high capacity cdna reverse transcription kit  (Thermo Fisher)


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    Name:
    High-Capacity cDNA Reverse Transcription Kit
    Description:
    The High Capacity cDNA Reverse Transcription Kit contains all components necessary for the quantitative conversion of up to 2 µg of total RNA to single-stranded cDNA in a single 20 µL reaction. The High Capacity cDNA Reverse Transcription Kit delivers extremely high-quality, single-stranded cDNA from 0.02 to 2 µg total RNA. Reactions can be scaled up to 100 µL to generate 10 µg of cDNA from a single reaction. Downstream applications include real-time PCR, standard PCR, and microarrays. The kit is ideal for generating cDNA archives.Features of this kit include:• Linear target amplification for real-time PCR• Higher yields and precision than other cDNA synthesis kits at a fraction of the cost• 10-fold greater dynamic range than other kitsExtensively tested with a variety of templates Quantitative first-strand synthesis of all RNA species is achieved with the use of the random primers. The kit has been extensively tested with a variety of RNA templates, including GC- and AU-rich targets and RNAs expressed at low levels. cRNA can also be efficiently generated from in vitro transcription of cDNA.
    Catalog Number:
    4368813
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Size:
    1 000 reactions
    Category:
    Kits and Assays, cDNA Synthesis, Library, & Cloning Kits, cDNA Synthesis Kits
    Score:
    85
    		Buy from Supplier

    Structured Review

    Thermo Fisher high capacity cdna reverse transcription kit
    Reactivation of LINE-1 by BaP is Effected via Canonical TGF-β1 Signaling Total <t>RNA</t> was isolated from BEAS-2B cells treated with 0.5 uM BaP for 8 hours, and 1 µg of RNA subjected to <t>cDNA</t> synthesis. Samples were analyzed by RT-qPCR using specific primers for ( A ) human LINE-1 (ORF1 and ORF2) or ( B ) TGF-β1. ( C ) cells pre-treated with TGF-β1 receptor inhibitor (LY2157299) or vehicle (DMSO) for 30 min before BaP challenge or ( D ) transfected with target-specific siRNAs to SMAD2, SMAD3, or scramble siRNA or no siRNA (mock) controls. Expression levels are shown as the mean of triplicates with SEM relative to controls. ( E ) Whole cell extracts from transfected cells were analyzed by immunoblotting for SMAD2, SMAD3, SMAD2/3 or GAPDH antibodies (loading control) to confirm target knockdown. Data are representative of two or more independent experiments. Points represent mean of triple samples with SE.
    The High Capacity cDNA Reverse Transcription Kit contains all components necessary for the quantitative conversion of up to 2 µg of total RNA to single-stranded cDNA in a single 20 µL reaction. The High Capacity cDNA Reverse Transcription Kit delivers extremely high-quality, single-stranded cDNA from 0.02 to 2 µg total RNA. Reactions can be scaled up to 100 µL to generate 10 µg of cDNA from a single reaction. Downstream applications include real-time PCR, standard PCR, and microarrays. The kit is ideal for generating cDNA archives.Features of this kit include:• Linear target amplification for real-time PCR• Higher yields and precision than other cDNA synthesis kits at a fraction of the cost• 10-fold greater dynamic range than other kitsExtensively tested with a variety of templates Quantitative first-strand synthesis of all RNA species is achieved with the use of the random primers. The kit has been extensively tested with a variety of RNA templates, including GC- and AU-rich targets and RNAs expressed at low levels. cRNA can also be efficiently generated from in vitro transcription of cDNA.
    https://www.bioz.com/result/high capacity cdna reverse transcription kit/product/Thermo Fisher
    Average 99 stars, based on 2431 article reviews
    Price from $9.99 to $1999.99
    high capacity cdna reverse transcription kit - by Bioz Stars, 2019-12
    99/100 stars

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    Images

    1) Product Images from "LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells"

    Article Title: LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21953

    Reactivation of LINE-1 by BaP is Effected via Canonical TGF-β1 Signaling Total RNA was isolated from BEAS-2B cells treated with 0.5 uM BaP for 8 hours, and 1 µg of RNA subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for ( A ) human LINE-1 (ORF1 and ORF2) or ( B ) TGF-β1. ( C ) cells pre-treated with TGF-β1 receptor inhibitor (LY2157299) or vehicle (DMSO) for 30 min before BaP challenge or ( D ) transfected with target-specific siRNAs to SMAD2, SMAD3, or scramble siRNA or no siRNA (mock) controls. Expression levels are shown as the mean of triplicates with SEM relative to controls. ( E ) Whole cell extracts from transfected cells were analyzed by immunoblotting for SMAD2, SMAD3, SMAD2/3 or GAPDH antibodies (loading control) to confirm target knockdown. Data are representative of two or more independent experiments. Points represent mean of triple samples with SE.
    Figure Legend Snippet: Reactivation of LINE-1 by BaP is Effected via Canonical TGF-β1 Signaling Total RNA was isolated from BEAS-2B cells treated with 0.5 uM BaP for 8 hours, and 1 µg of RNA subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for ( A ) human LINE-1 (ORF1 and ORF2) or ( B ) TGF-β1. ( C ) cells pre-treated with TGF-β1 receptor inhibitor (LY2157299) or vehicle (DMSO) for 30 min before BaP challenge or ( D ) transfected with target-specific siRNAs to SMAD2, SMAD3, or scramble siRNA or no siRNA (mock) controls. Expression levels are shown as the mean of triplicates with SEM relative to controls. ( E ) Whole cell extracts from transfected cells were analyzed by immunoblotting for SMAD2, SMAD3, SMAD2/3 or GAPDH antibodies (loading control) to confirm target knockdown. Data are representative of two or more independent experiments. Points represent mean of triple samples with SE.

