 reference genes, tyrosinase and (6) pAW109 control SYBR-Green qRT-PCR shows a single peak. (B) Agarose gel (2.5%) electrophoresis showing amplification of a specific qRT-PCR product of the expected size, (1) 16S rRNA, (2) histone H4, (3) NADH dehydrogenase subunit I, (4) tyrosinase 1, (5) tyrosinase 2 and pAW109 control. DNA ladder (M).
    Figure Legend Snippet: Specificity of qRT-PCR amplification. (A) Melting curves of the 3 reference genes, tyrosinase and (6) pAW109 control SYBR-Green qRT-PCR shows a single peak. (B) Agarose gel (2.5%) electrophoresis showing amplification of a specific qRT-PCR product of the expected size, (1) 16S rRNA, (2) histone H4, (3) NADH dehydrogenase subunit I, (4) tyrosinase 1, (5) tyrosinase 2 and pAW109 control. DNA ladder (M).

    Techniques Used: Quantitative RT-PCR, Amplification, SYBR Green Assay, Agarose Gel Electrophoresis, Electrophoresis

    50) Product Images from "Whole Genome Expression Analyses of miRNAs and mRNAs Suggest the Involvement of miR-320a and miR-155-3p and their Targeted Genes in Lithium Response in Bipolar Disorder"

    Article Title: Whole Genome Expression Analyses of miRNAs and mRNAs Suggest the Involvement of miR-320a and miR-155-3p and their Targeted Genes in Lithium Response in Bipolar Disorder

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20236040

    Workflow of the study. Abbreviations: DE, differentially expressed; ER, excellent responders; FDR, false discovery rate; HG, human genome, LCL, lymphoblastoid cell lines; Li, lithium; mRNA, messenger RNA, miRNA, microRNA; NGS, next generation sequencing; NR, non-responders; qRT-PCR, quantitative reverse transcription-PCR.
    Figure Legend Snippet: Workflow of the study. Abbreviations: DE, differentially expressed; ER, excellent responders; FDR, false discovery rate; HG, human genome, LCL, lymphoblastoid cell lines; Li, lithium; mRNA, messenger RNA, miRNA, microRNA; NGS, next generation sequencing; NR, non-responders; qRT-PCR, quantitative reverse transcription-PCR.

    Techniques Used: Next-Generation Sequencing, Quantitative RT-PCR, Polymerase Chain Reaction

    Results from qRT-PCR for miRNAs and target genes differentially expressed between lithium excellent responders and non-responders. Results from qRT-PCR for ( A ) miR-320 and its targets CAPNS1 and RGS16 and ( B ) miR-155-3p and its target SP4 . Abbreviations: ER, excellent responders; qRT-PCR, quantitative reverse transcription-PCR; NR, non-responders. *** p
    Figure Legend Snippet: Results from qRT-PCR for miRNAs and target genes differentially expressed between lithium excellent responders and non-responders. Results from qRT-PCR for ( A ) miR-320 and its targets CAPNS1 and RGS16 and ( B ) miR-155-3p and its target SP4 . Abbreviations: ER, excellent responders; qRT-PCR, quantitative reverse transcription-PCR; NR, non-responders. *** p

    Techniques Used: Quantitative RT-PCR, Polymerase Chain Reaction

    51) Product Images from "Linc-ROR induces epithelial-to-mesenchymal transition in ovarian cancer by increasing Wnt/β-catenin signaling"

    Article Title: Linc-ROR induces epithelial-to-mesenchymal transition in ovarian cancer by increasing Wnt/β-catenin signaling

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19545

    Linc-ROR mRNA expression in SKOV3 cells and its effects on proliferation qRT-PCR was used to detect linc-ROR expression after transfection with linc-ROR-targeting siRNA (A) or the linc-ROR expression plasmid (B) . GAPDH was used as an internal control. Linc-ROR knockdown reduced the proliferation rate of SKOV3 cells (C) . Linc-ROR overexpression promoted the proliferation rate of SKOV3 (D) . * P
    Figure Legend Snippet: Linc-ROR mRNA expression in SKOV3 cells and its effects on proliferation qRT-PCR was used to detect linc-ROR expression after transfection with linc-ROR-targeting siRNA (A) or the linc-ROR expression plasmid (B) . GAPDH was used as an internal control. Linc-ROR knockdown reduced the proliferation rate of SKOV3 cells (C) . Linc-ROR overexpression promoted the proliferation rate of SKOV3 (D) . * P

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

    Linc-ROR mRNA expression by qRT-PCR in normal ovarian and fallopian tube tissues and high-grade ovarian serous cancer tissues The level of linc-ROR expression in high-grade ovarian serous cancer tissues was significantly higher than in normal ovarian or normal fallopian tube tissues. ** P
    Figure Legend Snippet: Linc-ROR mRNA expression by qRT-PCR in normal ovarian and fallopian tube tissues and high-grade ovarian serous cancer tissues The level of linc-ROR expression in high-grade ovarian serous cancer tissues was significantly higher than in normal ovarian or normal fallopian tube tissues. ** P

    Techniques Used: Expressing, Quantitative RT-PCR

    52) Product Images from "Reconstitution of FOXP3+ regulatory T cells (Tregs) after CD25-depleted allotransplantion in elderly patients and association with acute graft-versus-host disease"

    Article Title: Reconstitution of FOXP3+ regulatory T cells (Tregs) after CD25-depleted allotransplantion in elderly patients and association with acute graft-versus-host disease

    Journal: Blood

    doi: 10.1182/blood-2007-03-079160

    T reg reconstitution in a representative patient (UPN 301) . T regs were determined by flow and qRT-PCR analysis before and 30, 60, and 90 days after transplantation (Pre, d30, d60, d90, respectively); donor (Don) and the CD25-depleted lymphocyte product (SD). (A) Flow analysis for FOXP3 and CD25 in the CD4 + subset. Percentages displayed represent the fraction of positive cells in CD4 + cells. Two T reg populations, a CD25 + CD4 + FOXP3 + and a CD25 − CD4 + FOXP3 + subset, could be identified before and after transplantation as well in the donor. The SD product was entirely CD25 − but contained a CD25 − CD4 + FOXP3 + subset of T regs . CD25 − CD4 + FOXP3 + and CD25 + CD4 + FOXP3 + T reg fractions increased after SCT. CD25 + CD4 + FOXP3 − T effs declined after SCT. (B) Total fraction of FOXP3 + cells in CD4 + cells. (C) Foxp3 mRNA expression in CD4-selected cells expressed as copy numbers per 1 × 10 6 copies of β- actin .
    Figure Legend Snippet: T reg reconstitution in a representative patient (UPN 301) . T regs were determined by flow and qRT-PCR analysis before and 30, 60, and 90 days after transplantation (Pre, d30, d60, d90, respectively); donor (Don) and the CD25-depleted lymphocyte product (SD). (A) Flow analysis for FOXP3 and CD25 in the CD4 + subset. Percentages displayed represent the fraction of positive cells in CD4 + cells. Two T reg populations, a CD25 + CD4 + FOXP3 + and a CD25 − CD4 + FOXP3 + subset, could be identified before and after transplantation as well in the donor. The SD product was entirely CD25 − but contained a CD25 − CD4 + FOXP3 + subset of T regs . CD25 − CD4 + FOXP3 + and CD25 + CD4 + FOXP3 + T reg fractions increased after SCT. CD25 + CD4 + FOXP3 − T effs declined after SCT. (B) Total fraction of FOXP3 + cells in CD4 + cells. (C) Foxp3 mRNA expression in CD4-selected cells expressed as copy numbers per 1 × 10 6 copies of β- actin .

    Techniques Used: Flow Cytometry, Quantitative RT-PCR, Transplantation Assay, Expressing

    Relative reconstitution CD25 + and FOXP3 + populations in CD4 + cells . Scatterplots indicate each cell population as a fraction of CD4 + cells (for flow cytometry: by gating on CD3 + CD4 + cells; for qRT-PCR: by magnetic selection of CD4 + cells); horizontal bars represent the median of each group. Red circles refer to patients with clinically significant (≥ grade II) aGvHD development. The gray-shaded area represents the interquartile (25%-75%) range of cell counts in donors. A 2-tailed, nonparametric Mann-Whitney test was applied to examine for differences among patient levels 30 and 60 days after transplantation. Statistically significant results are displayed. (A) PCR analysis: Pre indicates patients before transplantation (n = 12); Don, donor (n = 13); SD, selectively CD25-depleted lymphocyte product (n = 10); d30, d60, and d90, patients 30 (n = 14), 60 (n = 10), and 90 (n = 8) days after transplantation, respectively. (B-F) Flow cytometry: Pre (n = 11), Don (n = 13), SD (n = 9), d30 (n = 13), d60 (n = 8), and d90 (n = 5).
    Figure Legend Snippet: Relative reconstitution CD25 + and FOXP3 + populations in CD4 + cells . Scatterplots indicate each cell population as a fraction of CD4 + cells (for flow cytometry: by gating on CD3 + CD4 + cells; for qRT-PCR: by magnetic selection of CD4 + cells); horizontal bars represent the median of each group. Red circles refer to patients with clinically significant (≥ grade II) aGvHD development. The gray-shaded area represents the interquartile (25%-75%) range of cell counts in donors. A 2-tailed, nonparametric Mann-Whitney test was applied to examine for differences among patient levels 30 and 60 days after transplantation. Statistically significant results are displayed. (A) PCR analysis: Pre indicates patients before transplantation (n = 12); Don, donor (n = 13); SD, selectively CD25-depleted lymphocyte product (n = 10); d30, d60, and d90, patients 30 (n = 14), 60 (n = 10), and 90 (n = 8) days after transplantation, respectively. (B-F) Flow cytometry: Pre (n = 11), Don (n = 13), SD (n = 9), d30 (n = 13), d60 (n = 8), and d90 (n = 5).

