Structured Review

Roche qrt pcr analysis total rna
Quercetin increases uncoupling protein-1 ( Ucp1) gene expression specifically in subcutaneous white adipose tissue. Gene expression in sWAT ( A ) and BAT ( B ) was determined by <t>qRT-PCR.</t> Hematoxylin and eosin (H E) staining was performed on paraffin-embedded sWAT sections, and representative pictures are shown ( C ). Pictures were analyzed in ImageJ to determine the relative cell size ( D ). sWAT sections were stained for UCP-1 (arrows indicate UCP-1 positive cells, E ) as well. BAT sections were stained for H E ( F ) and used to quantify lipid droplet content in ImageJ ( G ). BAT sections were also stained for UCP-1 ( H ). Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β 2-microglobulin (sWAT), Gapdh , and Hprt (BAT), * p
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1) Product Images from "Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice"

Article Title: Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19061786

Quercetin increases uncoupling protein-1 ( Ucp1) gene expression specifically in subcutaneous white adipose tissue. Gene expression in sWAT ( A ) and BAT ( B ) was determined by qRT-PCR. Hematoxylin and eosin (H E) staining was performed on paraffin-embedded sWAT sections, and representative pictures are shown ( C ). Pictures were analyzed in ImageJ to determine the relative cell size ( D ). sWAT sections were stained for UCP-1 (arrows indicate UCP-1 positive cells, E ) as well. BAT sections were stained for H E ( F ) and used to quantify lipid droplet content in ImageJ ( G ). BAT sections were also stained for UCP-1 ( H ). Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β 2-microglobulin (sWAT), Gapdh , and Hprt (BAT), * p
Figure Legend Snippet: Quercetin increases uncoupling protein-1 ( Ucp1) gene expression specifically in subcutaneous white adipose tissue. Gene expression in sWAT ( A ) and BAT ( B ) was determined by qRT-PCR. Hematoxylin and eosin (H E) staining was performed on paraffin-embedded sWAT sections, and representative pictures are shown ( C ). Pictures were analyzed in ImageJ to determine the relative cell size ( D ). sWAT sections were stained for UCP-1 (arrows indicate UCP-1 positive cells, E ) as well. BAT sections were stained for H E ( F ) and used to quantify lipid droplet content in ImageJ ( G ). BAT sections were also stained for UCP-1 ( H ). Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β 2-microglobulin (sWAT), Gapdh , and Hprt (BAT), * p

Techniques Used: Expressing, Quantitative RT-PCR, Staining

Quercetin reduces hepatic apolipoprotein B ( Apob) expression and increases the uptake of triglycerides (TG)-derived fatty acid (FA) by subcutaneous white adipose tissue. In week 2 and week 10 of the intervention, 24 h feces was collected ( A ) and used to determine fecal free fatty acid (FFA) concentration ( B ). Gene expression in the liver was determined by qRT-PCR for acyl-CoA synthetase long-chain family member 1 ( Acsl1) , acetyl-CoA carboxylase 2 ( Acc2 ), microsomal triglyceride transfer protein ( Mttp ), and Apob ( C ). After 12 weeks, mice were injected with glycerol tri[ 3 H]oleate-labeled lipoprotein-like particles, and clearance from plasma ( D ) and uptake per gram organ ( E ) were determined by 3 H-activity analysis. Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β2-microglobulin , * p
Figure Legend Snippet: Quercetin reduces hepatic apolipoprotein B ( Apob) expression and increases the uptake of triglycerides (TG)-derived fatty acid (FA) by subcutaneous white adipose tissue. In week 2 and week 10 of the intervention, 24 h feces was collected ( A ) and used to determine fecal free fatty acid (FFA) concentration ( B ). Gene expression in the liver was determined by qRT-PCR for acyl-CoA synthetase long-chain family member 1 ( Acsl1) , acetyl-CoA carboxylase 2 ( Acc2 ), microsomal triglyceride transfer protein ( Mttp ), and Apob ( C ). After 12 weeks, mice were injected with glycerol tri[ 3 H]oleate-labeled lipoprotein-like particles, and clearance from plasma ( D ) and uptake per gram organ ( E ) were determined by 3 H-activity analysis. Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β2-microglobulin , * p

Techniques Used: Expressing, Derivative Assay, Concentration Assay, Quantitative RT-PCR, Mouse Assay, Injection, Labeling, Activity Assay

2) Product Images from "Mouse neutrophils express functional umami taste receptor T1R1/T1R3"

Article Title: Mouse neutrophils express functional umami taste receptor T1R1/T1R3

Journal: BMB Reports

doi: 10.5483/BMBRep.2014.47.11.185

Identification of the T1R1/T1R3 umami taste receptor in mouse neutrophils. Whole mouse tongue tissues, mouse neutrophils were isolated from the bone marrow of 8-week-old C57BL/6 mice femurs and tibias. T1R1/T1R3 and taste signaling-associated components ( GNAT3 and TRPM5 ) mRNA were quantified by qRT-PCR (n=6-9). Values are reported as the mean pM of mRNA±SEM and were quantified in reference to the known standard gene and normalized to eukaryotic Elongation Factor2 RNA.
Figure Legend Snippet: Identification of the T1R1/T1R3 umami taste receptor in mouse neutrophils. Whole mouse tongue tissues, mouse neutrophils were isolated from the bone marrow of 8-week-old C57BL/6 mice femurs and tibias. T1R1/T1R3 and taste signaling-associated components ( GNAT3 and TRPM5 ) mRNA were quantified by qRT-PCR (n=6-9). Values are reported as the mean pM of mRNA±SEM and were quantified in reference to the known standard gene and normalized to eukaryotic Elongation Factor2 RNA.

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

3) Product Images from "GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells *GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells * S⃞"

Article Title: GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells *GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M707117200

FST expression is preferentially induced by GLI2 in human keratinocytes. A , qRT-PCR analysis of FST mRNA levels in HaCaT keratinocytes expressing either GLI1 (GLI1-HaCaT) or GLI2act (GLI2act-HaCaT) under tetracycline control for the time indicated. As a control for GLI activity the known target gene PTCH is shown ( inset ). B, FST and PTCH mRNA levels in human N/TERT-1 keratinocytes retrovirally transduced with GLI1 or GLI2act measured by qRT-PCR. The -fold change refers to the mRNA ratios for induced to uninduced cells ( A ) and cells infected with a GLI1/2 to control EGFP expressing virus ( B ). C , Western blot analysis of FST protein levels in GLI2act-HaCaT cells. Samples were taken from tetracycline-treated and untreated GLI2act-HaCaT cells as indicated. The upper panel shows protein expression of GLI2act transgene. FST protein was detected using a specific antibody recognizing all FST isoforms ( lower panel ). For control HaCaT cells were transiently transfected with an expression plasmid expressing human FST isoform 344 (p4TO-FST344) or empty expression vector (p4TO). * , unspecific signal.
Figure Legend Snippet: FST expression is preferentially induced by GLI2 in human keratinocytes. A , qRT-PCR analysis of FST mRNA levels in HaCaT keratinocytes expressing either GLI1 (GLI1-HaCaT) or GLI2act (GLI2act-HaCaT) under tetracycline control for the time indicated. As a control for GLI activity the known target gene PTCH is shown ( inset ). B, FST and PTCH mRNA levels in human N/TERT-1 keratinocytes retrovirally transduced with GLI1 or GLI2act measured by qRT-PCR. The -fold change refers to the mRNA ratios for induced to uninduced cells ( A ) and cells infected with a GLI1/2 to control EGFP expressing virus ( B ). C , Western blot analysis of FST protein levels in GLI2act-HaCaT cells. Samples were taken from tetracycline-treated and untreated GLI2act-HaCaT cells as indicated. The upper panel shows protein expression of GLI2act transgene. FST protein was detected using a specific antibody recognizing all FST isoforms ( lower panel ). For control HaCaT cells were transiently transfected with an expression plasmid expressing human FST isoform 344 (p4TO-FST344) or empty expression vector (p4TO). * , unspecific signal.