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

    Specificity of TGF-β1/LINE-1 Interactions in Transformed Lung Epithelial Cell Lines NCI-H460, NCI-H520, or NCI-H1993 cell lines were challenged with BaP (0.5–2 uM) or 0.5% DMSO vehicle for 24 hours. Total RNA was isolated and 1 µg subjected to cDNA synthesis. Samples were analyzed by RT-PCR using specific primers for human TGF-β1 ( A ), LINE-1 (L1) ORF 1 ( B ) or GAPDH. Expression levels are presented relative to untreated cells. Each point represents the mean and SE of triplicate samples. The data are representative at two or more independent experiments. ( C ) Cells transfected with target-specific siRNAs to SNAIL or scramble siRNA or no siRNA (mock) were challenged with 3 ng/ml TGF-β1. Whole cell extracts were analyzed by immunoblotting for ORF1p, SNAIL, phospho(p)-SMAD2, or total SMAD2. Data are representative of two or more independent experiments.
    Figure Legend Snippet: Specificity of TGF-β1/LINE-1 Interactions in Transformed Lung Epithelial Cell Lines NCI-H460, NCI-H520, or NCI-H1993 cell lines were challenged with BaP (0.5–2 uM) or 0.5% DMSO vehicle for 24 hours. Total RNA was isolated and 1 µg subjected to cDNA synthesis. Samples were analyzed by RT-PCR using specific primers for human TGF-β1 ( A ), LINE-1 (L1) ORF 1 ( B ) or GAPDH. Expression levels are presented relative to untreated cells. Each point represents the mean and SE of triplicate samples. The data are representative at two or more independent experiments. ( C ) Cells transfected with target-specific siRNAs to SNAIL or scramble siRNA or no siRNA (mock) were challenged with 3 ng/ml TGF-β1. Whole cell extracts were analyzed by immunoblotting for ORF1p, SNAIL, phospho(p)-SMAD2, or total SMAD2. Data are representative of two or more independent experiments.

    Techniques Used: Transformation Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection

    Activation of EMT Programming by TGF-β1 is Associated with LINE-1 Expression in Human Bronchial Epithelial Cells ( A ) BEAS-2B whole cell lysates isolated from cells stimulated with 3 ng/mL TGF-β1 for 48 hours or control were analyzed by immunoblotting using antibodies against LINE1 (L1) ORF1 protein (ORF1p), E-cadherin, vimentin or GAPDH. ( B ) Total RNA from untreated or treated with 3 ng/ml TGF-β1 for 8 hours, and 1 µg of RNA was subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for human L1 (ORF1 and ORF2). Expression levels are shown as the mean of triplicates with SEM relative to controls. ( C ) Whole cell lysates from cells stimulated with different concentrations of TGF-β1 for 48 hours or control were analyzed for expression of L1 ORF1p by immunoblotting. Data are representative of two or more independent experiments.
    Figure Legend Snippet: Activation of EMT Programming by TGF-β1 is Associated with LINE-1 Expression in Human Bronchial Epithelial Cells ( A ) BEAS-2B whole cell lysates isolated from cells stimulated with 3 ng/mL TGF-β1 for 48 hours or control were analyzed by immunoblotting using antibodies against LINE1 (L1) ORF1 protein (ORF1p), E-cadherin, vimentin or GAPDH. ( B ) Total RNA from untreated or treated with 3 ng/ml TGF-β1 for 8 hours, and 1 µg of RNA was subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for human L1 (ORF1 and ORF2). Expression levels are shown as the mean of triplicates with SEM relative to controls. ( C ) Whole cell lysates from cells stimulated with different concentrations of TGF-β1 for 48 hours or control were analyzed for expression of L1 ORF1p by immunoblotting. Data are representative of two or more independent experiments.