    Techniques Used: Flow Cytometry, Cytometry, Quantitative RT-PCR, Selection, MANN-WHITNEY, Transplantation Assay, Polymerase Chain Reaction

    53) Product Images from "The mRNA and Proteins Expression Levels Analysis of TC-1 Cells Immune Response to H9N2 Avian Influenza Virus"

    Article Title: The mRNA and Proteins Expression Levels Analysis of TC-1 Cells Immune Response to H9N2 Avian Influenza Virus

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.01039

    TLR-3, TLR-7, and MDA-5 mRNA levels at various time points post-infection. TC-1 cells were infected with CK/SD/w3 and CK/SD/w4 at MOI = 1. qRT-PCR were used to examine the mRNA levels and fold-changes was calculated by 2 -ΔΔCt method as compared with non-infection cell control and using endogenous β-actin mRNA level for normalization. (A) The fold change of TLR-3 in mRNA levels; (B) The fold change of TLR-7 in mRNA levels; (C) The fold change of MDA-5 in mRNA levels. The data was shown the as mean ± SE from three sets of independent experiments. ∗ P
    Figure Legend Snippet: TLR-3, TLR-7, and MDA-5 mRNA levels at various time points post-infection. TC-1 cells were infected with CK/SD/w3 and CK/SD/w4 at MOI = 1. qRT-PCR were used to examine the mRNA levels and fold-changes was calculated by 2 -ΔΔCt method as compared with non-infection cell control and using endogenous β-actin mRNA level for normalization. (A) The fold change of TLR-3 in mRNA levels; (B) The fold change of TLR-7 in mRNA levels; (C) The fold change of MDA-5 in mRNA levels. The data was shown the as mean ± SE from three sets of independent experiments. ∗ P

    Techniques Used: Multiple Displacement Amplification, Infection, Quantitative RT-PCR

    Cytokine and chemokine mRNA levels at various time points post-infection. TC-1 cells were infected with CK/SD/w3 and CK/SD/w4 at MOI = 1. qRT-PCR were used to quantify the mRNA levels and fold-changes was calculated by 2 -ΔΔCt method as compared with non-infection cell control and using endogenous actin mRNA level for normalization. (A) The fold change of IL-1β in mRNA levels; (B) The fold change of IL-6 in mRNA levels; (C) The fold change of CCL-5 in mRNA levels; (D) The fold change of chemokine (C-X-C motif) ligand-10 (CXCL-10) in mRNA levels. The data was shown as the mean ± SE from three sets of independent experiments. ∗ P
    Figure Legend Snippet: Cytokine and chemokine mRNA levels at various time points post-infection. TC-1 cells were infected with CK/SD/w3 and CK/SD/w4 at MOI = 1. qRT-PCR were used to quantify the mRNA levels and fold-changes was calculated by 2 -ΔΔCt method as compared with non-infection cell control and using endogenous actin mRNA level for normalization. (A) The fold change of IL-1β in mRNA levels; (B) The fold change of IL-6 in mRNA levels; (C) The fold change of CCL-5 in mRNA levels; (D) The fold change of chemokine (C-X-C motif) ligand-10 (CXCL-10) in mRNA levels. The data was shown as the mean ± SE from three sets of independent experiments. ∗ P

    Techniques Used: Infection, Quantitative RT-PCR

    54) Product Images from "Transcriptomic Responses to Different Cry1Ac Selection Stresses in Helicoverpa armigera"

    Article Title: Transcriptomic Responses to Different Cry1Ac Selection Stresses in Helicoverpa armigera

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.01653

    The qRT-PCR analysis of transcript abundances among candidate Bt resistance trypsin genes within H. armigera -susceptible (LF) and -resistant (LF5, LF10, LF20, LF30, LF60, and LF120) strains. (A) The three genes ID numbers represent the three trypsin genes in NCBI; a pair of primers in the conserved region of these three trypsin genes were used to analyze transcript abundances. (B-G) Represent the relative expression levels of trypsin genes of XM_021340597, XM_021329499, XM_021333008, XM_021338512, XM_021340599 and XM_021340600 in resistance strains, respectively. Values shown are means and standard errors. Different letters indicate significant differences between treatments ( P
    Figure Legend Snippet: The qRT-PCR analysis of transcript abundances among candidate Bt resistance trypsin genes within H. armigera -susceptible (LF) and -resistant (LF5, LF10, LF20, LF30, LF60, and LF120) strains. (A) The three genes ID numbers represent the three trypsin genes in NCBI; a pair of primers in the conserved region of these three trypsin genes were used to analyze transcript abundances. (B-G) Represent the relative expression levels of trypsin genes of XM_021340597, XM_021329499, XM_021333008, XM_021338512, XM_021340599 and XM_021340600 in resistance strains, respectively. Values shown are means and standard errors. Different letters indicate significant differences between treatments ( P

    Techniques Used: Quantitative RT-PCR, Expressing

    The qRT-PCR estimates of transcript abundances among candidate Bt-resistance receptor genes within H. armigera -susceptible (LF) and -resistant (LF5, LF10, LF20, LF30, LF60, and LF120) strains. (A-D) Represent the relative expression levels of ALP-like, ALP2, APN5 and APN1 in resistance strains, respectively. Values shown are means and standard errors. Different letters indicate significant differences between treatments ( P
    Figure Legend Snippet: The qRT-PCR estimates of transcript abundances among candidate Bt-resistance receptor genes within H. armigera -susceptible (LF) and -resistant (LF5, LF10, LF20, LF30, LF60, and LF120) strains. (A-D) Represent the relative expression levels of ALP-like, ALP2, APN5 and APN1 in resistance strains, respectively. Values shown are means and standard errors. Different letters indicate significant differences between treatments ( P

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

    55) Product Images from "Investigating the role of filamin C in Belgian patients with frontotemporal dementia linked to GRN deficiency in FTLD-TDP brains"

    Article Title: Investigating the role of filamin C in Belgian patients with frontotemporal dementia linked to GRN deficiency in FTLD-TDP brains

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-015-0246-7

    Analysis of FLNC expression in FTD patients at transcript and protein level. a qRT-PCR analysis showed significantly increased levels of both the short and long isoform of FLNC in the frontal cortex of FTLD-TDP patients with GRN haploinsufficiency. The long isoform was also significantly increased in FTLD-TDP patients without a known mutation in causal FTD-ALS genes. b Increased expression levels of FLNC were confirmed on protein level using immunoblot analysis. In contrast to transcript levels, FLNC was also upregulated in VCP and FLNC variation carriers. Elevated phosphorylated FLNC levels at serines 2113 and 2213 (pSer 2113 and pSer 2213 ) were identified to a variable extent in GRN and VCP mutation carriers compared to controls. * P
    Figure Legend Snippet: Analysis of FLNC expression in FTD patients at transcript and protein level. a qRT-PCR analysis showed significantly increased levels of both the short and long isoform of FLNC in the frontal cortex of FTLD-TDP patients with GRN haploinsufficiency. The long isoform was also significantly increased in FTLD-TDP patients without a known mutation in causal FTD-ALS genes. b Increased expression levels of FLNC were confirmed on protein level using immunoblot analysis. In contrast to transcript levels, FLNC was also upregulated in VCP and FLNC variation carriers. Elevated phosphorylated FLNC levels at serines 2113 and 2213 (pSer 2113 and pSer 2213 ) were identified to a variable extent in GRN and VCP mutation carriers compared to controls. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Mutagenesis

    Analysis of Flnc expression in Grn knockout mice at transcript and protein level. qRT-PCR analysis of the long and short isoform of Flnc ( a ) measured in progranulin knockout mice of different ages. We analyzed the expression levels of both long and short isoforms of mouse Flnc in heterozygous Grn +/- and homozygous Grn -/- mice and wild-type (Wt) animals of 3 months ( n = 4), 9 months ( n = 4), 16-18 months ( n = 6) and 24 months ( n = 5) of age. b Increased FlnC expression levels were confirmed on protein level using quantitative immunoblot analysis. Two protein bands are detected around the height of mouse FLNC using the Kinasource AB152 anti FLNC antibody. The upper band is FLNC specific as determined by Western blotting using lysates from FlnC knockout mice (data not shown). The lower band is therefore considered as an aspecific protein band. * P
    Figure Legend Snippet: Analysis of Flnc expression in Grn knockout mice at transcript and protein level. qRT-PCR analysis of the long and short isoform of Flnc ( a ) measured in progranulin knockout mice of different ages. We analyzed the expression levels of both long and short isoforms of mouse Flnc in heterozygous Grn +/- and homozygous Grn -/- mice and wild-type (Wt) animals of 3 months ( n = 4), 9 months ( n = 4), 16-18 months ( n = 6) and 24 months ( n = 5) of age. b Increased FlnC expression levels were confirmed on protein level using quantitative immunoblot analysis. Two protein bands are detected around the height of mouse FLNC using the Kinasource AB152 anti FLNC antibody. The upper band is FLNC specific as determined by Western blotting using lysates from FlnC knockout mice (data not shown). The lower band is therefore considered as an aspecific protein band. * P

    Techniques Used: Expressing, Knock-Out, Mouse Assay, Quantitative RT-PCR, Western Blot

    56) Product Images from "The MicroRNA and MessengerRNA Profile of the RNA-Induced Silencing Complex in Human Primary Astrocyte and Astrocytoma Cells"

    Article Title: The MicroRNA and MessengerRNA Profile of the RNA-Induced Silencing Complex in Human Primary Astrocyte and Astrocytoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013445

    ( A ) MRNA microarray validation with qRT-PCR analysis in grouped RISC-IP U-87 astrocytoma and primary astrocytes samples. Grouped RISC-IP data were compared to the grouped global mRNA from U-87 astrocytoma and primary astrocytes samples. Eight mRNAs were selected from the grouped mRNA microarray dataset and examined by qRT-PCR. Fold change from the mRNA microarray are given by log2 values (left y-axis, light grey bars). Fold change from the qRT-PCR was determined using the 2 -ΔΔCt method and all mRNA expression values were normalized to the beta-actin endogenous control (right y-axis, dark grey bars). Error bars represent the standard deviation of the mean (SD). Importantly, the fold change (y-axis) cannot be directly compared between assays due to differences in calculation methods, but the general trend of up-regulation and down-regulation can be compared. ( B ) MRNAs in RISC compared to the global cellular milieu in U-87 astrocytoma cells. MRNA expression in U-87 astrocytoma cells were normalized to primary astrocytes mRNA expression. All mRNAs had a fold change > 2.5 and were significantly expressed (p
    Figure Legend Snippet: ( A ) MRNA microarray validation with qRT-PCR analysis in grouped RISC-IP U-87 astrocytoma and primary astrocytes samples. Grouped RISC-IP data were compared to the grouped global mRNA from U-87 astrocytoma and primary astrocytes samples. Eight mRNAs were selected from the grouped mRNA microarray dataset and examined by qRT-PCR. Fold change from the mRNA microarray are given by log2 values (left y-axis, light grey bars). Fold change from the qRT-PCR was determined using the 2 -ΔΔCt method and all mRNA expression values were normalized to the beta-actin endogenous control (right y-axis, dark grey bars). Error bars represent the standard deviation of the mean (SD). Importantly, the fold change (y-axis) cannot be directly compared between assays due to differences in calculation methods, but the general trend of up-regulation and down-regulation can be compared. ( B ) MRNAs in RISC compared to the global cellular milieu in U-87 astrocytoma cells. MRNA expression in U-87 astrocytoma cells were normalized to primary astrocytes mRNA expression. All mRNAs had a fold change > 2.5 and were significantly expressed (p

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

    MiRNA microarray validation with qRT-PCR analysis for (A) global miRNA and (B) RISC-specific miRNA. Nine random miRNAs were selected from the miRNA microarray datasets and examined by qRT-PCR. Fold change from the miRNA microarray are given by log2 values (left y-axis, light grey bars). Fold change from the qRT-PCR was determined using the 2 -ΔΔCt method and all miRNA expression values were normalized to the RNU6B endogenous control (right y-axis, dark grey bars). Error bars represent the standard deviation of the mean (SD). Note: only the general trend of up-regulation and down-regulation can be compared but the fold change (y-axis) cannot be directly compared between assays due to differences in calculation methods.
    Figure Legend Snippet: MiRNA microarray validation with qRT-PCR analysis for (A) global miRNA and (B) RISC-specific miRNA. Nine random miRNAs were selected from the miRNA microarray datasets and examined by qRT-PCR. Fold change from the miRNA microarray are given by log2 values (left y-axis, light grey bars). Fold change from the qRT-PCR was determined using the 2 -ΔΔCt method and all miRNA expression values were normalized to the RNU6B endogenous control (right y-axis, dark grey bars). Error bars represent the standard deviation of the mean (SD). Note: only the general trend of up-regulation and down-regulation can be compared but the fold change (y-axis) cannot be directly compared between assays due to differences in calculation methods.