Techniques Used: Expressing, Quantitative RT-PCR, Activity Assay, Transduction, Infection, Western Blot, Transfection, Plasmid Preparation

4) Product Images from "Coordination of meristem and boundary functions by transcription factors in the SHOOT MERISTEMLESS regulatory network"

Article Title: Coordination of meristem and boundary functions by transcription factors in the SHOOT MERISTEMLESS regulatory network

Journal: Development (Cambridge, England)

doi: 10.1242/dev.157081

Regulatory interactions among STM, CUC1, TCPs and miR164c. (A) qRT-PCR timecourse analysis of STM and CUC1 expression levels in DEX-induced CUC1-GR and STM-GR lines relative to mock-induced control lines. (B) qRT-PCR analysis of STM and CUC1 expression levels in long-term induced STM-GR and CUC1-GR lines relative to wild-type control lines. (C) qRT-PCR analysis of STM expression levels in response to induction of CUC1-GR with DEX versus mock induction or with cycloheximide (CHX) and DEX versus CHX. CHX-DEX treatment was discontinued after 16 h owing to toxicity effects. (D) pSTM:GUS (top) and pCUC1:GUS (bottom) activity (blue) in wild-type and STMoe or CUC1-GR (CUC1oe) seedlings treated with DEX. Arrows indicate GUS activity. (E) ChIP analysis of the upstream region (promoter) of STM . (Top) Schematic representation of positions of tested amplicons upstream of START codon. (Bottom) Quantification of ChIP amplicons in the IP sample relative to mock-IP sample. Results are based on two biological replicates. (F) qRT-PCR analysis of STM, CUC1 and pri-miR164c expression in STMoe samples 24 h after induction with DEX (STMoe 24 h DEX) and in plants treated with DEX from germination (STMoe constitutive DEX). (G) Expression of pmiR164c:GUS (top) and pmiR164c:VENUS (bottom) in wild-type and STMoe seedlings (arrow=SAM) treated with DEX. A pmiR164c:VENUS reporter is expressed ectopically in cotyledon bases in STMoe. Arrows indicate GUS or GFP activity. (H,I) Expression levels of STM , CUC1 , TCP3 and TCP4 in STMoe timecourse measured by ATH1 DNA microarray (H) and by qRT-PCR (I) in separate experiments. All experiments are based on at least three biological replicates unless otherwise stated. Error bars indicate s.d. All expression changes in H are significant ( P
Figure Legend Snippet: Regulatory interactions among STM, CUC1, TCPs and miR164c. (A) qRT-PCR timecourse analysis of STM and CUC1 expression levels in DEX-induced CUC1-GR and STM-GR lines relative to mock-induced control lines. (B) qRT-PCR analysis of STM and CUC1 expression levels in long-term induced STM-GR and CUC1-GR lines relative to wild-type control lines. (C) qRT-PCR analysis of STM expression levels in response to induction of CUC1-GR with DEX versus mock induction or with cycloheximide (CHX) and DEX versus CHX. CHX-DEX treatment was discontinued after 16 h owing to toxicity effects. (D) pSTM:GUS (top) and pCUC1:GUS (bottom) activity (blue) in wild-type and STMoe or CUC1-GR (CUC1oe) seedlings treated with DEX. Arrows indicate GUS activity. (E) ChIP analysis of the upstream region (promoter) of STM . (Top) Schematic representation of positions of tested amplicons upstream of START codon. (Bottom) Quantification of ChIP amplicons in the IP sample relative to mock-IP sample. Results are based on two biological replicates. (F) qRT-PCR analysis of STM, CUC1 and pri-miR164c expression in STMoe samples 24 h after induction with DEX (STMoe 24 h DEX) and in plants treated with DEX from germination (STMoe constitutive DEX). (G) Expression of pmiR164c:GUS (top) and pmiR164c:VENUS (bottom) in wild-type and STMoe seedlings (arrow=SAM) treated with DEX. A pmiR164c:VENUS reporter is expressed ectopically in cotyledon bases in STMoe. Arrows indicate GUS or GFP activity. (H,I) Expression levels of STM , CUC1 , TCP3 and TCP4 in STMoe timecourse measured by ATH1 DNA microarray (H) and by qRT-PCR (I) in separate experiments. All experiments are based on at least three biological replicates unless otherwise stated. Error bars indicate s.d. All expression changes in H are significant ( P

Techniques Used: Quantitative RT-PCR, Expressing, Activity Assay, Chromatin Immunoprecipitation, Microarray

5) Product Images from "Shade avoidance 6 encodes an Arabidopsis flap endonuclease required for maintenance of genome integrity and development"

Article Title: Shade avoidance 6 encodes an Arabidopsis flap endonuclease required for maintenance of genome integrity and development

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv1474

QC defects of sav6 correlates with elevated ERF115 and PSK5 signalling. ( A ) qRT-PCR results showing the elevated expression of PSK5, PSKR1 and ERF115 in root tips of seedlings grow in Wc for 3 days. As the expression of ERF115 was not detected in Col-0, the relative expression of ERF115 to the reference gene is shown here. N.D.: not detected. Error bars represent the SEM ( n = 3). ( B ) Long exposure to Zeocin abolished the expression of QC marker genes. QC46:GUS and WOX5 pro :GFP transgenic lines were sown and grown on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\frac{1}{2}$\end{document} MS medium supplemented with various concentrations of Zeocin for 7 days. GUS expression and GFP signals in root tips are shown. Scale bars represent 100 μM.
Figure Legend Snippet: QC defects of sav6 correlates with elevated ERF115 and PSK5 signalling. ( A ) qRT-PCR results showing the elevated expression of PSK5, PSKR1 and ERF115 in root tips of seedlings grow in Wc for 3 days. As the expression of ERF115 was not detected in Col-0, the relative expression of ERF115 to the reference gene is shown here. N.D.: not detected. Error bars represent the SEM ( n = 3). ( B ) Long exposure to Zeocin abolished the expression of QC marker genes. QC46:GUS and WOX5 pro :GFP transgenic lines were sown and grown on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\frac{1}{2}$\end{document} MS medium supplemented with various concentrations of Zeocin for 7 days. GUS expression and GFP signals in root tips are shown. Scale bars represent 100 μM.