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

    2) Product Images from "Anoctamin 1 (Ano1) is required for glucose-induced membrane potential oscillations and insulin secretion by murine β-cells"

    Article Title: Anoctamin 1 (Ano1) is required for glucose-induced membrane potential oscillations and insulin secretion by murine β-cells

    Journal: Pflugers Archiv

    doi: 10.1007/s00424-015-1758-5

    Detection of Ano1 in pancreas and pancreatic islets. a RT-PCR of cDNA prepared from mRNA extracted from rat and human tissues. Transcripts of the expected size for Ano1 are observed (rat: 223 bp; human 314 bp). The 300-bp band is shown in the molecular weight marker column (MWM). Positive control: kidney. Negative control (Neg. control): no DNA. The sequencing of PCR products confirmed 100 % identity with the reference sequence for rat Ano1 cDNA complementary of rat Ano1 mRNA. b Western blot of Ano1 in rat islets, from left to right : molecular weight column (MW) showing the 100-, 150-, and 250-kDa bands, 80 μg rat islet lysate, 30 μg rat islet lysate, and 30 μg human thyroid lysate (positive control). Ano1 is detected at 119 kDa. c Immunofluorescence staining of pancreas section . c1 Immunohistochemical labeling (green-fluorescent Tyramide Alexa 488) of Ano1 in a section photomicrograph of rat pancreas. Most of the islet cells and acinar cells (at the level of apical pole) are labeled. c2 Counterstaining labeling by hematoxylin–eosin performed on the slice used for c1. c3 Specificity control: immunohistochemical labeling of Ano1 in a section photomicrograph of rat pancreas. The primary goat Ano1 antibodies (sc-69343) were coincubated in the presence of Ano1 synthetic peptide (ab97423) in a ratio 1:8. The labeling disappears. c4 Counterstaining labeling by hematoxylin–eosin performed on the slice used for c3. Arrows show islets. Scale bar is 50 μm
    Figure Legend Snippet: Detection of Ano1 in pancreas and pancreatic islets. a RT-PCR of cDNA prepared from mRNA extracted from rat and human tissues. Transcripts of the expected size for Ano1 are observed (rat: 223 bp; human 314 bp). The 300-bp band is shown in the molecular weight marker column (MWM). Positive control: kidney. Negative control (Neg. control): no DNA. The sequencing of PCR products confirmed 100 % identity with the reference sequence for rat Ano1 cDNA complementary of rat Ano1 mRNA. b Western blot of Ano1 in rat islets, from left to right : molecular weight column (MW) showing the 100-, 150-, and 250-kDa bands, 80 μg rat islet lysate, 30 μg rat islet lysate, and 30 μg human thyroid lysate (positive control). Ano1 is detected at 119 kDa. c Immunofluorescence staining of pancreas section . c1 Immunohistochemical labeling (green-fluorescent Tyramide Alexa 488) of Ano1 in a section photomicrograph of rat pancreas. Most of the islet cells and acinar cells (at the level of apical pole) are labeled. c2 Counterstaining labeling by hematoxylin–eosin performed on the slice used for c1. c3 Specificity control: immunohistochemical labeling of Ano1 in a section photomicrograph of rat pancreas. The primary goat Ano1 antibodies (sc-69343) were coincubated in the presence of Ano1 synthetic peptide (ab97423) in a ratio 1:8. The labeling disappears. c4 Counterstaining labeling by hematoxylin–eosin performed on the slice used for c3. Arrows show islets. Scale bar is 50 μm

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker, Positive Control, Negative Control, Sequencing, Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Immunohistochemistry, Labeling

    3) Product Images from "Epstein-Barr Virus Latent Membrane Protein-1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells"

    Article Title: Epstein-Barr Virus Latent Membrane Protein-1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36312-4