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

    57) Product Images from "CD5 expression is regulated during human T-cell activation by alternative polyadenylation, PTBP1 and miR-204"

    Article Title: CD5 expression is regulated during human T-cell activation by alternative polyadenylation, PTBP1 and miR-204

    Journal: European journal of immunology

    doi: 10.1002/eji.201545663

    miR-204 is upregulated in activated T cells and targets the longer CD5 APA mRNA isoform ( A) The miR-204/211 putative binding sites were predicted on TargetScan using CD5 3′ UTR and the seed region sequences are indicated. Arrows represent the PAS location on CD5 3′ UTR. ( B ) Conservation analysis of miR-204/211 binding sites performed by alignment of the CD5 3′ UTR sequences of seven mammalian species using the Geneious v4.8 software. Pairwise percentage identities are indicated below. ( C and D ) qRT-PCR analysis of miR-204 expression in resting and activated human primary T cells and Jurkat cells. Data are shown as mean +SD (n= 3 replicates) and are pooled from 3 independent experiments. *p
    Figure Legend Snippet: miR-204 is upregulated in activated T cells and targets the longer CD5 APA mRNA isoform ( A) The miR-204/211 putative binding sites were predicted on TargetScan using CD5 3′ UTR and the seed region sequences are indicated. Arrows represent the PAS location on CD5 3′ UTR. ( B ) Conservation analysis of miR-204/211 binding sites performed by alignment of the CD5 3′ UTR sequences of seven mammalian species using the Geneious v4.8 software. Pairwise percentage identities are indicated below. ( C and D ) qRT-PCR analysis of miR-204 expression in resting and activated human primary T cells and Jurkat cells. Data are shown as mean +SD (n= 3 replicates) and are pooled from 3 independent experiments. *p

    Techniques Used: Binding Assay, Software, Quantitative RT-PCR, Expressing

    T-cell activation increases CD5 mRNAs and protein expression ( A-D ) Jurkat E6.1 cells were activated with anti-CD3 (OKT3) and anti-CD28 (28.2) or left untreated. (A) CD5 expression was determined by qRT-PCR in resting (0h) and upon 24h activation. Data is shown as mean +SD of three replicates pooled from 2 independent experiments. **p
    Figure Legend Snippet: T-cell activation increases CD5 mRNAs and protein expression ( A-D ) Jurkat E6.1 cells were activated with anti-CD3 (OKT3) and anti-CD28 (28.2) or left untreated. (A) CD5 expression was determined by qRT-PCR in resting (0h) and upon 24h activation. Data is shown as mean +SD of three replicates pooled from 2 independent experiments. **p

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

    58) Product Images from "Targeting nuclear RNA for in vivo correction of myotonic dystrophy"

    Article Title: Targeting nuclear RNA for in vivo correction of myotonic dystrophy

    Journal: Nature

    doi: 10.1038/nature11362

    Duration of ASO activity and in vivo targeting of human DMPK ( a-e ) Two month-old HSA LR mice received saline or indicated ASO by subcutaneous injection of 25 mg/kg twice weekly for 4 weeks ( n = 5 each ASO, n = 6 saline). Tissues were isolated 1 year after the final dose. a, qRT-PCR analysis of HSA LR transgene mRNA (± SD), normalized to the housekeeping gene Gtf2b mRNA. Results were similar when normalized to total RNA input. * P
    Figure Legend Snippet: Duration of ASO activity and in vivo targeting of human DMPK ( a-e ) Two month-old HSA LR mice received saline or indicated ASO by subcutaneous injection of 25 mg/kg twice weekly for 4 weeks ( n = 5 each ASO, n = 6 saline). Tissues were isolated 1 year after the final dose. a, qRT-PCR analysis of HSA LR transgene mRNA (± SD), normalized to the housekeeping gene Gtf2b mRNA. Results were similar when normalized to total RNA input. * P

    Techniques Used: Allele-specific Oligonucleotide, Activity Assay, In Vivo, Mouse Assay, Injection, Isolation, Quantitative RT-PCR

    59) Product Images from "Sperm mRNA Transcripts Are Indicators of Sub-Chronic Low Dose Testicular Injury in the Fischer 344 Rat"

    Article Title: Sperm mRNA Transcripts Are Indicators of Sub-Chronic Low Dose Testicular Injury in the Fischer 344 Rat

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044280

    Time course experiment: heatmap displaying heirarchical clustering of qRT-PCR data. The fold changes determined in the qRT-PCR analysis (relative to time point 0) for the 12 transcripts altered at any of the six time points (columns) were clustered using Manhattan Distance. The 12 transcripts (rows) were separated via the direction of fold change (downregulated = red dendrogram; upregulated = blue and orange dendrograms). The upregulated transcripts segregated into two groups based upon the temporal onset of statistically different transcript levels, with early changing transcripts (blue dendrogram) separated from late changing transcripts (orange dendrogram). The intensity of the color for each transcript at each time point is related to the transcript level, as depicted by the bar at the top of the figure.
    Figure Legend Snippet: Time course experiment: heatmap displaying heirarchical clustering of qRT-PCR data. The fold changes determined in the qRT-PCR analysis (relative to time point 0) for the 12 transcripts altered at any of the six time points (columns) were clustered using Manhattan Distance. The 12 transcripts (rows) were separated via the direction of fold change (downregulated = red dendrogram; upregulated = blue and orange dendrograms). The upregulated transcripts segregated into two groups based upon the temporal onset of statistically different transcript levels, with early changing transcripts (blue dendrogram) separated from late changing transcripts (orange dendrogram). The intensity of the color for each transcript at each time point is related to the transcript level, as depicted by the bar at the top of the figure.

    Techniques Used: Quantitative RT-PCR

    Time course experiment: transcript changes. Twelve of the 29 transcripts selected from the Preliminary Experiment showed significant alterations by qRT-PCR. ΔCT values were compared to time point 0 using one-way ANOVA with Dunnett’s correction for multiple comparisons (*** = p
    Figure Legend Snippet: Time course experiment: transcript changes. Twelve of the 29 transcripts selected from the Preliminary Experiment showed significant alterations by qRT-PCR. ΔCT values were compared to time point 0 using one-way ANOVA with Dunnett’s correction for multiple comparisons (*** = p

    Techniques Used: Quantitative RT-PCR

    60) Product Images from "RpoN Promotes Pseudomonas aeruginosa Survival in the Presence of Tobramycin"

    Article Title: RpoN Promotes Pseudomonas aeruginosa Survival in the Presence of Tobramycin

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.00839

    Expression of gacA (A) , rsmA (B) , rpoS (C) , and relA (D) in logarithmic phase wild-type PAO1 and the Δ rpoN , Δ rpoS , Δ rpoN Δ rpoS , and Δ rpoN / rpoN + mutants grown in the LB medium in the presence of 32 μg/ml tobramycin. The transcript levels were measured by qRT-PCR, were normalized to omlA expression, and are expressed relative to wild-type PAO1 at time = 0 h. The time points at which the cells were sampled for transcriptional analysis were t = 0 h and t = 3 h after the addition of tobramycin, as indicated. All results are the average of at least three independent experiments, and the error bars represent SDs. P ≤ 0.05 ( * ), ≤0.01 ( ** ).
    Figure Legend Snippet: Expression of gacA (A) , rsmA (B) , rpoS (C) , and relA (D) in logarithmic phase wild-type PAO1 and the Δ rpoN , Δ rpoS , Δ rpoN Δ rpoS , and Δ rpoN / rpoN + mutants grown in the LB medium in the presence of 32 μg/ml tobramycin. The transcript levels were measured by qRT-PCR, were normalized to omlA expression, and are expressed relative to wild-type PAO1 at time = 0 h. The time points at which the cells were sampled for transcriptional analysis were t = 0 h and t = 3 h after the addition of tobramycin, as indicated. All results are the average of at least three independent experiments, and the error bars represent SDs. P ≤ 0.05 ( * ), ≤0.01 ( ** ).

    Techniques Used: Expressing, Quantitative RT-PCR

    Expression of gacA , rsmA , relA , and rpoS genes in stationary phase wild-type PAO1 and the Δ rpoN , Δ rpoS , Δ rpoN Δ rpoS , and Δ rpoN / rpoN + mutants grown in the LB medium (A,C,E,G) and in AB minimal medium (B,D,F,H) . The gacA, rsmA, relA , and rpoS transcript levels were measured by qRT-PCR, were normalized to omlA expression, and the levels are expressed relative to the wild-type PAO1 at time = 0 h. The time points at which the cells were sampled for transcriptional analysis were t = 0 h and t = 24 h after the addition of 32 μg/ml tobramycin, as indicated. All results are the average of at least three independent experiments, and the error bars represent SDs. P ≤ 0.05 ( * ), ≤0.01 ( ** ).
    Figure Legend Snippet: Expression of gacA , rsmA , relA , and rpoS genes in stationary phase wild-type PAO1 and the Δ rpoN , Δ rpoS , Δ rpoN Δ rpoS , and Δ rpoN / rpoN + mutants grown in the LB medium (A,C,E,G) and in AB minimal medium (B,D,F,H) . The gacA, rsmA, relA , and rpoS transcript levels were measured by qRT-PCR, were normalized to omlA expression, and the levels are expressed relative to the wild-type PAO1 at time = 0 h. The time points at which the cells were sampled for transcriptional analysis were t = 0 h and t = 24 h after the addition of 32 μg/ml tobramycin, as indicated. All results are the average of at least three independent experiments, and the error bars represent SDs. P ≤ 0.05 ( * ), ≤0.01 ( ** ).