Techniques Used: Quantitative RT-PCR, Expressing, Marker, Transgenic Assay, Mass Spectrometry

Elevated SMR7 expression in sav6 may affect RAM development. ( A ) qRT-PCR results showing the relative expression (normalized using the reference gene) of WEE1, SMR4, 5 and 7 in Col-0 and sav6 ( n = 3). ( B ) SMR7 -overexpression leads to reduced RAM size. Left panel: representative figures of PI-stained Col-0, sav6 and 35S:SMR7:3XFLAG root tips (arrowheads mark the RAM upper border); the arrowhead in the inset marks the QC cells; right panel: number of cortex cells in the RAM ( n ≥ 11); scale bars represent 50 μM. ( C ) Knocking down the expression of SMR7 in sav6 increases the susceptibility of the transgenic line ( 35S:amiR172-SMR7/sav6 ) to Zeocin. Dead cells were detected by PI staining. Left panel: examples of no staining (-), mild staining (+) and strong staining (++); right panel: percentage of cells in each category. The experiment was repeated three times in total and similar patterns were observed each time. Error bars represent the SEM.
Figure Legend Snippet: Elevated SMR7 expression in sav6 may affect RAM development. ( A ) qRT-PCR results showing the relative expression (normalized using the reference gene) of WEE1, SMR4, 5 and 7 in Col-0 and sav6 ( n = 3). ( B ) SMR7 -overexpression leads to reduced RAM size. Left panel: representative figures of PI-stained Col-0, sav6 and 35S:SMR7:3XFLAG root tips (arrowheads mark the RAM upper border); the arrowhead in the inset marks the QC cells; right panel: number of cortex cells in the RAM ( n ≥ 11); scale bars represent 50 μM. ( C ) Knocking down the expression of SMR7 in sav6 increases the susceptibility of the transgenic line ( 35S:amiR172-SMR7/sav6 ) to Zeocin. Dead cells were detected by PI staining. Left panel: examples of no staining (-), mild staining (+) and strong staining (++); right panel: percentage of cells in each category. The experiment was repeated three times in total and similar patterns were observed each time. Error bars represent the SEM.

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

6) Product Images from "Targeted regulation of fibroblast state by CRISPR-mediated CEBPA expression"

Article Title: Targeted regulation of fibroblast state by CRISPR-mediated CEBPA expression

Journal: Respiratory Research

doi: 10.1186/s12931-019-1253-1

CEBPA overexpression in IPF-derived fibroblasts reduces their fibrogenic activation. a ) qRT-PCR analysis of Cebpa expression in the C/EBPα plasmid-overexpressing IPF fibroblasts compared to empty vector transfected control. b ) Western blotting analysis of C/EBPα protein in the C/EBPα-overexpressing IPF fibroblasts compared and empty vector transfected control. c-g ) qRT-PCR analysis showing CEBPA , ACTA2 , COL1A1 , FN1 and CTGF transcript levels in the C/EBPα-overexpressing IPF fibroblasts compared and empty vector transfected control. h-i ) ECM deposition assay shows collagen I and fibronectin production in the C/EBPα-overexpressing IPF fibroblasts compared to empty vector transfected control. j-k ) Immunostaining for αSMA expression in the C/EBPα-overexpressing IPF fibroblasts compared to empty vector transfected control. Scale bar = 100 μm. l) Western blot analysis of phospho-SMAD2/3 (pSMAD2/3) and total SMAD2/3 and GAPDH protein expression in the C/EBPα-overexpressing IPF fibroblasts and empty vector transfected control with TGF-β for the indicated periods. Time point 1–5: 0 min, 30 min, 60 min, 120 min, 240 min. m) Quantification of the pSMAD2/3 expression to total SMAD2/3 ratio from two independent experiment. Data are expressed as mean ± SD (* p
Figure Legend Snippet: CEBPA overexpression in IPF-derived fibroblasts reduces their fibrogenic activation. a ) qRT-PCR analysis of Cebpa expression in the C/EBPα plasmid-overexpressing IPF fibroblasts compared to empty vector transfected control. b ) Western blotting analysis of C/EBPα protein in the C/EBPα-overexpressing IPF fibroblasts compared and empty vector transfected control. c-g ) qRT-PCR analysis showing CEBPA , ACTA2 , COL1A1 , FN1 and CTGF transcript levels in the C/EBPα-overexpressing IPF fibroblasts compared and empty vector transfected control. h-i ) ECM deposition assay shows collagen I and fibronectin production in the C/EBPα-overexpressing IPF fibroblasts compared to empty vector transfected control. j-k ) Immunostaining for αSMA expression in the C/EBPα-overexpressing IPF fibroblasts compared to empty vector transfected control. Scale bar = 100 μm. l) Western blot analysis of phospho-SMAD2/3 (pSMAD2/3) and total SMAD2/3 and GAPDH protein expression in the C/EBPα-overexpressing IPF fibroblasts and empty vector transfected control with TGF-β for the indicated periods. Time point 1–5: 0 min, 30 min, 60 min, 120 min, 240 min. m) Quantification of the pSMAD2/3 expression to total SMAD2/3 ratio from two independent experiment. Data are expressed as mean ± SD (* p

Techniques Used: Over Expression, Derivative Assay, Activation Assay, Quantitative RT-PCR, Expressing, Plasmid Preparation, Transfection, Western Blot, Immunostaining

CEBPA expression is enhanced by a G9a inhibitor and partially mediates its anti-fibrotic effects. a ) qRT-PCR analysis of CEBPA expression in CEBPA knock down lung fibroblasts and their control with or without BIX01294 for 48 h. b-d ) Oil Red O staining and their quantification in IPF fibroblasts with or without BIX01294 in the adipogenic medium for 10 days. e-h ) qRT-PCR analysis showing ACTA2 , COL1A1 , FN1 and CTGF transcript levels in CEBPA knock down lung fibroblasts and their control with or without BIX01294 for 48 h. Data are expressed as mean ± SD (* p
Figure Legend Snippet: CEBPA expression is enhanced by a G9a inhibitor and partially mediates its anti-fibrotic effects. a ) qRT-PCR analysis of CEBPA expression in CEBPA knock down lung fibroblasts and their control with or without BIX01294 for 48 h. b-d ) Oil Red O staining and their quantification in IPF fibroblasts with or without BIX01294 in the adipogenic medium for 10 days. e-h ) qRT-PCR analysis showing ACTA2 , COL1A1 , FN1 and CTGF transcript levels in CEBPA knock down lung fibroblasts and their control with or without BIX01294 for 48 h. Data are expressed as mean ± SD (* p

Techniques Used: Expressing, Quantitative RT-PCR, Staining

CEBPA expression promotes a lipofibroblast phenotype. a-b ) Oil Red O staining and quantification in the Cebpa-overexpressing IPF fibroblasts and empty vector transfected control with (AD: adipogenic medium) or without adipogenic medium induction ( c : control medium). Scale bar = 100 μm. c-f ) qRT-PCR analysis of PLIN2 , PPARA , PPARG1 and PPARGC1A transcript levels in Cebpa-overexpressing IPF fibroblasts and empty vector transfected control with or without TGF-β1 treatment for 48 h. Data are expressed as mean ± SD (* p
Figure Legend Snippet: CEBPA expression promotes a lipofibroblast phenotype. a-b ) Oil Red O staining and quantification in the Cebpa-overexpressing IPF fibroblasts and empty vector transfected control with (AD: adipogenic medium) or without adipogenic medium induction ( c : control medium). Scale bar = 100 μm. c-f ) qRT-PCR analysis of PLIN2 , PPARA , PPARG1 and PPARGC1A transcript levels in Cebpa-overexpressing IPF fibroblasts and empty vector transfected control with or without TGF-β1 treatment for 48 h. Data are expressed as mean ± SD (* p