    LMP1 CTAR1 and CTAR2 contributed to LMP1-mediated increase in sumo levels via the activation of NF-κB. ( a , b ) 293 cells were transfected with 1 μg of a LMP1-expression plasmid, select LMP1 mutant-expression plasmid, or a pcDNA3 control plasmid. ( a ) RNA was harvested and cDNA was prepared. Real-time PCR was performed to quantitate relative sumo-1 and sumo-2/3 levels (relative to gapdh ), The fold change in sumo- 1 and sumo-2/3 levels (compared to control-expressing cells) was determined. Results are shown as the mean ± the standard deviation for samples performed in triplicate and independent experiments performed in triplicate. ( b ) Slot blots were performed to detect SUMO-1 and SUMO-2/3 levels. Actin was used as a loading control. Representative blots for experiments performed in triplicate are shown. ( c , d ) EBV-transformed LCLs were treated with Bay 11–7082 (1 μM), LY294002 (5 μM), or DMSO (the vehicle control; Control) for 24 hours. ( c ) RNA was harvested and cDNA was prepared. Real-time PCR was performed to quantitate relative sumo-1 , and sumo-2/3 expression (relative to gapdh ), and the fold change in sumo-1 and sumo-2/3 levels (relative to control-treated cells) was determined. Results are shown as the mean ± the standard deviation for samples performed in triplicate and independent experiments performed in triplicate. ( d ) Slot immunoblots and Western blots analyses were performed to detect SUMO-1 and SUMO-2/3 levels. GAPDH was used as a loading control. Representative blots for experiments performed in triplicate are shown. ( e ) cDNA generated from RNA harvested from BL41 EBV-negative, BL41 EBV WT, and BL41 EBV mut (P3HR1) cells that were treated with DMSO (the vehicle control; control) or Bay 11–7085 (1 μM) for 24 hours. Real-time PCR was performed was performed to quantitate relative sumo-1 , and sumo-2/3 levels (relative to gapdh ). The fold change in sumo-1 and sumo-2/3 levels (relative to control-treated BL41 EBV WT cells) was determined. Results are shown as the mean ± the standard deviation for samples performed in triplicate and independent experiments performed in triplicate.
    Figure Legend Snippet: LMP1 CTAR1 and CTAR2 contributed to LMP1-mediated increase in sumo levels via the activation of NF-κB. ( a , b ) 293 cells were transfected with 1 μg of a LMP1-expression plasmid, select LMP1 mutant-expression plasmid, or a pcDNA3 control plasmid. ( a ) RNA was harvested and cDNA was prepared. Real-time PCR was performed to quantitate relative sumo-1 and sumo-2/3 levels (relative to gapdh ), The fold change in sumo- 1 and sumo-2/3 levels (compared to control-expressing cells) was determined. Results are shown as the mean ± the standard deviation for samples performed in triplicate and independent experiments performed in triplicate. ( b ) Slot blots were performed to detect SUMO-1 and SUMO-2/3 levels. Actin was used as a loading control. Representative blots for experiments performed in triplicate are shown. ( c , d ) EBV-transformed LCLs were treated with Bay 11–7082 (1 μM), LY294002 (5 μM), or DMSO (the vehicle control; Control) for 24 hours. ( c ) RNA was harvested and cDNA was prepared. Real-time PCR was performed to quantitate relative sumo-1 , and sumo-2/3 expression (relative to gapdh ), and the fold change in sumo-1 and sumo-2/3 levels (relative to control-treated cells) was determined. Results are shown as the mean ± the standard deviation for samples performed in triplicate and independent experiments performed in triplicate. ( d ) Slot immunoblots and Western blots analyses were performed to detect SUMO-1 and SUMO-2/3 levels. GAPDH was used as a loading control. Representative blots for experiments performed in triplicate are shown. ( e ) cDNA generated from RNA harvested from BL41 EBV-negative, BL41 EBV WT, and BL41 EBV mut (P3HR1) cells that were treated with DMSO (the vehicle control; control) or Bay 11–7085 (1 μM) for 24 hours. Real-time PCR was performed was performed to quantitate relative sumo-1 , and sumo-2/3 levels (relative to gapdh ). The fold change in sumo-1 and sumo-2/3 levels (relative to control-treated BL41 EBV WT cells) was determined. Results are shown as the mean ± the standard deviation for samples performed in triplicate and independent experiments performed in triplicate.

    Techniques Used: Activation Assay, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Real-time Polymerase Chain Reaction, Standard Deviation, Transformation Assay, Western Blot, Generated

    Increased sumo-1/2/3 levels were detected in LMP1-positive lymphoma tissues in an LMP1-dependent manner. ( a , b ) RNA was extracted from 42 biopsy tissues and cDNA was made. Real-time PCR was performed to quantitate gapdh , LMP1, and sumo-1 levels. RNA yields allowed sumo-2/3 levels to be tested in 17 of the 42 samples. ( a ) Relative LMP1, sumo-1 and sumo-2/3 levels (relative to gapdh ) were determined. The detection of LMP1 RNA levels determined is tissues were LMP1-negative (LMP1-neg) and LMP1-positive (LMP1-pos). Results are shown as the mean relative sumo-1 and sumo-2/3 levels for individual samples (shapes). Horizontal lines represent man sumo-1 and sumo-2/2 levels for the collective LMP1-neg and LMP1-pos samples. ( b ) Of the 19 LMP1-positive tissue samples, relative sumo- 1 levels were determined in all 19 samples but relative sumo-2/ 3 levels were determined in only 9 samples. Results are shown as the mean relative LMP1, sumo- 1, and sumo-2/ 3 leves of samples run in duplicate. Regression analysis was performed.
    Figure Legend Snippet: Increased sumo-1/2/3 levels were detected in LMP1-positive lymphoma tissues in an LMP1-dependent manner. ( a , b ) RNA was extracted from 42 biopsy tissues and cDNA was made. Real-time PCR was performed to quantitate gapdh , LMP1, and sumo-1 levels. RNA yields allowed sumo-2/3 levels to be tested in 17 of the 42 samples. ( a ) Relative LMP1, sumo-1 and sumo-2/3 levels (relative to gapdh ) were determined. The detection of LMP1 RNA levels determined is tissues were LMP1-negative (LMP1-neg) and LMP1-positive (LMP1-pos). Results are shown as the mean relative sumo-1 and sumo-2/3 levels for individual samples (shapes). Horizontal lines represent man sumo-1 and sumo-2/2 levels for the collective LMP1-neg and LMP1-pos samples. ( b ) Of the 19 LMP1-positive tissue samples, relative sumo- 1 levels were determined in all 19 samples but relative sumo-2/ 3 levels were determined in only 9 samples. Results are shown as the mean relative LMP1, sumo- 1, and sumo-2/ 3 leves of samples run in duplicate. Regression analysis was performed.