    Techniques Used: Expressing, Quantitative RT-PCR

    61) Product Images from "WIPF1 antagonizes the tumor suppressive effect of miR-141/200c and is associated with poor survival in patients with PDAC"

    Article Title: WIPF1 antagonizes the tumor suppressive effect of miR-141/200c and is associated with poor survival in patients with PDAC

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0848-6

    CpG hypermethylation is responsible for the silencing of miR-141 and miR-200c. a Expression of miR-141 and miR-200c in 10 paired PDAC and their surrounding non-cancerous tissues. The mRNA expression was measured by qRT–PCR. U6 was used as internal reference. b Correlation between the expression of miRNA-141 and its CpG methylation level in 37 PDAC tissues using Spearman’s correlation analysis. c Correlation between the expression of miRNA-200c and its CpG methylation level in 37 PDAC tissues using Spearman’s correlation analysis. d Levels of methylation of miR-141 and miR-200c promoter region in human pancreatic ductal epithelial cells (HPDE) and pancreatic cancer cell lines (PANC-1, BxPC-3, HPAF-II, and SW1990). Note the levels of methylation in the pancreatic cell lines were all dramatically higher than HPDE cells. The data were derived from three sets of experiments. Error bars are represented as the mean +/− SD. e Change of methylation levels of miR-141 and miR-200c promoter in pancreatic cancer cell lines in response to 5-Aza-dC treatment. f Relative levels of miR-141 and miR-200c expression in response to 5-Aza-dC treatment in the pancreatic cancer cell lines. For both ( e ) and ( f ), Data were derived from three sets of experiments. Error bars are repreented as the mean +/− SD. * P
    Figure Legend Snippet: CpG hypermethylation is responsible for the silencing of miR-141 and miR-200c. a Expression of miR-141 and miR-200c in 10 paired PDAC and their surrounding non-cancerous tissues. The mRNA expression was measured by qRT–PCR. U6 was used as internal reference. b Correlation between the expression of miRNA-141 and its CpG methylation level in 37 PDAC tissues using Spearman’s correlation analysis. c Correlation between the expression of miRNA-200c and its CpG methylation level in 37 PDAC tissues using Spearman’s correlation analysis. d Levels of methylation of miR-141 and miR-200c promoter region in human pancreatic ductal epithelial cells (HPDE) and pancreatic cancer cell lines (PANC-1, BxPC-3, HPAF-II, and SW1990). Note the levels of methylation in the pancreatic cell lines were all dramatically higher than HPDE cells. The data were derived from three sets of experiments. Error bars are represented as the mean +/− SD. e Change of methylation levels of miR-141 and miR-200c promoter in pancreatic cancer cell lines in response to 5-Aza-dC treatment. f Relative levels of miR-141 and miR-200c expression in response to 5-Aza-dC treatment in the pancreatic cancer cell lines. For both ( e ) and ( f ), Data were derived from three sets of experiments. Error bars are repreented as the mean +/− SD. * P

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

    62) Product Images from "SMAD proteins directly suppress PAX2 transcription downstream of transforming growth factor-beta 1 (TGF-β1) signalling in renal cell carcinoma"

    Article Title: SMAD proteins directly suppress PAX2 transcription downstream of transforming growth factor-beta 1 (TGF-β1) signalling in renal cell carcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25516

    TGF-β1 treatment suppresses PAX2 mRNA and protein expression in CC-RCC cell lines (A) Western blot analysis of PAX2 protein expression in RCC cell lines. GAPDH was used as a loading control. (B) QRT-PCR analysis of the relative level of endogenous PAX2 mRNA in HEK293 and three CC-RCC human cell lines. The data represent three separate experiments. (C) Relative PAX2 mRNA expression level, and (D) PAX2 protein following treatment of 786-O and A498 CC-RCC cells with 10 ng/ml TGF-β1 (labelled “+”, black columns) versus vehicle treated controls (labelled “-“, white columns). The results from three separate experiments after normalisation with GAPDH are shown. (E) Western blot of PAX2 protein expression relative to GAPDH following no treatment (0 h) of 786-O cells, or treatment for 6, 24 or 48 h with 10 ng/ml TGF-β1. Data are represented as mean ± S.E.M. The data were analysed by Student's t test using GraphPad Prism 5.01, ** p
    Figure Legend Snippet: TGF-β1 treatment suppresses PAX2 mRNA and protein expression in CC-RCC cell lines (A) Western blot analysis of PAX2 protein expression in RCC cell lines. GAPDH was used as a loading control. (B) QRT-PCR analysis of the relative level of endogenous PAX2 mRNA in HEK293 and three CC-RCC human cell lines. The data represent three separate experiments. (C) Relative PAX2 mRNA expression level, and (D) PAX2 protein following treatment of 786-O and A498 CC-RCC cells with 10 ng/ml TGF-β1 (labelled “+”, black columns) versus vehicle treated controls (labelled “-“, white columns). The results from three separate experiments after normalisation with GAPDH are shown. (E) Western blot of PAX2 protein expression relative to GAPDH following no treatment (0 h) of 786-O cells, or treatment for 6, 24 or 48 h with 10 ng/ml TGF-β1. Data are represented as mean ± S.E.M. The data were analysed by Student's t test using GraphPad Prism 5.01, ** p

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

    63) Product Images from "Inhibition of Plasmodium Hepatic Infection by Antiretroviral Compounds"

    Article Title: Inhibition of Plasmodium Hepatic Infection by Antiretroviral Compounds

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00329

    Antiretroviral drugs decrease liver Plasmodium infection in vivo . (A) Parasite infection loads measured by qRT-PCR in the livers of mice treated with the various compounds, relative to those of DMSO-treated controls, at 46 hpi. (B) Effect of drug treatment on parasite size as a surrogate of parasite development at 46 hpi. Analysis was performed by immunofluorescence microscopy through quantification of EEF area. (C) Representative confocal microscopy images of liver parasites in treated and control mice. Red, Pb UIS4 labeling showing the PVM; blue, Hoechst nuclear staining. Scale bars, 10 μm. (D) Parasite density per square millimeter of mouse liver sections following treatment with selected drug combinations, or with the vehicle, at 46 hpi. Plots represent the mean values of at least two independent experiments with error bars indicating SEM. One-way ANOVA with post-test Dunnett. ns, Not significant, * p
    Figure Legend Snippet: Antiretroviral drugs decrease liver Plasmodium infection in vivo . (A) Parasite infection loads measured by qRT-PCR in the livers of mice treated with the various compounds, relative to those of DMSO-treated controls, at 46 hpi. (B) Effect of drug treatment on parasite size as a surrogate of parasite development at 46 hpi. Analysis was performed by immunofluorescence microscopy through quantification of EEF area. (C) Representative confocal microscopy images of liver parasites in treated and control mice. Red, Pb UIS4 labeling showing the PVM; blue, Hoechst nuclear staining. Scale bars, 10 μm. (D) Parasite density per square millimeter of mouse liver sections following treatment with selected drug combinations, or with the vehicle, at 46 hpi. Plots represent the mean values of at least two independent experiments with error bars indicating SEM. One-way ANOVA with post-test Dunnett. ns, Not significant, * p

    Techniques Used: Infection, In Vivo, Quantitative RT-PCR, Mouse Assay, Immunofluorescence, Microscopy, Confocal Microscopy, Labeling, Staining

    64) Product Images from "WIPF1 antagonizes the tumor suppressive effect of miR-141/200c and is associated with poor survival in patients with PDAC"

    Article Title: WIPF1 antagonizes the tumor suppressive effect of miR-141/200c and is associated with poor survival in patients with PDAC

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0848-6

    CpG hypermethylation is responsible for the silencing of miR-141 and miR-200c. a Expression of miR-141 and miR-200c in 10 paired PDAC and their surrounding non-cancerous tissues. The mRNA expression was measured by qRT–PCR. U6 was used as internal reference. b Correlation between the expression of miRNA-141 and its CpG methylation level in 37 PDAC tissues using Spearman’s correlation analysis. c Correlation between the expression of miRNA-200c and its CpG methylation level in 37 PDAC tissues using Spearman’s correlation analysis. d Levels of methylation of miR-141 and miR-200c promoter region in human pancreatic ductal epithelial cells (HPDE) and pancreatic cancer cell lines (PANC-1, BxPC-3, HPAF-II, and SW1990). Note the levels of methylation in the pancreatic cell lines were all dramatically higher than HPDE cells. The data were derived from three sets of experiments. Error bars are represented as the mean +/− SD. e Change of methylation levels of miR-141 and miR-200c promoter in pancreatic cancer cell lines in response to 5-Aza-dC treatment. f Relative levels of miR-141 and miR-200c expression in response to 5-Aza-dC treatment in the pancreatic cancer cell lines. For both ( e ) and ( f ), Data were derived from three sets of experiments. Error bars are repreented as the mean +/− SD. * P
    Figure Legend Snippet: CpG hypermethylation is responsible for the silencing of miR-141 and miR-200c. a Expression of miR-141 and miR-200c in 10 paired PDAC and their surrounding non-cancerous tissues. The mRNA expression was measured by qRT–PCR. U6 was used as internal reference. b Correlation between the expression of miRNA-141 and its CpG methylation level in 37 PDAC tissues using Spearman’s correlation analysis. c Correlation between the expression of miRNA-200c and its CpG methylation level in 37 PDAC tissues using Spearman’s correlation analysis. d Levels of methylation of miR-141 and miR-200c promoter region in human pancreatic ductal epithelial cells (HPDE) and pancreatic cancer cell lines (PANC-1, BxPC-3, HPAF-II, and SW1990). Note the levels of methylation in the pancreatic cell lines were all dramatically higher than HPDE cells. The data were derived from three sets of experiments. Error bars are represented as the mean +/− SD. e Change of methylation levels of miR-141 and miR-200c promoter in pancreatic cancer cell lines in response to 5-Aza-dC treatment. f Relative levels of miR-141 and miR-200c expression in response to 5-Aza-dC treatment in the pancreatic cancer cell lines. For both ( e ) and ( f ), Data were derived from three sets of experiments. Error bars are repreented as the mean +/− SD. * P

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

    65) Product Images from "Gene expression profiling of periodontitis-affected gingival tissue by spatial transcriptomics"

    Article Title: Gene expression profiling of periodontitis-affected gingival tissue by spatial transcriptomics

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-27627-3

    Gene expression validation in gingival tissue biopsies using qRT-PCR. The four most highly upregulated genes in connective tissue with inflammatory cells, compared to connective tissue without inflammatory cells, as identified by spatial transcriptomics, were validated using qRT-PCR in RNA from six gingival tissues from patients with periodontitis and four control samples without periodontitis. Gene expression is shown as log2 fold change (FC) of periodontitis samples relative to healthy controls and was calculated according to the ΔΔCt method, where each periodontitis sample was normalized to the control samples and to GAPDH (reference gene) of corresponding periodontitis sample. The reactions were run in duplicates and experiment was repeated with similar results. IGLL5 : immunoglobulin lambda-like polypeptide 5; SSR4 : signal sequence receptor subunit 4; MZB1 : marginal zone B and B1 cell specific protein; XBP1 : X-box binding protein 1.
    Figure Legend Snippet: Gene expression validation in gingival tissue biopsies using qRT-PCR. The four most highly upregulated genes in connective tissue with inflammatory cells, compared to connective tissue without inflammatory cells, as identified by spatial transcriptomics, were validated using qRT-PCR in RNA from six gingival tissues from patients with periodontitis and four control samples without periodontitis. Gene expression is shown as log2 fold change (FC) of periodontitis samples relative to healthy controls and was calculated according to the ΔΔCt method, where each periodontitis sample was normalized to the control samples and to GAPDH (reference gene) of corresponding periodontitis sample. The reactions were run in duplicates and experiment was repeated with similar results. IGLL5 : immunoglobulin lambda-like polypeptide 5; SSR4 : signal sequence receptor subunit 4; MZB1 : marginal zone B and B1 cell specific protein; XBP1 : X-box binding protein 1.