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

Loss of CEBPA in normal lung fibroblasts enhanced ECM deposition. a ) qRT-PCR analysis showing CEBPA expression in IPF-derived fibroblasts ( N = 8) and healthy fibroblasts (N = 8). b ) Western blotting analysis showing CEBPA protein expression in IPF-derived fibroblasts ( N = 5) and healthy fibroblasts ( N = 4). c ) Western blotting analysis and d ) qRT-PCR confirms the knock down of CEBPA expression. e-h ) qRT-PCR analysis showing ACTA2 , COL1A1 , FN1 and CTGF transcript levels in CEBPA knock down normal fibroblasts and their control. i-j ) ECM deposition assay shows collagen I and fibronectin production in CEBPA knockdown lung fibroblasts and their control. k ) Immunostaining and l) quantification for αSMA expression in both CEBPA knockdown lung fibroblasts and control lung fibroblasts. Scale bar = 100 μm. Data are expressed as mean ± SD (* p
Figure Legend Snippet: Loss of CEBPA in normal lung fibroblasts enhanced ECM deposition. a ) qRT-PCR analysis showing CEBPA expression in IPF-derived fibroblasts ( N = 8) and healthy fibroblasts (N = 8). b ) Western blotting analysis showing CEBPA protein expression in IPF-derived fibroblasts ( N = 5) and healthy fibroblasts ( N = 4). c ) Western blotting analysis and d ) qRT-PCR confirms the knock down of CEBPA expression. e-h ) qRT-PCR analysis showing ACTA2 , COL1A1 , FN1 and CTGF transcript levels in CEBPA knock down normal fibroblasts and their control. i-j ) ECM deposition assay shows collagen I and fibronectin production in CEBPA knockdown lung fibroblasts and their control. k ) Immunostaining and l) quantification for αSMA expression in both CEBPA knockdown lung fibroblasts and control lung fibroblasts. Scale bar = 100 μm. Data are expressed as mean ± SD (* p

Techniques Used: Quantitative RT-PCR, Expressing, Derivative Assay, Western Blot, Immunostaining

CEBPA expression can be restored by CRISPR activation. a ) Schematic illustration of the process of CRISPR gene activation (reproduced image is from Horizon Discovery and used with their permission) b ) qRT-PCR analysis of CEBPA expression in the dCAS9 expressing IPF fibroblasts with non-targeting gRNA or CEBPA gRNA or CEBPA gRNA CEBPA siRNA together for 3 days. c-d ) Western blot analysis and quantification of Fibronectin and αSMA expression in the dCAS9 expressing IPF fibroblasts with non-targeting gRNA or CEBPA gRNA or CEBPA gRNA CEBPA siRNA together for 3 days. e-i ) qRT-PCR analysis showing ACTA2 , COL1A1 , FN1 , CTGF and PLIN2 transcript levels in the dCAS9 expressing IPF fibroblasts with non-targeting gRNA or CEBPA gRNA or CEBPA gRNA CEBPA siRNA together for 3 days. Data are expressed as mean ± SD (* p
Figure Legend Snippet: CEBPA expression can be restored by CRISPR activation. a ) Schematic illustration of the process of CRISPR gene activation (reproduced image is from Horizon Discovery and used with their permission) b ) qRT-PCR analysis of CEBPA expression in the dCAS9 expressing IPF fibroblasts with non-targeting gRNA or CEBPA gRNA or CEBPA gRNA CEBPA siRNA together for 3 days. c-d ) Western blot analysis and quantification of Fibronectin and αSMA expression in the dCAS9 expressing IPF fibroblasts with non-targeting gRNA or CEBPA gRNA or CEBPA gRNA CEBPA siRNA together for 3 days. e-i ) qRT-PCR analysis showing ACTA2 , COL1A1 , FN1 , CTGF and PLIN2 transcript levels in the dCAS9 expressing IPF fibroblasts with non-targeting gRNA or CEBPA gRNA or CEBPA gRNA CEBPA siRNA together for 3 days. Data are expressed as mean ± SD (* p

Techniques Used: Expressing, CRISPR, Activation Assay, Quantitative RT-PCR, Western Blot

7) Product Images from "Multisignal control of expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum"

Article Title: Multisignal control of expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/erw198

Effect of iron starvation on P. tricornutum LHCX expression and photophysiology. Experiments were performed on cells grown in iron-replete (11 µM, +Fe) or iron-limited (5nM iron+100 µM FerroZine™, –Fe) conditions: (A) qRT-PCR analysis of LHCX transcripts in –Fe, normalized against the +Fe condition and using RPS and H4 as reference genes. (B) Immunoblot analysis of the LHCX, D2, and PsaF proteins, using βCF1 as loading control. NPQ capacity (C) and relative electron transfer rates (rETR PSII ) (D) of cells grown in +Fe or –Fe. The horizontal bar in (C) indicates when the actinic light was on (white) or off (black). rETR PSII was measured at different light intensities (20, 170, 260, 320, 520, and 950 µmol m −2 s −1 ). In (A), (C), and (D), error bars represent ±SD of three biological replicates. (This figure is available in colour at JXB online).
Figure Legend Snippet: Effect of iron starvation on P. tricornutum LHCX expression and photophysiology. Experiments were performed on cells grown in iron-replete (11 µM, +Fe) or iron-limited (5nM iron+100 µM FerroZine™, –Fe) conditions: (A) qRT-PCR analysis of LHCX transcripts in –Fe, normalized against the +Fe condition and using RPS and H4 as reference genes. (B) Immunoblot analysis of the LHCX, D2, and PsaF proteins, using βCF1 as loading control. NPQ capacity (C) and relative electron transfer rates (rETR PSII ) (D) of cells grown in +Fe or –Fe. The horizontal bar in (C) indicates when the actinic light was on (white) or off (black). rETR PSII was measured at different light intensities (20, 170, 260, 320, 520, and 950 µmol m −2 s −1 ). In (A), (C), and (D), error bars represent ±SD of three biological replicates. (This figure is available in colour at JXB online).

Techniques Used: Expressing, Quantitative RT-PCR

Light and dark regulation of P. tricornutum LHCXs. Analysis of the four LHCX transcripts by qRT-PCR (A) and of LHCX proteins (B) by western blotting in cells adapted to low light (LL) (12L/12D cycles), after exposure to LL for 2h then to high light (HL) for 30min, 1h, 3h, or 5h. mRNA levels were quantified by using RPS as the reference gene (A). Proteins were detected using the anti-LHCSR antibody which recognizes all the Pt LHCXs (arrowheads) and the anti-βCF1 antibody as loading control (B). Cells adapted to darkness for 60h were compared with those grown in LL for the analysis of LHCX transcripts (C), proteins (D), and NPQ (E). Relative transcript levels were determined using RPS as a reference, and values were normalized to gene expression levels in LL. LHCX proteins were detected as in (B). The horizontal bar in (E) indicates when the actinic light was on (white) or off (black). (F) LHCX4 mRNAs in 60h dark-adapted cells (Time 0) and in response to 10min, 30min, or 1h of blue light (1 µmol m −2 s −1 ), in the presence (black) or absence (grey) of the inhibitor DCMU. Transcript levels were quantified by using RPS as the reference, and normalized to gene expression levels in the dark. Error bars represent ±SD of three technical replicates from one representative experiment in (A), and ±SD of three biological replicates in (C), (E), and (F). (G) Alignment of regions 1 and 2 of the Chlamydomonas reinhardtii LHCSR3 and P. tricornutum LHCX1, 2, 3, and 4 protein sequences. The boxes indicate the pH-sensing residues conserved between the LHCXs and LHCSR3. (This figure is available in colour at JXB online).
Figure Legend Snippet: Light and dark regulation of P. tricornutum LHCXs. Analysis of the four LHCX transcripts by qRT-PCR (A) and of LHCX proteins (B) by western blotting in cells adapted to low light (LL) (12L/12D cycles), after exposure to LL for 2h then to high light (HL) for 30min, 1h, 3h, or 5h. mRNA levels were quantified by using RPS as the reference gene (A). Proteins were detected using the anti-LHCSR antibody which recognizes all the Pt LHCXs (arrowheads) and the anti-βCF1 antibody as loading control (B). Cells adapted to darkness for 60h were compared with those grown in LL for the analysis of LHCX transcripts (C), proteins (D), and NPQ (E). Relative transcript levels were determined using RPS as a reference, and values were normalized to gene expression levels in LL. LHCX proteins were detected as in (B). The horizontal bar in (E) indicates when the actinic light was on (white) or off (black). (F) LHCX4 mRNAs in 60h dark-adapted cells (Time 0) and in response to 10min, 30min, or 1h of blue light (1 µmol m −2 s −1 ), in the presence (black) or absence (grey) of the inhibitor DCMU. Transcript levels were quantified by using RPS as the reference, and normalized to gene expression levels in the dark. Error bars represent ±SD of three technical replicates from one representative experiment in (A), and ±SD of three biological replicates in (C), (E), and (F). (G) Alignment of regions 1 and 2 of the Chlamydomonas reinhardtii LHCSR3 and P. tricornutum LHCX1, 2, 3, and 4 protein sequences. The boxes indicate the pH-sensing residues conserved between the LHCXs and LHCSR3. (This figure is available in colour at JXB online).