    Techniques Used: Real-time Polymerase Chain Reaction

    LMP1 was necessary and sufficient to increase sumo-1/2/3 and SUMO-1/2/3 levels in vivo . ( a ) Real-time PCR was performed using cDNA generated from RNA harvested from BL41 EBV-negative, BL41 EBV WT, and BL41 EBV mut (P3HR1) cells. The fold change in relative sumo- 1 and sumo- 2/3 expression (relative to gapdh ) was determined. Results are shown as the mean ± the standard deviation samples run in triplicate and independent experiments performed in triplicate. ( b – c ) 293 cells were serum-starved for 24 hours and then transfected with graduated amounts of an LMP1-expression vector or a control vector in serum-free media. ( b ) RNA was harvested, cDNA prepared, and real-time PCR performed. Relative LMP1, sumo-1 , and sumo-2/3 levels were determined (relative to gapdh ). Regression analysis of LMP1 and sumo-1 or sumo-2/3 levels was performed. Samples were run in triplicate and independent experiments performed in triplicate. ( c ) Slot blots were performed to detect SUMO-1, SUMO-2/3, and LMP1 levels. GAPDH was used as a loading control. Representative blots for experiments performed in triplicate are shown.
    Figure Legend Snippet: LMP1 was necessary and sufficient to increase sumo-1/2/3 and SUMO-1/2/3 levels in vivo . ( a ) Real-time PCR was performed using cDNA generated from RNA harvested from BL41 EBV-negative, BL41 EBV WT, and BL41 EBV mut (P3HR1) cells. The fold change in relative sumo- 1 and sumo- 2/3 expression (relative to gapdh ) was determined. Results are shown as the mean ± the standard deviation samples run in triplicate and independent experiments performed in triplicate. ( b – c ) 293 cells were serum-starved for 24 hours and then transfected with graduated amounts of an LMP1-expression vector or a control vector in serum-free media. ( b ) RNA was harvested, cDNA prepared, and real-time PCR performed. Relative LMP1, sumo-1 , and sumo-2/3 levels were determined (relative to gapdh ). Regression analysis of LMP1 and sumo-1 or sumo-2/3 levels was performed. Samples were run in triplicate and independent experiments performed in triplicate. ( c ) Slot blots were performed to detect SUMO-1, SUMO-2/3, and LMP1 levels. GAPDH was used as a loading control. Representative blots for experiments performed in triplicate are shown.

    Techniques Used: In Vivo, Real-time Polymerase Chain Reaction, Generated, Expressing, Standard Deviation, Transfection, Plasmid Preparation

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056737

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

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

    Expression analysis of CaZF in response to various stresses in chickpea. (A) Expression patterns of CaZF gene induced under different treatments. Total RNA was isolated from 10-d-old chickpea seedling treated with distilled water (control, Con), 100 µM abscisic acid (ABA), 4°C (Cold), dehydration (DH), 50 µM methyl-jasmonate (MeJA), 5 mM salicylic acid (SA), 250 mM NaCl (Salt) and wound for 5 h. (B) Time-course of accumulation of CaZF mRNA under dehydration, cold and salt treatments for 0.5 h, 1 h and 3 h. (C) Organ-specific expression of CaZF in root (R), shoot (S) and leaf (L) after cold, salt and dehydration treatments for 3 h. 20 µg of total RNA was electrophoresed on formaldehyde denaturing gel, blotted onto nylon membrane and hybridized with 32 P-radiolabeled CaZF cDNA. The lower panel in each figure shows ethidium bromide-stained ribosomal RNAs as loading controls. The relative intensities of the bands were quantitated by densitometry in PhosphorImager scanner and are presented in the form of fold changes below each blot.
    Figure Legend Snippet: Expression analysis of CaZF in response to various stresses in chickpea. (A) Expression patterns of CaZF gene induced under different treatments. Total RNA was isolated from 10-d-old chickpea seedling treated with distilled water (control, Con), 100 µM abscisic acid (ABA), 4°C (Cold), dehydration (DH), 50 µM methyl-jasmonate (MeJA), 5 mM salicylic acid (SA), 250 mM NaCl (Salt) and wound for 5 h. (B) Time-course of accumulation of CaZF mRNA under dehydration, cold and salt treatments for 0.5 h, 1 h and 3 h. (C) Organ-specific expression of CaZF in root (R), shoot (S) and leaf (L) after cold, salt and dehydration treatments for 3 h. 20 µg of total RNA was electrophoresed on formaldehyde denaturing gel, blotted onto nylon membrane and hybridized with 32 P-radiolabeled CaZF cDNA. The lower panel in each figure shows ethidium bromide-stained ribosomal RNAs as loading controls. The relative intensities of the bands were quantitated by densitometry in PhosphorImager scanner and are presented in the form of fold changes below each blot.