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

    66) Product Images from "Gene expression profiling of periodontitis-affected gingival tissue by spatial transcriptomics"

    Article Title: Gene expression profiling of periodontitis-affected gingival tissue by spatial transcriptomics

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-27627-3

    Gene expression validation in gingival tissue biopsies using qRT-PCR. The four most highly upregulated genes in connective tissue with inflammatory cells, compared to connective tissue without inflammatory cells, as identified by spatial transcriptomics, were validated using qRT-PCR in RNA from six gingival tissues from patients with periodontitis and four control samples without periodontitis. Gene expression is shown as log2 fold change (FC) of periodontitis samples relative to healthy controls and was calculated according to the ΔΔCt method, where each periodontitis sample was normalized to the control samples and to GAPDH (reference gene) of corresponding periodontitis sample. The reactions were run in duplicates and experiment was repeated with similar results. IGLL5 : immunoglobulin lambda-like polypeptide 5; SSR4 : signal sequence receptor subunit 4; MZB1 : marginal zone B and B1 cell specific protein; XBP1 : X-box binding protein 1.
    Figure Legend Snippet: Gene expression validation in gingival tissue biopsies using qRT-PCR. The four most highly upregulated genes in connective tissue with inflammatory cells, compared to connective tissue without inflammatory cells, as identified by spatial transcriptomics, were validated using qRT-PCR in RNA from six gingival tissues from patients with periodontitis and four control samples without periodontitis. Gene expression is shown as log2 fold change (FC) of periodontitis samples relative to healthy controls and was calculated according to the ΔΔCt method, where each periodontitis sample was normalized to the control samples and to GAPDH (reference gene) of corresponding periodontitis sample. The reactions were run in duplicates and experiment was repeated with similar results. IGLL5 : immunoglobulin lambda-like polypeptide 5; SSR4 : signal sequence receptor subunit 4; MZB1 : marginal zone B and B1 cell specific protein; XBP1 : X-box binding protein 1.

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

    67) Product Images from "Chemotherapy-induced hyaluronan production: a novel chemoresistance mechanism in ovarian cancer"

    Article Title: Chemotherapy-induced hyaluronan production: a novel chemoresistance mechanism in ovarian cancer

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-13-476

    Chemotherapy treatment increases ABCC2 expression in ovarian cancer cells. A . qRT-PCR for ABCC2 expression. Ovarian cancer cells treated with LD 50 CBP for 72 hr. OVCAR-5 cells also treated in presence or absence of HA oligomers (HYA oligo, 250 μg/ml). Relative gene expression was determined by calibration against no CBP control and normalized to the house keeping gene β-actin using the 2 -∆∆CT method. Data represents mean ± SEM from 3 independent experiments performed in triplicate. B . OVCAR-5 treated with LD 50 CBP for 72 hr. Fixed cells were incubated with biotinylated HABP and ABCC2 mouse monoclonal antibody and visualized with Cy3-conjugated streptavidin (red) and FITC-conjugated donkey anti-mouse immunoglobulins (green) respectively. Nuclei are counterstained with DAPI dye (blue).
    Figure Legend Snippet: Chemotherapy treatment increases ABCC2 expression in ovarian cancer cells. A . qRT-PCR for ABCC2 expression. Ovarian cancer cells treated with LD 50 CBP for 72 hr. OVCAR-5 cells also treated in presence or absence of HA oligomers (HYA oligo, 250 μg/ml). Relative gene expression was determined by calibration against no CBP control and normalized to the house keeping gene β-actin using the 2 -∆∆CT method. Data represents mean ± SEM from 3 independent experiments performed in triplicate. B . OVCAR-5 treated with LD 50 CBP for 72 hr. Fixed cells were incubated with biotinylated HABP and ABCC2 mouse monoclonal antibody and visualized with Cy3-conjugated streptavidin (red) and FITC-conjugated donkey anti-mouse immunoglobulins (green) respectively. Nuclei are counterstained with DAPI dye (blue).

    Techniques Used: Expressing, Quantitative RT-PCR, Incubation

    68) Product Images from "Enhancing titres of therapeutic viral vectors using the transgene repression in vector production (TRiP) system"

    Article Title: Enhancing titres of therapeutic viral vectors using the transgene repression in vector production (TRiP) system

    Journal: Nature Communications

    doi: 10.1038/ncomms14834

    The TRAP-tbs configuration confers a translational block resulting in potent repression of GFP expression in HEK293T cells. ( a ) The GFP reporter plasmid containing the tbs within the 5′UTR of the transcription unit and the TRAP-expression plasmid used throughout the study. B. subtilis TRAP was codon-optimised for human cell expression and C-terminally Hisx6 tagged. ( b ) 2-D plots (FSC v FL1/GFP) of HEK293T cells co-transfected with GFP reporter plasmids and TRAP or control plasmids, analysed by flow cytometry (MFI, median fluoresence intensity [arbitrary units]). ( c ) GFP Expression scores for the co-transfections were generated (percent GFP × MFI; plotted on left y-axis) and qRT-PCR data of cytoplasmic GFP RNA copies detected plotted on the right y-axis; grey bars—RT-positive, white bars—no RT (residual pDNA control). All data are mean average values±s.d. [log 10 -transformed data] ( n =8); * P
    Figure Legend Snippet: The TRAP-tbs configuration confers a translational block resulting in potent repression of GFP expression in HEK293T cells. ( a ) The GFP reporter plasmid containing the tbs within the 5′UTR of the transcription unit and the TRAP-expression plasmid used throughout the study. B. subtilis TRAP was codon-optimised for human cell expression and C-terminally Hisx6 tagged. ( b ) 2-D plots (FSC v FL1/GFP) of HEK293T cells co-transfected with GFP reporter plasmids and TRAP or control plasmids, analysed by flow cytometry (MFI, median fluoresence intensity [arbitrary units]). ( c ) GFP Expression scores for the co-transfections were generated (percent GFP × MFI; plotted on left y-axis) and qRT-PCR data of cytoplasmic GFP RNA copies detected plotted on the right y-axis; grey bars—RT-positive, white bars—no RT (residual pDNA control). All data are mean average values±s.d. [log 10 -transformed data] ( n =8); * P

    Techniques Used: Blocking Assay, Expressing, Plasmid Preparation, Transfection, Flow Cytometry, Cytometry, Generated, Quantitative RT-PCR, Transformation Assay

    69) Product Images from "miR-182 suppresses invadopodia formation and metastasis in non-small cell lung cancer by targeting cortactin gene"

    Article Title: miR-182 suppresses invadopodia formation and metastasis in non-small cell lung cancer by targeting cortactin gene

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0824-1

    Cortactin promoted the migratory and invasive potential of NSCLC cells in vitro. a A549 and H1299 cells were transfected with cortactin small interfering RNA (siRNA) or the siRNA control oligonucleotides, followed by qRT-PCR analysis of cortactin levels in the differently treated cells. b Protein expression levels of cortactin were measured by Western blotting. c Wound healing assay in A549 and H1299 cells transfected with cortactin siRNA or siRNA control oligonucleotides. After 32 h, wound healing assays were performed in triplicate. Representative images are displayed Scale bar, 800 μm, 4× magnification. The values shown are the mean ± SD, * P
    Figure Legend Snippet: Cortactin promoted the migratory and invasive potential of NSCLC cells in vitro. a A549 and H1299 cells were transfected with cortactin small interfering RNA (siRNA) or the siRNA control oligonucleotides, followed by qRT-PCR analysis of cortactin levels in the differently treated cells. b Protein expression levels of cortactin were measured by Western blotting. c Wound healing assay in A549 and H1299 cells transfected with cortactin siRNA or siRNA control oligonucleotides. After 32 h, wound healing assays were performed in triplicate. Representative images are displayed Scale bar, 800 μm, 4× magnification. The values shown are the mean ± SD, * P

    Techniques Used: In Vitro, Transfection, Small Interfering RNA, Quantitative RT-PCR, Expressing, Western Blot, Wound Healing Assay

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

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

    Journal: Scientific Reports

    doi: 10.1038/srep17975

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

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

    71) Product Images from "Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promote Disease Progression and Induce the Production of Different Cytokines in Macrophages and B-1 Cells"

    Article Title: Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promote Disease Progression and Induce the Production of Different Cytokines in Macrophages and B-1 Cells

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.03056

    Cytokine expression in BMDMs and B-1 cells stimulated with EVs released by L. amazonensis promastigotes. After 48 h of stimulation with EVs, RNA was extracted, and the expression of IL-6, IL-10, and TNF- α was determined by qRT-PCR. (A) IL-6, (B) IL-10, and (C) TNF-α expression in BMDMs; (D) IL-6, (E) IL-10, and (F) TNF-α expression in B-1 cells. NC, negative control (unstimulated macrophages); PC, positive control (cells infected with live parasites). The bars show the average of three measurements, and the error bars denote the SD. The data are representative of three independent experiments. ANOVA followed by a post hoc Tukey’s test ( ∗ P
    Figure Legend Snippet: Cytokine expression in BMDMs and B-1 cells stimulated with EVs released by L. amazonensis promastigotes. After 48 h of stimulation with EVs, RNA was extracted, and the expression of IL-6, IL-10, and TNF- α was determined by qRT-PCR. (A) IL-6, (B) IL-10, and (C) TNF-α expression in BMDMs; (D) IL-6, (E) IL-10, and (F) TNF-α expression in B-1 cells. NC, negative control (unstimulated macrophages); PC, positive control (cells infected with live parasites). The bars show the average of three measurements, and the error bars denote the SD. The data are representative of three independent experiments. ANOVA followed by a post hoc Tukey’s test ( ∗ P

    Techniques Used: Expressing, Quantitative RT-PCR, Negative Control, Positive Control, Infection

    72) Product Images from "Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promote Disease Progression and Induce the Production of Different Cytokines in Macrophages and B-1 Cells"

    Article Title: Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promote Disease Progression and Induce the Production of Different Cytokines in Macrophages and B-1 Cells

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.03056

    Cytokine expression in BMDMs and B-1 cells stimulated with EVs released by L. amazonensis promastigotes. After 48 h of stimulation with EVs, RNA was extracted, and the expression of IL-6, IL-10, and TNF- α was determined by qRT-PCR. (A) IL-6, (B) IL-10, and (C) TNF-α expression in BMDMs; (D) IL-6, (E) IL-10, and (F) TNF-α expression in B-1 cells. NC, negative control (unstimulated macrophages); PC, positive control (cells infected with live parasites). The bars show the average of three measurements, and the error bars denote the SD. The data are representative of three independent experiments. ANOVA followed by a post hoc Tukey’s test ( ∗ P
    Figure Legend Snippet: Cytokine expression in BMDMs and B-1 cells stimulated with EVs released by L. amazonensis promastigotes. After 48 h of stimulation with EVs, RNA was extracted, and the expression of IL-6, IL-10, and TNF- α was determined by qRT-PCR. (A) IL-6, (B) IL-10, and (C) TNF-α expression in BMDMs; (D) IL-6, (E) IL-10, and (F) TNF-α expression in B-1 cells. NC, negative control (unstimulated macrophages); PC, positive control (cells infected with live parasites). The bars show the average of three measurements, and the error bars denote the SD. The data are representative of three independent experiments. ANOVA followed by a post hoc Tukey’s test ( ∗ P

    Techniques Used: Expressing, Quantitative RT-PCR, Negative Control, Positive Control, Infection

    73) Product Images from "Age-Dependent Oxidative DNA Damage Does Not Correlate with Reduced Proliferation of Cardiomyocytes in Humans"

    Article Title: Age-Dependent Oxidative DNA Damage Does Not Correlate with Reduced Proliferation of Cardiomyocytes in Humans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0170351

    Ki67 and cyclin D2 mRNA is significantly decreased relative to age. qRT-PCR was used to analyze Ki67 and cyclin D2 mRNA levels. Both Ki67 (A) and cyclin D2 (B) were decreased with age. GAPDH served as a control. Bars indicate mean ±standard deviation. * p
    Figure Legend Snippet: Ki67 and cyclin D2 mRNA is significantly decreased relative to age. qRT-PCR was used to analyze Ki67 and cyclin D2 mRNA levels. Both Ki67 (A) and cyclin D2 (B) were decreased with age. GAPDH served as a control. Bars indicate mean ±standard deviation. * p