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

Phaeodactylum tricornutum Pt4 ecotype lines overexpressing the LHCX genes. (A), (C), (E) LHCX transcript (upper panels) and protein (lower panels) analyses in the Pt4 wild type and transgenic strains overexpressing HA-tagged LHCX2 (A), LHCX3 (C), or LHCX4 (E). Transcript abundance was measured by qRT-PCR using RPS as the reference gene and normalized to the wild type expression value. Tagged proteins were detected by immunoblot using an anti-HA antibody, and an anti-CPF1 antibody as loading control. Bands are taken from the same blots but from non-adjacent lanes. (B), (D), (F) NPQ max capacity in the Pt4 wild type, in a transgenic strain expressing the vector for antibiotic resistance (transformation control, T.c.), and in independent transgenic lines overexpressing LHCX1 (OE1), LHCX2 (OE2), LHCX3 (OE3), and LHCX4 (OE4) genes. Asterisks indicate the results of two-tailed Student t -tests: ** p
Figure Legend Snippet: Phaeodactylum tricornutum Pt4 ecotype lines overexpressing the LHCX genes. (A), (C), (E) LHCX transcript (upper panels) and protein (lower panels) analyses in the Pt4 wild type and transgenic strains overexpressing HA-tagged LHCX2 (A), LHCX3 (C), or LHCX4 (E). Transcript abundance was measured by qRT-PCR using RPS as the reference gene and normalized to the wild type expression value. Tagged proteins were detected by immunoblot using an anti-HA antibody, and an anti-CPF1 antibody as loading control. Bands are taken from the same blots but from non-adjacent lanes. (B), (D), (F) NPQ max capacity in the Pt4 wild type, in a transgenic strain expressing the vector for antibiotic resistance (transformation control, T.c.), and in independent transgenic lines overexpressing LHCX1 (OE1), LHCX2 (OE2), LHCX3 (OE3), and LHCX4 (OE4) genes. Asterisks indicate the results of two-tailed Student t -tests: ** p

Techniques Used: Transgenic Assay, Quantitative RT-PCR, Expressing, Plasmid Preparation, Transformation Assay, Two Tailed Test

Effect of nitrogen starvation on P. tricornutum LHCX expression and photophysiology. Experiments were performed on cells grown in nitrogen-replete (1mM, +NO 3 – ) or nitrogen starvation (50 µM, –NO 3 – ) conditions: (A) qRT-PCR analysis of LHCX transcripts in –NO 3 – , normalized against the values in the +NO 3 – condition, and using RPS and H4 as reference genes. (B) Immunoblot analysis of the LHCX, D2, and PsaF proteins, using βCF1 as loading control. NPQ capacity (C) and relative electron transfer rates (rETR PSII ) (D) of cells grown in +NO 3 – and –NO 3 – conditions. The horizontal bar in (C) indicates when the actinic light was on (white) or off (black). rETR PSII was measured at different light intensities (20, 170, 260, 320, 520, and 950 µmol m −2 s −1 ). In (A), (C), and (D), error bars represent ±SD of three biological replicates. (This figure is available in colour at JXB online).
Figure Legend Snippet: Effect of nitrogen starvation on P. tricornutum LHCX expression and photophysiology. Experiments were performed on cells grown in nitrogen-replete (1mM, +NO 3 – ) or nitrogen starvation (50 µM, –NO 3 – ) conditions: (A) qRT-PCR analysis of LHCX transcripts in –NO 3 – , normalized against the values in the +NO 3 – condition, and using RPS and H4 as reference genes. (B) Immunoblot analysis of the LHCX, D2, and PsaF proteins, using βCF1 as loading control. NPQ capacity (C) and relative electron transfer rates (rETR PSII ) (D) of cells grown in +NO 3 – and –NO 3 – conditions. The horizontal bar in (C) indicates when the actinic light was on (white) or off (black). rETR PSII was measured at different light intensities (20, 170, 260, 320, 520, and 950 µmol m −2 s −1 ). In (A), (C), and (D), error bars represent ±SD of three biological replicates. (This figure is available in colour at JXB online).

Techniques Used: Expressing, Quantitative RT-PCR

8) Product Images from "Sorafenib suppresses extrahepatic metastasis de novo in hepatocellular carcinoma through inhibition of mesenchymal cancer stem cells characterized by the expression of CD90"

Article Title: Sorafenib suppresses extrahepatic metastasis de novo in hepatocellular carcinoma through inhibition of mesenchymal cancer stem cells characterized by the expression of CD90

Journal: Scientific Reports

doi: 10.1038/s41598-017-11848-z

Sorafenib targets CD90 + cells to suppress EV secretion. ( A ) Cell motility of HuH7 cells (green) co-cultured with HLF cells (blue) with/without sorafenib was monitored in real-time by time-lapse image analysis. See also Supplementary Movies 2 and 3 . ( B ) qRT-PCR analysis of CD63 and TGFB1 obtained from the EVs secreted from HLF and HuH7 cells treated with vehicle or sorafenib (2.5 μM). ( C ) Immunofluorescence analysis of HuH7 cells (green) cultured with CD63-labeled HLF cells (red). Sorafenib suppressed the number of HuH7 cells capturing EVs secreted from HLF cells (merge cells yellow). ( D ) Percentages of HuH7 cells (green), HLF cells (red), and HuH7 cells capturing EVs secreted from HLF cells (yellow). The number of green, red, and yellow cells was counted in triplicate at three independent areas. Sorafenib treatment significantly reduced the percentage of HuH7 cells trapping EVs produced by HLF cells.
Figure Legend Snippet: Sorafenib targets CD90 + cells to suppress EV secretion. ( A ) Cell motility of HuH7 cells (green) co-cultured with HLF cells (blue) with/without sorafenib was monitored in real-time by time-lapse image analysis. See also Supplementary Movies 2 and 3 . ( B ) qRT-PCR analysis of CD63 and TGFB1 obtained from the EVs secreted from HLF and HuH7 cells treated with vehicle or sorafenib (2.5 μM). ( C ) Immunofluorescence analysis of HuH7 cells (green) cultured with CD63-labeled HLF cells (red). Sorafenib suppressed the number of HuH7 cells capturing EVs secreted from HLF cells (merge cells yellow). ( D ) Percentages of HuH7 cells (green), HLF cells (red), and HuH7 cells capturing EVs secreted from HLF cells (yellow). The number of green, red, and yellow cells was counted in triplicate at three independent areas. Sorafenib treatment significantly reduced the percentage of HuH7 cells trapping EVs produced by HLF cells.