    Techniques Used: Expressing, Isolation, Staining

    5) Product Images from "Modeling Acute ER stress in vivo and in vitro"

    Article Title: Modeling Acute ER stress in vivo and in vitro

    Journal:

    doi: 10.1097/SHK.0000000000000759

    Tunicamycin is a more robust inducer of ER stress in Adipocytes A) Oil Red O staining confirming full differentiation of 3T3-L1 cells to mature adipocytes before treatments were initiated. B-E) 3T3-L1 cells were exposed to either TG (25nM, 50nM, 100nM) or TUN (2.5ug, 5ug, 10ug) for 24 Hrs, and total RNA was extracted and transcribed to cDNA. Relative mRNA levels of the indicated ER stress/UPR genes were quantified using real-time PCR and normalized to 18S rRNA. F) Immunostaining of cleaved-ATF6 (red), ER (green), and nuclei (blue) upon stimulation with vehicle or TUN (5ug) for 24hrs. Thapsigargin (TG); tunicamycin (Tun). Data shown are in mean ± SEM, *p < 0.05 vs vehicle, # p < 0.05 vs TG. (n=6).
    Figure Legend Snippet: Tunicamycin is a more robust inducer of ER stress in Adipocytes A) Oil Red O staining confirming full differentiation of 3T3-L1 cells to mature adipocytes before treatments were initiated. B-E) 3T3-L1 cells were exposed to either TG (25nM, 50nM, 100nM) or TUN (2.5ug, 5ug, 10ug) for 24 Hrs, and total RNA was extracted and transcribed to cDNA. Relative mRNA levels of the indicated ER stress/UPR genes were quantified using real-time PCR and normalized to 18S rRNA. F) Immunostaining of cleaved-ATF6 (red), ER (green), and nuclei (blue) upon stimulation with vehicle or TUN (5ug) for 24hrs. Thapsigargin (TG); tunicamycin (Tun). Data shown are in mean ± SEM, *p < 0.05 vs vehicle, # p < 0.05 vs TG. (n=6).

    Techniques Used: Staining, Real-time Polymerase Chain Reaction, Immunostaining

    Tunicamycin and thapsigargin are equally induce ER stress in Hepatocytes A-D) HepG2 cells were exposed to either TG (25nM, 50nM, 100nM) or TUN (2.5ug, 5ug, 10ug) for 24 Hrs, and total RNA was extracted and transcribed to cDNA. Relative mRNA levels of the indicated ER stress/UPR genes were quantified using real-time PCR and normalized to 18S rRNA. E) Immunostaining of cleaved-ATF6 (red), ER (green), and nuclei (blue) upon stimulation with vehicle or TG (100 mM) for 24hrs. F) Cell viability assay were performed after HepG2s were incubated with various doses of TG or TUN based on quantitation of the ATP present. Thapsigargin TG); tunicamycin (Tun). Data shown are in mean ± SEM, *p < 0.05 vs vehicle, # p < 0.05 vs TUN (5ug) (n=6).
    Figure Legend Snippet: Tunicamycin and thapsigargin are equally induce ER stress in Hepatocytes A-D) HepG2 cells were exposed to either TG (25nM, 50nM, 100nM) or TUN (2.5ug, 5ug, 10ug) for 24 Hrs, and total RNA was extracted and transcribed to cDNA. Relative mRNA levels of the indicated ER stress/UPR genes were quantified using real-time PCR and normalized to 18S rRNA. E) Immunostaining of cleaved-ATF6 (red), ER (green), and nuclei (blue) upon stimulation with vehicle or TG (100 mM) for 24hrs. F) Cell viability assay were performed after HepG2s were incubated with various doses of TG or TUN based on quantitation of the ATP present. Thapsigargin TG); tunicamycin (Tun). Data shown are in mean ± SEM, *p < 0.05 vs vehicle, # p < 0.05 vs TUN (5ug) (n=6).

    Techniques Used: Real-time Polymerase Chain Reaction, Immunostaining, Viability Assay, Incubation, Quantitation Assay

    6) Product Images from "Validation of Tuba1a as Appropriate Internal Control for Normalization of Gene Expression Analysis during Mouse Lung Development"

    Article Title: Validation of Tuba1a as Appropriate Internal Control for Normalization of Gene Expression Analysis during Mouse Lung Development