    Techniques Used: Quantitative RT-PCR, Standard Deviation

    74) Product Images from "The microRNA-200 family targets multiple non-small cell lung cancer prognostic markers in H1299 cells and BEAS-2B cells"

    Article Title: The microRNA-200 family targets multiple non-small cell lung cancer prognostic markers in H1299 cells and BEAS-2B cells

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2013.1963

    Relative mRNA levels of the predicted targets of miR-200 in H1299. (A) Relative mRNA expression of predicted miR-200 target genes in H1299 cells infected with miR-scrambled and miR-200a, -200b and -200c. These targets were predicted with microRNA.org based on 3′-UTR and downregulation after microRNA transduction. (B) Relative mRNA expression level of E-cadherin in H1299 infected with miR-200a and -200c. Cells infected with miR-scr were used as a negative control. The mRNA expression levels were determined using qRT-PCR as described in Materials and methods. * Statistically significant at p≤0.05, n=3 (biological replicates). Data presented as mean ± SEM. Relative mRNA level was calculated after normalization to endogenous gene, UBC and relative to negative control, miR-scr.
    Figure Legend Snippet: Relative mRNA levels of the predicted targets of miR-200 in H1299. (A) Relative mRNA expression of predicted miR-200 target genes in H1299 cells infected with miR-scrambled and miR-200a, -200b and -200c. These targets were predicted with microRNA.org based on 3′-UTR and downregulation after microRNA transduction. (B) Relative mRNA expression level of E-cadherin in H1299 infected with miR-200a and -200c. Cells infected with miR-scr were used as a negative control. The mRNA expression levels were determined using qRT-PCR as described in Materials and methods. * Statistically significant at p≤0.05, n=3 (biological replicates). Data presented as mean ± SEM. Relative mRNA level was calculated after normalization to endogenous gene, UBC and relative to negative control, miR-scr.

    Techniques Used: Expressing, Infection, Transduction, Negative Control, Quantitative RT-PCR

    75) Product Images from "A T7 autogene-based hybrid mRNA/DNA system for long-term shRNA expression in cytoplasm without inefficient nuclear entry"

    Article Title: A T7 autogene-based hybrid mRNA/DNA system for long-term shRNA expression in cytoplasm without inefficient nuclear entry

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39407-8

    Prolonged silencing effects on target RFP gene in auto_shRFP@LS-transfected B16F10/RFP cells. ( A ) Sustained decrease of RFP mRNA in auto_shRFP@LS-transfected B16F10/RFP cells. For the indicated period of time, B16F10/RFP cells were transfected with synthetic siRFP@LS (corresponding to 50 nM of siRFP), auto_shRFP@LS (15 μg/mL), or auto(−)_shRFP@LS (14.04 μg/mL; corresponding to T7 autogene plasmid-lacking auto_shRFP system). For each RNA samples, the amounts of RFP mRNA were quantitatively estimated by qRT-PCR method, and then plotted against incubation time after transfection. For comparison, each amount of amplified RFP DNA products was normalized with respect to that of amplified β-actin DNA products. Data represent the mean ± s.d. (n = 5). ( B ) Sustained suppression of RFP expression in auto_shRFP@LS-transfected B16F10/RFP cells. After B16F10/RFP cells were transfected with auto_shRFP@LS or synthetic siRFP@LS for the specified period of time, the amounts of RFP protein within each protein samples were determined by immunoblotting method, and then plotted against incubation time after transfection. Data represent the mean ± s.d. (n = 5). The full blot images are presented in Supplementary Fig. S5 . ( C ) Flow cytometry profiles demonstrating the sustained suppression of RFP expression. For the indicated period of time, B16F10/RFP cells were transfected in the same manner as ( A ). RFP-expressing cells were sorted with respect to a prefixed gate region for RFP fluorescence, and then their percentage was plotted against incubation time after transfection. Data represent the mean ± s.d. (n = 3). ( D , E ) A comparison of duration of silencing effect between auto_shRFP system and nuclear shRNA expression system. B16F10/RFP cells were transfected with auto_shRFP@LS (15 μg/mL) or pSuper_shRFP plasmid@LS (15 μg/mL; corresponding to nuclear H1 promoter-driven shRFP expression system), and then relative amounts of RFP mRNA within the cells were plotted versus transfection time ( D ). The representative FACS profiles on 3 days and 4 days post-transfection are presented ( E ).
    Figure Legend Snippet: Prolonged silencing effects on target RFP gene in auto_shRFP@LS-transfected B16F10/RFP cells. ( A ) Sustained decrease of RFP mRNA in auto_shRFP@LS-transfected B16F10/RFP cells. For the indicated period of time, B16F10/RFP cells were transfected with synthetic siRFP@LS (corresponding to 50 nM of siRFP), auto_shRFP@LS (15 μg/mL), or auto(−)_shRFP@LS (14.04 μg/mL; corresponding to T7 autogene plasmid-lacking auto_shRFP system). For each RNA samples, the amounts of RFP mRNA were quantitatively estimated by qRT-PCR method, and then plotted against incubation time after transfection. For comparison, each amount of amplified RFP DNA products was normalized with respect to that of amplified β-actin DNA products. Data represent the mean ± s.d. (n = 5). ( B ) Sustained suppression of RFP expression in auto_shRFP@LS-transfected B16F10/RFP cells. After B16F10/RFP cells were transfected with auto_shRFP@LS or synthetic siRFP@LS for the specified period of time, the amounts of RFP protein within each protein samples were determined by immunoblotting method, and then plotted against incubation time after transfection. Data represent the mean ± s.d. (n = 5). The full blot images are presented in Supplementary Fig. S5 . ( C ) Flow cytometry profiles demonstrating the sustained suppression of RFP expression. For the indicated period of time, B16F10/RFP cells were transfected in the same manner as ( A ). RFP-expressing cells were sorted with respect to a prefixed gate region for RFP fluorescence, and then their percentage was plotted against incubation time after transfection. Data represent the mean ± s.d. (n = 3). ( D , E ) A comparison of duration of silencing effect between auto_shRFP system and nuclear shRNA expression system. B16F10/RFP cells were transfected with auto_shRFP@LS (15 μg/mL) or pSuper_shRFP plasmid@LS (15 μg/mL; corresponding to nuclear H1 promoter-driven shRFP expression system), and then relative amounts of RFP mRNA within the cells were plotted versus transfection time ( D ). The representative FACS profiles on 3 days and 4 days post-transfection are presented ( E ).

    Techniques Used: Transfection, Plasmid Preparation, Quantitative RT-PCR, Incubation, Amplification, Expressing, Flow Cytometry, Cytometry, Fluorescence, shRNA, FACS

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    Amplification:

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    Synthesized:

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    Quantitative RT-PCR:

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    Article Title: Evaluation of Schistosome Promoter Expression for Transgenesis and Genetic Analysis
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    SYBR Green Assay:

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    Quantitation Assay:

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    Significance Assay:

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    Expressing:

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    Article Title: Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells
    Article Snippet: The expression of β-actin was used as an internal control for both conventional RT-PCR and qRT-PCR. .. All qRT-PCR reactions were carried out on a 7500 Fast Real-Time PCR system (Applied Biosystems).

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    Article Title: Antisense oligonucleotide treatment ameliorates IFN-γ–induced proteinuria in APOL1-transgenic mice
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    Modification:

    Article Title: Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells
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    Transfection:

    Article Title: Evaluation of Schistosome Promoter Expression for Transgenesis and Genetic Analysis
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    Cell Culture:

    Article Title: Antisense oligonucleotide treatment ameliorates IFN-γ–induced proteinuria in APOL1-transgenic mice
    Article Snippet: Paragraph title: Cell culture experiments. ... Briefly, 5 μL qRT-PCR reactions containing 1 μL RNA were run using AgPath-ID One-Step RT-PCR Reagent (Life Technologies) following the manufacturer’s recommendations.

    Generated:

    Article Title: Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells
    Article Snippet: First-strand cDNA was generated using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer's instructions. .. All qRT-PCR reactions were carried out on a 7500 Fast Real-Time PCR system (Applied Biosystems).

    Article Title: Survivin-targeting miR-542-3p overcomes HER3 signaling-induced chemoresistance and enhances the antitumor activity of paclitaxel against HER2-overexpressing breast cancer
    Article Snippet: First-strand cDNA was generated using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) following the manufacturer’s instructions. .. All qRT-PCR reactions were carried out on a 7500 Fast Real-Time PCR system (Applied Biosystems).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Biosynthetic CircRNA_001160 induced by PTBP1 regulates the permeability of BTB via the CircRNA_001160/miR-195-5p/ETV1 axis
    Article Snippet: .. All qRT-PCR reactions were performed using the 7500 Fast RT-PCR System (Applied Biosystems, USA). ..

    Article Title: Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells
    Article Snippet: Paragraph title: Reverse transcription (RT)-PCR and quantitative real-time (qRT)-PCR ... All qRT-PCR reactions were carried out on a 7500 Fast Real-Time PCR system (Applied Biosystems).

    Article Title: Survivin-targeting miR-542-3p overcomes HER3 signaling-induced chemoresistance and enhances the antitumor activity of paclitaxel against HER2-overexpressing breast cancer
    Article Snippet: Paragraph title: 2.6. Reverse transcription (RT)-PCR and quantitative real-time (qRT)-PCR ... All qRT-PCR reactions were carried out on a 7500 Fast Real-Time PCR system (Applied Biosystems).

    Article Title: Antisense oligonucleotide treatment ameliorates IFN-γ–induced proteinuria in APOL1-transgenic mice
    Article Snippet: .. Briefly, 5 μL qRT-PCR reactions containing 1 μL RNA were run using AgPath-ID One-Step RT-PCR Reagent (Life Technologies) following the manufacturer’s recommendations. ..

    Article Title: Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis
    Article Snippet: Analysis of gene transcript levels Extraction of total RNA samples from appropriate plant materials and RT-PCR conditions have been described previously [ ]. .. RNA sample preparation, reverse transcription, and quantitative PCR were conducted according to the rules that have been proposed to ensure reproducible and accurate measurements [ ]. qRT-PCR reactions were performed in 96-well blocks using an Applied Biosystems 7500 Real-Time PCR System (Foster City, CA) using the SYBR Green I master mix in a volume of 20 μl.

    Isolation:

    Article Title: In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGF?-signaling and WT1
    Article Snippet: Paragraph title: mRNA isolation and quantitative RT-PCR analysis ... Primer sequences and annealing temperatures are available on request. qRT-PCR was performed for the following factors: WT1 isoform A, WT1 isoform D, TGFβ1 , 2 , and 3 , ALK1 , ALK5 , VCAM -1 , E -cadherin , endoglin and Snai1 . qRT-PCR reactions were run on a 7900HT Applied Biosystems.

    Article Title: Gene Expression and Yeast Two-Hybrid Studies of 1R-MYB Transcription Factor Mediating Drought Stress Response in Chickpea (Cicer arietinum L.)
    Article Snippet: The DNase-treated total RNA isolated from the yeast cells, were used for synthesizing cDNA using SuperScript® III First-Strand Synthesis SuperMix (Invitrogen™, USA). .. The qRT-PCR reactions were conducted using SYBR green chemistry (Applied Biosystems, USA) in two technical replicates and two biological replicates.