Techniques Used: Cell Culture, Quantitative RT-PCR, Immunofluorescence, Labeling, Produced

Differential activation of c-Kit signaling in EpCAM + and CD90 + HCC cell lines. ( A ) qRT-PCR analysis of EpCAM + (Hep3B, HuH7, and HuH1) and CD90 + (HLE, HLF, SK-Hep-1) HCC cell lines. ( B ) Western blot analysis of c-Kit expression in EpCAM + (Hep3B, HuH7, and HuH1) and CD90 + (HLE, HLF, SK-Hep-1) HCC cell lines. ( C ) Western blot analysis of c-Kit and phospho-c-Kit in HLF cells treated with SCF-1 and sorafenib for 24 h. ( D ) Cell proliferation assay of EpCAM + (Hep3B, HuH7, and HuH1) and CD90 + (HLE, HLF, SK-Hep-1) HCC cell lines treated with vehicle (0.1% DMSO) or sorafenib (5 μM).
Figure Legend Snippet: Differential activation of c-Kit signaling in EpCAM + and CD90 + HCC cell lines. ( A ) qRT-PCR analysis of EpCAM + (Hep3B, HuH7, and HuH1) and CD90 + (HLE, HLF, SK-Hep-1) HCC cell lines. ( B ) Western blot analysis of c-Kit expression in EpCAM + (Hep3B, HuH7, and HuH1) and CD90 + (HLE, HLF, SK-Hep-1) HCC cell lines. ( C ) Western blot analysis of c-Kit and phospho-c-Kit in HLF cells treated with SCF-1 and sorafenib for 24 h. ( D ) Cell proliferation assay of EpCAM + (Hep3B, HuH7, and HuH1) and CD90 + (HLE, HLF, SK-Hep-1) HCC cell lines treated with vehicle (0.1% DMSO) or sorafenib (5 μM).

Techniques Used: Activation Assay, Quantitative RT-PCR, Western Blot, Expressing, Proliferation Assay

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SYBR Green Assay:

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RNA Extraction:

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RNA Sequencing Assay:

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Synthesized:

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Isolation:

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Quantitative RT-PCR:

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Article Snippet: .. RNA extraction and qRT-PCR analysis Total RNA was isolated with RNeasy Plus Mini kit. cDNA was synthesized with SuperScript™ IV Reverse Transcriptase. qRT-PCR was carried out using the FastStart Essential DNA Green Master (Roche, 06402712001) for SYBR Green I-based real-time PCR on the Lightcycler 96 Real-Time PCR System (Roche) according to the manufacturer’s instructions. qRT-PCR was performed by incubating the plates at 95 °C for 10 min and then cycling 40 times at 95 °C for 10 s, 60 °C for 10s, and 72 °C for 10s. ..

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Article Title: Shade avoidance 6 encodes an Arabidopsis flap endonuclease required for maintenance of genome integrity and development
Article Snippet: .. RNA isolation and qRT-PCR analysis Total RNA was extracted from root tips that were shorter than 5 mm using the TriPure (Roche) reagent. .. The RNA was used for reverse transcription (cDNA Synthesis Kit K1622; Thermo Scientific). qRT-PCR was carried out using SYBR green reagents and a Stratagene Mx3000p real-time PCR system (AGILENT Technologies).

Article Title: Coordination of meristem and boundary functions by transcription factors in the SHOOT MERISTEMLESS regulatory network
Article Snippet: .. qRT-PCR analysis Total RNA was isolated with Tripure (Roche) and cDNA synthesis was performed using the Ambion Retroscript kit. qRT-PCR (Rotorgene 6000; Corbett Research) used Abgene SYBR green mix (Thermo Fisher) and ACTIN2 as reference. ..

Article Title: Sorafenib suppresses extrahepatic metastasis de novo in hepatocellular carcinoma through inhibition of mesenchymal cancer stem cells characterized by the expression of CD90
Article Snippet: .. qRT-PCR analysis Total RNA was extracted using a High Pure RNA Isolation Kit (Roche Diagnostics K.K., Tokyo, Japan) according to the manufacturer’s instructions. .. The expression of selected genes was determined in triplicate using the 7900 Sequence Detection System (Applied Biosystems, Foster City, CA).

Purification:

Article Title: GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells *GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells * S⃞
Article Snippet: .. Slides were counterstained with hematoxylin. qRT-PCR Analysis —Total RNA was isolated and purified (High Pure RNA Isolation Kit, Roche Applied Science), and cDNA was synthesized from 4 μg of total RNA with Superscript II (RNase H- ) reverse transcriptase (Invitrogen) using oligo(dT) primers, according to the manufacturer's instructions. qRT-PCR analysis was performed on a Rotor-Gene 3000 (Corbett Research) using iQ™ SYBR Green Supermix (Bio-Rad). .. Human large ribosomal protein P0 (RPLP0) or mouse acidic ribosomal phosphoprotein P0 (Arbp) was used for normalization in qRT-PCR analysis ( ).

Real-time Polymerase Chain Reaction:

Article Title: Targeted regulation of fibroblast state by CRISPR-mediated CEBPA expression
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    Roche qrt pcr analysis total rna
    Quercetin increases uncoupling protein-1 ( Ucp1) gene expression specifically in subcutaneous white adipose tissue. Gene expression in sWAT ( A ) and BAT ( B ) was determined by <t>qRT-PCR.</t> Hematoxylin and eosin (H E) staining was performed on paraffin-embedded sWAT sections, and representative pictures are shown ( C ). Pictures were analyzed in ImageJ to determine the relative cell size ( D ). sWAT sections were stained for UCP-1 (arrows indicate UCP-1 positive cells, E ) as well. BAT sections were stained for H E ( F ) and used to quantify lipid droplet content in ImageJ ( G ). BAT sections were also stained for UCP-1 ( H ). Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β 2-microglobulin (sWAT), Gapdh , and Hprt (BAT), * p
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    Quercetin increases uncoupling protein-1 ( Ucp1) gene expression specifically in subcutaneous white adipose tissue. Gene expression in sWAT ( A ) and BAT ( B ) was determined by qRT-PCR. Hematoxylin and eosin (H E) staining was performed on paraffin-embedded sWAT sections, and representative pictures are shown ( C ). Pictures were analyzed in ImageJ to determine the relative cell size ( D ). sWAT sections were stained for UCP-1 (arrows indicate UCP-1 positive cells, E ) as well. BAT sections were stained for H E ( F ) and used to quantify lipid droplet content in ImageJ ( G ). BAT sections were also stained for UCP-1 ( H ). Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β 2-microglobulin (sWAT), Gapdh , and Hprt (BAT), * p

    Journal: International Journal of Molecular Sciences

    Article Title: Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice

    doi: 10.3390/ijms19061786

    Figure Lengend Snippet: Quercetin increases uncoupling protein-1 ( Ucp1) gene expression specifically in subcutaneous white adipose tissue. Gene expression in sWAT ( A ) and BAT ( B ) was determined by qRT-PCR. Hematoxylin and eosin (H E) staining was performed on paraffin-embedded sWAT sections, and representative pictures are shown ( C ). Pictures were analyzed in ImageJ to determine the relative cell size ( D ). sWAT sections were stained for UCP-1 (arrows indicate UCP-1 positive cells, E ) as well. BAT sections were stained for H E ( F ) and used to quantify lipid droplet content in ImageJ ( G ). BAT sections were also stained for UCP-1 ( H ). Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β 2-microglobulin (sWAT), Gapdh , and Hprt (BAT), * p

    Article Snippet: RNA Isolation and qRT-PCR Analysis Total RNA was isolated using TriPure Isolation reagent (Roche obtained via Sigma, St. Louis, MO, USA ) following the manufacturer’s protocol. cDNA was made using Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Leiden, The Netherlands).