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms16034492

    Characterization of primer pairs designed for expression analysis of the internal control during mouse lung development. ( A ) Amplification efficiency for each primer pair was calculated using 10-fold serial dilutions of a cDNA template. Primer amplification efficiency was assessed by plotting the cycle threshold ( C T ) value for each concentration against the logarithm (base 10) of the fold dilution (log 10 (Quantity)). Efficiency was calculated using the slope of the linear function; ( B ) Dissociation curve analysis of primer specific products was performed by constantly monitoring the fluorescence with increasing temperatures from 60 to 95 °C. Melt curves were generated by plotting the negative first derivative of the fluorescence (−d/d T (Fluorescence) 520 nm) versus temperature (degree Celsius, °C); ( C ) Agarose gel electrophoresis after qRT-PCR indicates a specific product for each of the primer pairs. +RT, template is cDNA; −RT, template is RNA without reverse transcriptase, W, water control.
    Figure Legend Snippet: Characterization of primer pairs designed for expression analysis of the internal control during mouse lung development. ( A ) Amplification efficiency for each primer pair was calculated using 10-fold serial dilutions of a cDNA template. Primer amplification efficiency was assessed by plotting the cycle threshold ( C T ) value for each concentration against the logarithm (base 10) of the fold dilution (log 10 (Quantity)). Efficiency was calculated using the slope of the linear function; ( B ) Dissociation curve analysis of primer specific products was performed by constantly monitoring the fluorescence with increasing temperatures from 60 to 95 °C. Melt curves were generated by plotting the negative first derivative of the fluorescence (−d/d T (Fluorescence) 520 nm) versus temperature (degree Celsius, °C); ( C ) Agarose gel electrophoresis after qRT-PCR indicates a specific product for each of the primer pairs. +RT, template is cDNA; −RT, template is RNA without reverse transcriptase, W, water control.

    Techniques Used: Expressing, Amplification, Concentration Assay, Fluorescence, Generated, Agarose Gel Electrophoresis, Electrophoresis, Quantitative RT-PCR

    7) Product Images from "Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury. Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury"

    Article Title: Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury. Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury

    Journal: Physiological Reports

    doi: 10.14814/phy2.13753

    Compound 1 treatment of human lung fibroblasts cell cultures increases intracellular cAMP and decreases TGF ‐ β ‐mediated induction of fibrotic gene expression. WI ‐38 human lung fibroblasts were seeded on 24‐well plates and treated with increasing concentrations of Compound 1 (1 × 10 −10 mol/L to 1 × 10 −5 mol/L). After 30 min, cells were stimulated by 1 μ mol/L forskolin for 30 min and cell lysates were analyzed for cAMP concentration (A). To analyze fibroblast gene expression, WI ‐38 human lung fibroblasts were cultured in media containing 0.5% FBS for 24 h and treated with increasing concentrations of Compound 1 (1 × 10 −10 mol/L to 1 × 10 −5 mol/L) for 1 h followed by TGF ‐ β (3 ng/mL) and forskolin (1 μ mol/L) for 24 h. Total RNA was extracted from cell lysates, cDNA was amplified, and target gene mRNA was measured. The target gene expression levels of Col1a1 (Type‐1 collagen), Fn (Fibronectin), CTGF (connective tissue growth factor), and PAI ‐1 (plasminogen activator inhibitor‐1) was normalized to Glyceraldehyde 3‐phosphate dehydrogenase ( GAPDH ). Data are reported as mean ± SD with individual data points shown ( n = 3 per group). # P ≤ 0.05 versus forskolin group by two‐tailed Williams’ test.
    Figure Legend Snippet: Compound 1 treatment of human lung fibroblasts cell cultures increases intracellular cAMP and decreases TGF ‐ β ‐mediated induction of fibrotic gene expression. WI ‐38 human lung fibroblasts were seeded on 24‐well plates and treated with increasing concentrations of Compound 1 (1 × 10 −10 mol/L to 1 × 10 −5 mol/L). After 30 min, cells were stimulated by 1 μ mol/L forskolin for 30 min and cell lysates were analyzed for cAMP concentration (A). To analyze fibroblast gene expression, WI ‐38 human lung fibroblasts were cultured in media containing 0.5% FBS for 24 h and treated with increasing concentrations of Compound 1 (1 × 10 −10 mol/L to 1 × 10 −5 mol/L) for 1 h followed by TGF ‐ β (3 ng/mL) and forskolin (1 μ mol/L) for 24 h. Total RNA was extracted from cell lysates, cDNA was amplified, and target gene mRNA was measured. The target gene expression levels of Col1a1 (Type‐1 collagen), Fn (Fibronectin), CTGF (connective tissue growth factor), and PAI ‐1 (plasminogen activator inhibitor‐1) was normalized to Glyceraldehyde 3‐phosphate dehydrogenase ( GAPDH ). Data are reported as mean ± SD with individual data points shown ( n = 3 per group). # P ≤ 0.05 versus forskolin group by two‐tailed Williams’ test.

    Techniques Used: Expressing, Concentration Assay, Cell Culture, Amplification, Two Tailed Test