    Article Title: Antisense oligonucleotide treatment ameliorates IFN-γ–induced proteinuria in APOL1-transgenic mice
    Article Snippet: After 48 hours, cells were washed once with PBS before lysing for RNA isolation. .. Briefly, 5 μL qRT-PCR reactions containing 1 μL RNA were run using AgPath-ID One-Step RT-PCR Reagent (Life Technologies) following the manufacturer’s recommendations.

    Article Title: Effect of Radiation Dose-Rate on Hematopoietic Cell Engraftment in Adult Zebrafish
    Article Snippet: .. RNA isolation and DNA removal were achieved with the RNeasy Mini Kit (Qiagen). cDNA was synthesized using SuperScript III 1st strand synthesis kit (Invitrogen). qRT-PCR was performed using Taqman Primers (listed in supplemental data) and reagents (Applied Biosystems). qRT-PCR reactions were performed in technical triplicates on a StepOnePlus qRT-PCR system (Applied Biosystems). ..

    Article Title: A novel mechanism for the transcriptional regulation of Wnt signaling in development
    Article Snippet: .. Total RNA was isolated using Trizol, reverse transcription was performed using SuperScript III, and 5′ RACE was performed using the GeneRacer kit (all Invitrogen). qRT–PCR reactions were performed in triplicate using SYBR Green PCR master mix (Applied Biosystems) and normalized against expression of Hprt and GAPDH. .. The 5′ RACE and mouse qPCR primers are listed in Supplemental Tables S2 and S3, respectively.

    Size-exclusion Chromatography:

    Article Title: Glypican-3 induces a mesenchymal to epithelial transition in human breast cancer cells
    Article Snippet: The resulting cDNA was subsequently treated with 1 Unit of RNase H (GE Healthcare, Little Chalfont, Buckinghamshire, UK). qRT-PCR reactions contained: 3 μl cDNA (1:10), 6 μl 2X SYBR Green Master Mix (Applied Biosystems Life Technologies, Carlsbad, CA, USA), and forward and reverse primers for GPC3, E-Cadherin, N-Cadherin, vimentin, SNAIL1, SNAIL2 (SLUG), ZEB1 and GAPDH (Table ). .. Cycle conditions were: 50°C 2 min, 95°C 10 min and 40 cycles at 95°C for 15 sec and 60°C during 1 min. 2−ΔΔCt was used to calculate relative gene/GAPDH expression.

    Article Title: Evaluation of Schistosome Promoter Expression for Transgenesis and Genetic Analysis
    Article Snippet: Gene Transcript Analysis Total RNA (∼600 ng) from each transfected sample was converted to cDNA using the First Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). qRT-PCR reactions were carried out using Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and the endogenous cyclophilin gene as an internal control, as previously described . .. The qPCR was performed on a StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA) programmed for 42 cycles of the following temperature schedule: 94°C 15 sec, 60°C 30 sec, 72°C 30 sec.

    Purification:

    Article Title: Antisense oligonucleotide treatment ameliorates IFN-γ–induced proteinuria in APOL1-transgenic mice
    Article Snippet: RNA was purified using a glass fiber filter plate (Pall) and chaotropic salts. mRNA expression levels were quantitated with quantitative reverse transcription PCR (qRT-PCR) on the 7900HT and QuantStudio 7 instruments (Applied Biosystems) using the following primer probe sets: human APOL1 , forward primer 5′-GCTACTCCTGCTGACTGATAATG-3′, reverse primer 5′-AAGGTTGTCCAGAGCTTTACG-3′, probe 5′-FAM-TGCCCAGGAATGAGGCAGATGAG-IOWA-BLACK-3′; human APOL2 , forward primer 5′-GATCCACACAGCTCAGAACA-3′, reverse primer 5′-TTCCTCTTCCCTCACTCTCA-3′, probe 5′-FAM-TTGGACAAGAGGACCCTGCCTTG-IOWA-BLACK-3′; human APOL3 , forward primer 5′-CTGAATTGCCCAGGGATGA-3′, reverse primer 5′-CTCATCTTTCTGCTGCACATATTC-3′, probe 5′-FAM-AGAGCTTCGTAGAGAGCATCTGCC-IOWA-BLACK-3′; and human GAPDH , forward primer 5′-GAAGGTGAAGGTCGGAGTC-3′, reverse primer 5′-GAAGATGGTGATGGGATTTC-3′, probe 5′-VIC-CAAGCTTCCCGTTCTCAGCC-TAMRA-3′. .. Briefly, 5 μL qRT-PCR reactions containing 1 μL RNA were run using AgPath-ID One-Step RT-PCR Reagent (Life Technologies) following the manufacturer’s recommendations.

    Polymerase Chain Reaction:

    Article Title: A Site-Specific, Single-Copy Transgenesis Strategy to Identify 5? Regulatory Sequences of the Mouse Testis-Determining Gene Sry
    Article Snippet: .. All qRT-PCR reactions were performed in triplicate using SYBR Green PCR master mix (Invitrogen) and 150 nM each of forward and reverse primers, and analyzed on a Viia7™ Real-Time PCR System (Invitrogen). .. Sry cDNA was amplified with primers 5′-TTATGGTGTGGTCCCGTGGT and 5′-GGCCTTTTTTCGGCTTCTGT and Sox9 cDNA was amplified with primers 5′-AGTACCCGCATCTGCACAAC and 5′-TACTTGTAATCGGGGTGGTCT .

    Article Title: MiR-148b suppresses cell proliferation and invasion in hepatocellular carcinoma by targeting WNT1/β-catenin pathway
    Article Snippet: After reverse transcription, qRT-PCR was performed using the quantitative SYBR Green PCR kit (TaKaRa Bio). .. All qRT-PCR reactions were run in an Applied Biosystems 7500 Real-Time PCR System and performed in triplicate.

    Article Title: Antisense oligonucleotide treatment ameliorates IFN-γ–induced proteinuria in APOL1-transgenic mice
    Article Snippet: RNA was purified using a glass fiber filter plate (Pall) and chaotropic salts. mRNA expression levels were quantitated with quantitative reverse transcription PCR (qRT-PCR) on the 7900HT and QuantStudio 7 instruments (Applied Biosystems) using the following primer probe sets: human APOL1 , forward primer 5′-GCTACTCCTGCTGACTGATAATG-3′, reverse primer 5′-AAGGTTGTCCAGAGCTTTACG-3′, probe 5′-FAM-TGCCCAGGAATGAGGCAGATGAG-IOWA-BLACK-3′; human APOL2 , forward primer 5′-GATCCACACAGCTCAGAACA-3′, reverse primer 5′-TTCCTCTTCCCTCACTCTCA-3′, probe 5′-FAM-TTGGACAAGAGGACCCTGCCTTG-IOWA-BLACK-3′; human APOL3 , forward primer 5′-CTGAATTGCCCAGGGATGA-3′, reverse primer 5′-CTCATCTTTCTGCTGCACATATTC-3′, probe 5′-FAM-AGAGCTTCGTAGAGAGCATCTGCC-IOWA-BLACK-3′; and human GAPDH , forward primer 5′-GAAGGTGAAGGTCGGAGTC-3′, reverse primer 5′-GAAGATGGTGATGGGATTTC-3′, probe 5′-VIC-CAAGCTTCCCGTTCTCAGCC-TAMRA-3′. .. Briefly, 5 μL qRT-PCR reactions containing 1 μL RNA were run using AgPath-ID One-Step RT-PCR Reagent (Life Technologies) following the manufacturer’s recommendations.

    Article Title: TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis
    Article Snippet: 1 μg of RNA was reversely transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Waltham, MA, USA) and TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR reactions were performed to detect TP53TG1, miR-18a and PTEN mRNA expression using SuperScript Platinum SYBR™ Green One-Step qRT-PCR Kit (Invitrogrn) on an 7900HT Fast Real-Time PCR System (Applied Biosystems). .. Primers for the PCR analysis were listed as follows: TP53TG1: 5′-ACGAAGGTACCCAACCCTCT-3′ (sense), and 5′-GGTGTAAGTGTTCGCCTGGT-3′ (antisense); miR-18a: 5′-GGTAAGGTGCATCTAGTG-3′ (sense), and 5′-GACTGTTCCTCTCTTCCTC-3′ (antisense); PTEN: 5′-CGGCAGCATCAAATGTTTCAG-3′ (sense), and 5′-AACTGGCAGGTAGAAGGCAACTC-3′ (antisense); GAPDH: 5′-GCACCGTCAAGGCTGAGAAC-3′ (sense), and 5′-TGGTGAAGACGCCAGTGGA-3′ (antisense); U6: 5′-GCTTCGGCAGCACATATACTAAAAT-3′(sense), and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (antisense).

    Article Title: Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis
    Article Snippet: RNA sample preparation, reverse transcription, and quantitative PCR were conducted according to the rules that have been proposed to ensure reproducible and accurate measurements [ ]. qRT-PCR reactions were performed in 96-well blocks using an Applied Biosystems 7500 Real-Time PCR System (Foster City, CA) using the SYBR Green I master mix in a volume of 20 μl. .. The PCR primers were designed using the Primer Express Software installed in the system and listed in Additional file .

    Article Title: A novel mechanism for the transcriptional regulation of Wnt signaling in development
    Article Snippet: .. Total RNA was isolated using Trizol, reverse transcription was performed using SuperScript III, and 5′ RACE was performed using the GeneRacer kit (all Invitrogen). qRT–PCR reactions were performed in triplicate using SYBR Green PCR master mix (Applied Biosystems) and normalized against expression of Hprt and GAPDH. .. The 5′ RACE and mouse qPCR primers are listed in Supplemental Tables S2 and S3, respectively.

    Software:

    Article Title: Evaluation of Schistosome Promoter Expression for Transgenesis and Genetic Analysis
    Article Snippet: Gene Transcript Analysis Total RNA (∼600 ng) from each transfected sample was converted to cDNA using the First Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). qRT-PCR reactions were carried out using Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and the endogenous cyclophilin gene as an internal control, as previously described . .. Detection of the SYBR Green fluorescent intensity occurred at 72°C of each cycle and was analyzed by StepOne System software (Applied Biosystems, Foster City, CA) using ΔΔCt method. qRT-PCR data were verified by t-test, and a p-value less than 0.05 was set as a statistically significant criterion .

    Article Title: Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis
    Article Snippet: RNA sample preparation, reverse transcription, and quantitative PCR were conducted according to the rules that have been proposed to ensure reproducible and accurate measurements [ ]. qRT-PCR reactions were performed in 96-well blocks using an Applied Biosystems 7500 Real-Time PCR System (Foster City, CA) using the SYBR Green I master mix in a volume of 20 μl. .. The PCR primers were designed using the Primer Express Software installed in the system and listed in Additional file .