    Techniques: Expressing, Quantitative RT-PCR, Staining

    Quercetin reduces hepatic apolipoprotein B ( Apob) expression and increases the uptake of triglycerides (TG)-derived fatty acid (FA) by subcutaneous white adipose tissue. In week 2 and week 10 of the intervention, 24 h feces was collected ( A ) and used to determine fecal free fatty acid (FFA) concentration ( B ). Gene expression in the liver was determined by qRT-PCR for acyl-CoA synthetase long-chain family member 1 ( Acsl1) , acetyl-CoA carboxylase 2 ( Acc2 ), microsomal triglyceride transfer protein ( Mttp ), and Apob ( C ). After 12 weeks, mice were injected with glycerol tri[ 3 H]oleate-labeled lipoprotein-like particles, and clearance from plasma ( D ) and uptake per gram organ ( E ) were determined by 3 H-activity analysis. Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β2-microglobulin , * p

    Journal: International Journal of Molecular Sciences

    Article Title: Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice

    doi: 10.3390/ijms19061786

    Figure Lengend Snippet: Quercetin reduces hepatic apolipoprotein B ( Apob) expression and increases the uptake of triglycerides (TG)-derived fatty acid (FA) by subcutaneous white adipose tissue. In week 2 and week 10 of the intervention, 24 h feces was collected ( A ) and used to determine fecal free fatty acid (FFA) concentration ( B ). Gene expression in the liver was determined by qRT-PCR for acyl-CoA synthetase long-chain family member 1 ( Acsl1) , acetyl-CoA carboxylase 2 ( Acc2 ), microsomal triglyceride transfer protein ( Mttp ), and Apob ( C ). After 12 weeks, mice were injected with glycerol tri[ 3 H]oleate-labeled lipoprotein-like particles, and clearance from plasma ( D ) and uptake per gram organ ( E ) were determined by 3 H-activity analysis. Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β2-microglobulin , * p

    Article Snippet: RNA Isolation and qRT-PCR Analysis Total RNA was isolated using TriPure Isolation reagent (Roche obtained via Sigma, St. Louis, MO, USA ) following the manufacturer’s protocol. cDNA was made using Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Leiden, The Netherlands).

    Techniques: Expressing, Derivative Assay, Concentration Assay, Quantitative RT-PCR, Mouse Assay, Injection, Labeling, Activity Assay

    Knockdown of APOA4 -AS in ob/ob mice decreases APOA4 expression and plasma TG and TC levels without affecting steatosis. ob/ob mice (8 weeks old) were tail-vein injected ad-scramble or ad-sh APOA4 -AS1 purified adenovirus. Mice were sacrificed at 10–12 days after adenovirus injection. ( A ) APOA4 and tubulin were measured by western blot. ( B ) APOA4 -AS, APOA4, FASN, SCD1, APOB, MTP, CPT1a and MCAD transcripts in the liver were measured by qRT-PCR. ( C ) Liver TG level. ( D ) H E staining of liver sections. ( E ) Plasma TG levels. ( F ) Plasma TC levels. n = 5–6. * P

    Journal: Nucleic Acids Research

    Article Title: A long non-coding RNA, APOA4-AS, regulates APOA4 expression depending on HuR in mice

    doi: 10.1093/nar/gkw341

    Figure Lengend Snippet: Knockdown of APOA4 -AS in ob/ob mice decreases APOA4 expression and plasma TG and TC levels without affecting steatosis. ob/ob mice (8 weeks old) were tail-vein injected ad-scramble or ad-sh APOA4 -AS1 purified adenovirus. Mice were sacrificed at 10–12 days after adenovirus injection. ( A ) APOA4 and tubulin were measured by western blot. ( B ) APOA4 -AS, APOA4, FASN, SCD1, APOB, MTP, CPT1a and MCAD transcripts in the liver were measured by qRT-PCR. ( C ) Liver TG level. ( D ) H E staining of liver sections. ( E ) Plasma TG levels. ( F ) Plasma TC levels. n = 5–6. * P

    Article Snippet: qRT-PCR analysis Total RNAs were extracted using TriPure Isolation Reagent (Roche, Mannheim, Germany).

    Techniques: Mouse Assay, Expressing, Injection, Purification, Western Blot, Quantitative RT-PCR, Staining

    APOA4 -AS transcript interacts with mRNA stabilizing protein HuR. ( A ) Semi-quantitative RT-PCR (left) analyses of APOA4 -AS in RNA isolated from a-flag immunocomplex (top) or input (bottom) from transiently transfected HEK293T cells. qRT-PCR analyses (right) were also performed by using the same samples and normalized by input. ( B ) Flag-HuR was co-transfected with APOA4 -AS WT or two truncated APOA4 -AS (T1 or T2) in HEK293T cells. APOA4 -AS enrichment in flag immunocomplex was present by RIP/IgG. ( C ) and ( D ) qRT-PCR analyses of endogenous APOA4 -AS and APOA4 transcripts in the HuR immunocomplex from mouse liver. ( E ) Immunoblots of Flag-HuR after strepavidin-APOA4-AS (StA-APOA4-AS) precipitation from transfected HEK293T cell lysates. Schematic diagrams of StA-APOA4-AS are shown on the right. n = 3–5. * P

    Journal: Nucleic Acids Research

    Article Title: A long non-coding RNA, APOA4-AS, regulates APOA4 expression depending on HuR in mice

    doi: 10.1093/nar/gkw341

    Figure Lengend Snippet: APOA4 -AS transcript interacts with mRNA stabilizing protein HuR. ( A ) Semi-quantitative RT-PCR (left) analyses of APOA4 -AS in RNA isolated from a-flag immunocomplex (top) or input (bottom) from transiently transfected HEK293T cells. qRT-PCR analyses (right) were also performed by using the same samples and normalized by input. ( B ) Flag-HuR was co-transfected with APOA4 -AS WT or two truncated APOA4 -AS (T1 or T2) in HEK293T cells. APOA4 -AS enrichment in flag immunocomplex was present by RIP/IgG. ( C ) and ( D ) qRT-PCR analyses of endogenous APOA4 -AS and APOA4 transcripts in the HuR immunocomplex from mouse liver. ( E ) Immunoblots of Flag-HuR after strepavidin-APOA4-AS (StA-APOA4-AS) precipitation from transfected HEK293T cell lysates. Schematic diagrams of StA-APOA4-AS are shown on the right. n = 3–5. * P

    Article Snippet: qRT-PCR analysis Total RNAs were extracted using TriPure Isolation Reagent (Roche, Mannheim, Germany).

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

    Knockdown of APOA4 -AS transcript decreases APOA4 mRNA and protein levels both in vitro and in vivo . ( A–C ) Primary hepatocytes were isolated from C57BL/6 mice. Cells were infected with ad-scramble or two different ad-shRNAs (sh APOA4 -AS1 or sh APOA4 -AS2) adenovirus targeting to APOA4 -AS transcript. APOA4 -AS (A) and APOA4 (B) transcripts were measured by qRT-PCR. APOA4 protein level was measured by western blot (C). ( D–F ) C57BL/6 mice (8 weeks old) were tail-vein injected with ad-scramble or two different ad-shRNAs (sh APOA4 -AS1 or sh APOA4 -AS2) adenovirus targeting APOA4 -AS transcript. APOA4 -AS (D) and APOA4 transcripts (E) in the liver were measured by qRT-PCR. APOA4 protein levels in the liver and plasma were measured by western blot (F). n = 3–6. * P

    Journal: Nucleic Acids Research

    Article Title: A long non-coding RNA, APOA4-AS, regulates APOA4 expression depending on HuR in mice

    doi: 10.1093/nar/gkw341

    Figure Lengend Snippet: Knockdown of APOA4 -AS transcript decreases APOA4 mRNA and protein levels both in vitro and in vivo . ( A–C ) Primary hepatocytes were isolated from C57BL/6 mice. Cells were infected with ad-scramble or two different ad-shRNAs (sh APOA4 -AS1 or sh APOA4 -AS2) adenovirus targeting to APOA4 -AS transcript. APOA4 -AS (A) and APOA4 (B) transcripts were measured by qRT-PCR. APOA4 protein level was measured by western blot (C). ( D–F ) C57BL/6 mice (8 weeks old) were tail-vein injected with ad-scramble or two different ad-shRNAs (sh APOA4 -AS1 or sh APOA4 -AS2) adenovirus targeting APOA4 -AS transcript. APOA4 -AS (D) and APOA4 transcripts (E) in the liver were measured by qRT-PCR. APOA4 protein levels in the liver and plasma were measured by western blot (F). n = 3–6. * P

    Article Snippet: qRT-PCR analysis Total RNAs were extracted using TriPure Isolation Reagent (Roche, Mannheim, Germany).

    Techniques: In Vitro, In Vivo, Isolation, Mouse Assay, Infection, Quantitative RT-PCR, Western Blot, Injection

    Deletion of HuR decreases both APOA4 -AS and APOA4 mRNA in primary hepatocytes. Primary hepatocytes were isolated from C57BL/6 mice. Cells were infected with ad-Cas9 and two different ad-sgRNAs adenovirus targeting to HuR . ( A ) HuR and β-actin protein levels were measured by western blot. ( B ) APOA4- AS and APOA4 transcripts were measured by qRT-PCR. ( C ) and ( D ) Hepatocytes with HuR deletion were infected with APOA4 -AS adenovirus. APOA4 (C) and APOA4- AS (D) transcripts were measured by qRT-PCR. n = 4–6. * P

    Journal: Nucleic Acids Research

    Article Title: A long non-coding RNA, APOA4-AS, regulates APOA4 expression depending on HuR in mice

    doi: 10.1093/nar/gkw341

    Figure Lengend Snippet: Deletion of HuR decreases both APOA4 -AS and APOA4 mRNA in primary hepatocytes. Primary hepatocytes were isolated from C57BL/6 mice. Cells were infected with ad-Cas9 and two different ad-sgRNAs adenovirus targeting to HuR . ( A ) HuR and β-actin protein levels were measured by western blot. ( B ) APOA4- AS and APOA4 transcripts were measured by qRT-PCR. ( C ) and ( D ) Hepatocytes with HuR deletion were infected with APOA4 -AS adenovirus. APOA4 (C) and APOA4- AS (D) transcripts were measured by qRT-PCR. n = 4–6. * P

    Article Snippet: qRT-PCR analysis Total RNAs were extracted using TriPure Isolation Reagent (Roche, Mannheim, Germany).

    Techniques: Isolation, Mouse Assay, Infection, Western Blot, Quantitative RT-PCR

    Identification of the T1R1/T1R3 umami taste receptor in mouse neutrophils. Whole mouse tongue tissues, mouse neutrophils were isolated from the bone marrow of 8-week-old C57BL/6 mice femurs and tibias. T1R1/T1R3 and taste signaling-associated components ( GNAT3 and TRPM5 ) mRNA were quantified by qRT-PCR (n=6-9). Values are reported as the mean pM of mRNA±SEM and were quantified in reference to the known standard gene and normalized to eukaryotic Elongation Factor2 RNA.

    Journal: BMB Reports

    Article Title: Mouse neutrophils express functional umami taste receptor T1R1/T1R3

    doi: 10.5483/BMBRep.2014.47.11.185

    Figure Lengend Snippet: Identification of the T1R1/T1R3 umami taste receptor in mouse neutrophils. Whole mouse tongue tissues, mouse neutrophils were isolated from the bone marrow of 8-week-old C57BL/6 mice femurs and tibias. T1R1/T1R3 and taste signaling-associated components ( GNAT3 and TRPM5 ) mRNA were quantified by qRT-PCR (n=6-9). Values are reported as the mean pM of mRNA±SEM and were quantified in reference to the known standard gene and normalized to eukaryotic Elongation Factor2 RNA.

    Article Snippet: RNA isolation, RNA sequencing, and qRT-PCR analysis Total RNA was extracted using a MagNA lyser (Roche Molecular Diagnostics GmbH, Penzburg, Germany) with TRIzol reagent (Invitrogen, Carlsbad, CA, USA).

    Techniques: Isolation, Mouse Assay, Quantitative RT-PCR

    FST expression is preferentially induced by GLI2 in human keratinocytes. A , qRT-PCR analysis of FST mRNA levels in HaCaT keratinocytes expressing either GLI1 (GLI1-HaCaT) or GLI2act (GLI2act-HaCaT) under tetracycline control for the time indicated. As a control for GLI activity the known target gene PTCH is shown ( inset ). B, FST and PTCH mRNA levels in human N/TERT-1 keratinocytes retrovirally transduced with GLI1 or GLI2act measured by qRT-PCR. The -fold change refers to the mRNA ratios for induced to uninduced cells ( A ) and cells infected with a GLI1/2 to control EGFP expressing virus ( B ). C , Western blot analysis of FST protein levels in GLI2act-HaCaT cells. Samples were taken from tetracycline-treated and untreated GLI2act-HaCaT cells as indicated. The upper panel shows protein expression of GLI2act transgene. FST protein was detected using a specific antibody recognizing all FST isoforms ( lower panel ). For control HaCaT cells were transiently transfected with an expression plasmid expressing human FST isoform 344 (p4TO-FST344) or empty expression vector (p4TO). * , unspecific signal.

    Journal: The Journal of Biological Chemistry

    Article Title: GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells *GLI2-specific Transcriptional Activation of the Bone Morphogenetic Protein/Activin Antagonist Follistatin in Human Epidermal Cells * S⃞

    doi: 10.1074/jbc.M707117200

    Figure Lengend Snippet: FST expression is preferentially induced by GLI2 in human keratinocytes. A , qRT-PCR analysis of FST mRNA levels in HaCaT keratinocytes expressing either GLI1 (GLI1-HaCaT) or GLI2act (GLI2act-HaCaT) under tetracycline control for the time indicated. As a control for GLI activity the known target gene PTCH is shown ( inset ). B, FST and PTCH mRNA levels in human N/TERT-1 keratinocytes retrovirally transduced with GLI1 or GLI2act measured by qRT-PCR. The -fold change refers to the mRNA ratios for induced to uninduced cells ( A ) and cells infected with a GLI1/2 to control EGFP expressing virus ( B ). C , Western blot analysis of FST protein levels in GLI2act-HaCaT cells. Samples were taken from tetracycline-treated and untreated GLI2act-HaCaT cells as indicated. The upper panel shows protein expression of GLI2act transgene. FST protein was detected using a specific antibody recognizing all FST isoforms ( lower panel ). For control HaCaT cells were transiently transfected with an expression plasmid expressing human FST isoform 344 (p4TO-FST344) or empty expression vector (p4TO). * , unspecific signal.

    Article Snippet: Slides were counterstained with hematoxylin. qRT-PCR Analysis —Total RNA was isolated and purified (High Pure RNA Isolation Kit, Roche Applied Science), and cDNA was synthesized from 4 μg of total RNA with Superscript II (RNase H- ) reverse transcriptase (Invitrogen) using oligo(dT) primers, according to the manufacturer's instructions. qRT-PCR analysis was performed on a Rotor-Gene 3000 (Corbett Research) using iQ™ SYBR Green Supermix (Bio-Rad).

    Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Transduction, Infection, Western Blot, Transfection, Plasmid Preparation