    Real-time Polymerase Chain Reaction:

    Article Title: A Site-Specific, Single-Copy Transgenesis Strategy to Identify 5? Regulatory Sequences of the Mouse Testis-Determining Gene Sry
    Article Snippet: .. All qRT-PCR reactions were performed in triplicate using SYBR Green PCR master mix (Invitrogen) and 150 nM each of forward and reverse primers, and analyzed on a Viia7™ Real-Time PCR System (Invitrogen). .. Sry cDNA was amplified with primers 5′-TTATGGTGTGGTCCCGTGGT and 5′-GGCCTTTTTTCGGCTTCTGT and Sox9 cDNA was amplified with primers 5′-AGTACCCGCATCTGCACAAC and 5′-TACTTGTAATCGGGGTGGTCT .

    Article Title: MiR-148b suppresses cell proliferation and invasion in hepatocellular carcinoma by targeting WNT1/β-catenin pathway
    Article Snippet: .. All qRT-PCR reactions were run in an Applied Biosystems 7500 Real-Time PCR System and performed in triplicate. .. Statistical Analysis MiR-148b expression in HCC tissues and cells were compared by the unpaired Student's t test.

    Article Title: Glypican-3 induces a mesenchymal to epithelial transition in human breast cancer cells
    Article Snippet: Paragraph title: Quantitative Real-Time PCR (qRT-PCR) ... The resulting cDNA was subsequently treated with 1 Unit of RNase H (GE Healthcare, Little Chalfont, Buckinghamshire, UK). qRT-PCR reactions contained: 3 μl cDNA (1:10), 6 μl 2X SYBR Green Master Mix (Applied Biosystems Life Technologies, Carlsbad, CA, USA), and forward and reverse primers for GPC3, E-Cadherin, N-Cadherin, vimentin, SNAIL1, SNAIL2 (SLUG), ZEB1 and GAPDH (Table ).

    Article Title: Role of the venus kinase receptor in the female reproductive physiology of the desert locust, Schistocerca gregaria
    Article Snippet: Paragraph title: Quantitative real-time PCR (qRT-PCR) ... All qRT-PCR reactions were performed in duplicate in 96-well plates on a StepOne System (ABI Prism, Applied Biosystems).

    Article Title: Biosynthetic CircRNA_001160 induced by PTBP1 regulates the permeability of BTB via the CircRNA_001160/miR-195-5p/ETV1 axis
    Article Snippet: Paragraph title: Quantitative real-time PCR (qRT-PCR) assays ... All qRT-PCR reactions were performed using the 7500 Fast RT-PCR System (Applied Biosystems, USA).

    Article Title: Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells
    Article Snippet: .. All qRT-PCR reactions were carried out on a 7500 Fast Real-Time PCR system (Applied Biosystems). .. Analysis of miRNA expression Total RNA, including small RNA, was extracted and purified using the miRNeasy Mini Kit (QIAGEN Inc., Valencia, CA, USA) following the manufacturer's instructions.

    Article Title: Survivin-targeting miR-542-3p overcomes HER3 signaling-induced chemoresistance and enhances the antitumor activity of paclitaxel against HER2-overexpressing breast cancer
    Article Snippet: .. All qRT-PCR reactions were carried out on a 7500 Fast Real-Time PCR system (Applied Biosystems). .. The expression levels of miRNAs were determined as described previously [ , ].

    Article Title: Evaluation of Schistosome Promoter Expression for Transgenesis and Genetic Analysis
    Article Snippet: Gene Transcript Analysis Total RNA (∼600 ng) from each transfected sample was converted to cDNA using the First Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). qRT-PCR reactions were carried out using Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and the endogenous cyclophilin gene as an internal control, as previously described . .. The qPCR was performed on a StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA) programmed for 42 cycles of the following temperature schedule: 94°C 15 sec, 60°C 30 sec, 72°C 30 sec.

    Article Title: TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis
    Article Snippet: .. 1 μg of RNA was reversely transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Waltham, MA, USA) and TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR reactions were performed to detect TP53TG1, miR-18a and PTEN mRNA expression using SuperScript Platinum SYBR™ Green One-Step qRT-PCR Kit (Invitrogrn) on an 7900HT Fast Real-Time PCR System (Applied Biosystems). ..

    Article Title: Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis
    Article Snippet: .. RNA sample preparation, reverse transcription, and quantitative PCR were conducted according to the rules that have been proposed to ensure reproducible and accurate measurements [ ]. qRT-PCR reactions were performed in 96-well blocks using an Applied Biosystems 7500 Real-Time PCR System (Foster City, CA) using the SYBR Green I master mix in a volume of 20 μl. .. The PCR primers were designed using the Primer Express Software installed in the system and listed in Additional file .

    Article Title: A novel mechanism for the transcriptional regulation of Wnt signaling in development
    Article Snippet: Total RNA was isolated using Trizol, reverse transcription was performed using SuperScript III, and 5′ RACE was performed using the GeneRacer kit (all Invitrogen). qRT–PCR reactions were performed in triplicate using SYBR Green PCR master mix (Applied Biosystems) and normalized against expression of Hprt and GAPDH. .. The 5′ RACE and mouse qPCR primers are listed in Supplemental Tables S2 and S3, respectively.

    RNA Extraction:

    Article Title: A Site-Specific, Single-Copy Transgenesis Strategy to Identify 5? Regulatory Sequences of the Mouse Testis-Determining Gene Sry
    Article Snippet: Paragraph title: RNA extraction, cDNA synthesis, and quantitative real-time RT-PCR ... All qRT-PCR reactions were performed in triplicate using SYBR Green PCR master mix (Invitrogen) and 150 nM each of forward and reverse primers, and analyzed on a Viia7™ Real-Time PCR System (Invitrogen).

    Article Title: TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis
    Article Snippet: Paragraph title: RNA extraction and quantitative real-time PCR (qRT-PCR) ... 1 μg of RNA was reversely transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Waltham, MA, USA) and TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR reactions were performed to detect TP53TG1, miR-18a and PTEN mRNA expression using SuperScript Platinum SYBR™ Green One-Step qRT-PCR Kit (Invitrogrn) on an 7900HT Fast Real-Time PCR System (Applied Biosystems).

    Sample Prep:

    Article Title: Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis
    Article Snippet: .. RNA sample preparation, reverse transcription, and quantitative PCR were conducted according to the rules that have been proposed to ensure reproducible and accurate measurements [ ]. qRT-PCR reactions were performed in 96-well blocks using an Applied Biosystems 7500 Real-Time PCR System (Foster City, CA) using the SYBR Green I master mix in a volume of 20 μl. .. The PCR primers were designed using the Primer Express Software installed in the system and listed in Additional file .

    Transgenic Assay:

    Article Title: A Site-Specific, Single-Copy Transgenesis Strategy to Identify 5? Regulatory Sequences of the Mouse Testis-Determining Gene Sry
    Article Snippet: Total RNA was extracted and subjected to DNase treatment using an RNeasy Micro kit (Qiagen) in accordance with the manufacturer's instructions, and quantified with a NanoDrop spectrophotometer (NanoDrop Technologies). cDNA was synthesized by reverse transcription with SuperScript III and random hexamers (Invitrogen), from 100 ng total RNA (for analyses of precisely-staged embryos between 8–30 ts, within a single transgenic line) or from 300 ng total RNA (for all other analyses, at 11.5 or 13.5 dpc). .. All qRT-PCR reactions were performed in triplicate using SYBR Green PCR master mix (Invitrogen) and 150 nM each of forward and reverse primers, and analyzed on a Viia7™ Real-Time PCR System (Invitrogen).

    Spectrophotometry:

    Article Title: A Site-Specific, Single-Copy Transgenesis Strategy to Identify 5? Regulatory Sequences of the Mouse Testis-Determining Gene Sry
    Article Snippet: Total RNA was extracted and subjected to DNase treatment using an RNeasy Micro kit (Qiagen) in accordance with the manufacturer's instructions, and quantified with a NanoDrop spectrophotometer (NanoDrop Technologies). cDNA was synthesized by reverse transcription with SuperScript III and random hexamers (Invitrogen), from 100 ng total RNA (for analyses of precisely-staged embryos between 8–30 ts, within a single transgenic line) or from 300 ng total RNA (for all other analyses, at 11.5 or 13.5 dpc). .. All qRT-PCR reactions were performed in triplicate using SYBR Green PCR master mix (Invitrogen) and 150 nM each of forward and reverse primers, and analyzed on a Viia7™ Real-Time PCR System (Invitrogen).

    Article Title: Glypican-3 induces a mesenchymal to epithelial transition in human breast cancer cells
    Article Snippet: RNA was quantified in a Nanodrop (Thermo 2000 spectrophotometer) and cDNA was synthesized from 1 μg of RNA previously treated with 10 Units of DNAsa I (Invitrogen Life Technologies, Carlsbad, CA, USA), using iScript cDNA synthesis kit (Bio-Rad Life Science, Hercules, CA, USA). .. The resulting cDNA was subsequently treated with 1 Unit of RNase H (GE Healthcare, Little Chalfont, Buckinghamshire, UK). qRT-PCR reactions contained: 3 μl cDNA (1:10), 6 μl 2X SYBR Green Master Mix (Applied Biosystems Life Technologies, Carlsbad, CA, USA), and forward and reverse primers for GPC3, E-Cadherin, N-Cadherin, vimentin, SNAIL1, SNAIL2 (SLUG), ZEB1 and GAPDH (Table ).

    Article Title: TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis
    Article Snippet: Total RNA was extracted from tissues and cell lines using TRIzol™ Reagent (Invitrogen, Waltham, MA, USA), and then quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. 1 μg of RNA was reversely transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Waltham, MA, USA) and TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR reactions were performed to detect TP53TG1, miR-18a and PTEN mRNA expression using SuperScript Platinum SYBR™ Green One-Step qRT-PCR Kit (Invitrogrn) on an 7900HT Fast Real-Time PCR System (Applied Biosystems).

    Concentration Assay:

    Article Title: In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGF?-signaling and WT1
    Article Snippet: mRNA isolation and quantitative RT-PCR analysis Total RNA from cEPDCs stimulated as described was isolated using TriPure (Roche, Almere, The Netherlands) following the manufacturer’s protocol. cDNA was synthesized from 750 ng RNA per sample, using iScript cDNA synthesis kit (Fermentas, Leon-Rot, Germany). cDNA samples were subjected to quantitative RT-PCR (qRT-PCR) by using SYBRgreen (Roche) and a primer concentration of 10 μM. .. Primer sequences and annealing temperatures are available on request. qRT-PCR was performed for the following factors: WT1 isoform A, WT1 isoform D, TGFβ1 , 2 , and 3 , ALK1 , ALK5 , VCAM -1 , E -cadherin , endoglin and Snai1 . qRT-PCR reactions were run on a 7900HT Applied Biosystems.

    T-Test:

    Article Title: Evaluation of Schistosome Promoter Expression for Transgenesis and Genetic Analysis
    Article Snippet: Gene Transcript Analysis Total RNA (∼600 ng) from each transfected sample was converted to cDNA using the First Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). qRT-PCR reactions were carried out using Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and the endogenous cyclophilin gene as an internal control, as previously described . .. Detection of the SYBR Green fluorescent intensity occurred at 72°C of each cycle and was analyzed by StepOne System software (Applied Biosystems, Foster City, CA) using ΔΔCt method. qRT-PCR data were verified by t-test, and a p-value less than 0.05 was set as a statistically significant criterion .

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    Thermo Fisher taqmangene expression assay
    Taqmangene Expression Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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