    Therapeutic administration of Compound 1 to targeted type II AEC ‐injured mice reduces the expression of TNF α within the lung. DTR ‐expressing mice ( DTR +) were administered daily I.P. PBS or DT from day 0 through Day 14. Subsets of the DTR +: DT ‐treated animals were treated by oral gavage once daily beginning on day 11 with vehicle or Compound 1 at 5.0 mg/kg. On day 21, the left lung was harvested and homogenized, and total RNA was extracted. First‐strand cDNA was synthesized and mRNA levels for Col1a1, Fibronectin, CTGF TNF α and PAI ‐1 (plasminogen activator inhibitor‐1) were assessed using SYBR Green‐based detection. The expression levels were normalized to GAPDH using the following formula: % GAPDH expression = 100/2‐ΔΔ CT . Data are presented as an average ± SEM ( n = 9 per DTR +: DT vehicle‐ and drug‐treated groups).
    Figure Legend Snippet: Therapeutic administration of Compound 1 to targeted type II AEC ‐injured mice reduces the expression of TNF α within the lung. DTR ‐expressing mice ( DTR +) were administered daily I.P. PBS or DT from day 0 through Day 14. Subsets of the DTR +: DT ‐treated animals were treated by oral gavage once daily beginning on day 11 with vehicle or Compound 1 at 5.0 mg/kg. On day 21, the left lung was harvested and homogenized, and total RNA was extracted. First‐strand cDNA was synthesized and mRNA levels for Col1a1, Fibronectin, CTGF TNF α and PAI ‐1 (plasminogen activator inhibitor‐1) were assessed using SYBR Green‐based detection. The expression levels were normalized to GAPDH using the following formula: % GAPDH expression = 100/2‐ΔΔ CT . Data are presented as an average ± SEM ( n = 9 per DTR +: DT vehicle‐ and drug‐treated groups).

    Techniques Used: Mouse Assay, Expressing, Synthesized, SYBR Green Assay

    8) Product Images from "Gene Expression Analysis of Parthenogenetic Embryonic Development of the Pea Aphid, Acyrthosiphon pisum, Suggests That Aphid Parthenogenesis Evolved from Meiotic Oogenesis"

    Article Title: Gene Expression Analysis of Parthenogenetic Embryonic Development of the Pea Aphid, Acyrthosiphon pisum, Suggests That Aphid Parthenogenesis Evolved from Meiotic Oogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115099

    Spo11 is spliced less in asexual aphids than in sexual aphids. A. Pea aphid Spo11 gene structure is shown. Boxes refer to exons and lines refer to introns, with bp lengths indicated inside or below, respectively. The third intron includes a 1720 bp SINE2 retrotransposon, indicated by the arrowhead. PCR primer sets used in reactions shown in Panels B , C and D are indicated by a numbered dashed line and pair of arrows. The scale bar indicates 100 bp. B. Unspliced Spo11 is detected in asexual aphids. Shown are PCRs from: genomic DNA (g); cDNA pools synthesized from RNA from sexual (“Sex”) LSR1.G1.AC ovaries, asexual (“Asex”) LSR1.G1.AC ovaries (+) and Tucson (“TUC”) pea aphid ovaries; and the RT-absent control template (-). Upper panel shows PCRs products from the second and third exons of Spo11 . The bottom panel shows PCR products of the last four ( left) or last six ( right ) exons of Spo11 . DNA size standards are indicated on the marker (M) lanes of these gels. C. Spo11 intron RNA is more abundant in asexually reproducing aphids than in sexual aphids. Shown are PCRs from: genomic DNA (g); cDNA pools synthesized from RNA from Tucson ovaries, asexual or sexual LSR1.G1.AC ovaries (+); or the RT-absent control template (-). Upper panel shows PCRs products from the fourth intron of Spo11 . The bottom panel shows PCR products of full-length Hop2 using the same template amounts. D. Spo11 is expressed in asexual embryos in utero . Top panel shows Spo11 PCR products using primer set 2, and the bottom panel shows Pms1 PCR product. Pools of cDNA (+) or control template from cDNA synthesis reactions lacking RT (-) were created from the tissue sources listed above the lanes.
    Figure Legend Snippet: Spo11 is spliced less in asexual aphids than in sexual aphids. A. Pea aphid Spo11 gene structure is shown. Boxes refer to exons and lines refer to introns, with bp lengths indicated inside or below, respectively. The third intron includes a 1720 bp SINE2 retrotransposon, indicated by the arrowhead. PCR primer sets used in reactions shown in Panels B , C and D are indicated by a numbered dashed line and pair of arrows. The scale bar indicates 100 bp. B. Unspliced Spo11 is detected in asexual aphids. Shown are PCRs from: genomic DNA (g); cDNA pools synthesized from RNA from sexual (“Sex”) LSR1.G1.AC ovaries, asexual (“Asex”) LSR1.G1.AC ovaries (+) and Tucson (“TUC”) pea aphid ovaries; and the RT-absent control template (-). Upper panel shows PCRs products from the second and third exons of Spo11 . The bottom panel shows PCR products of the last four ( left) or last six ( right ) exons of Spo11 . DNA size standards are indicated on the marker (M) lanes of these gels. C. Spo11 intron RNA is more abundant in asexually reproducing aphids than in sexual aphids. Shown are PCRs from: genomic DNA (g); cDNA pools synthesized from RNA from Tucson ovaries, asexual or sexual LSR1.G1.AC ovaries (+); or the RT-absent control template (-). Upper panel shows PCRs products from the fourth intron of Spo11 . The bottom panel shows PCR products of full-length Hop2 using the same template amounts. D. Spo11 is expressed in asexual embryos in utero . Top panel shows Spo11 PCR products using primer set 2, and the bottom panel shows Pms1 PCR product. Pools of cDNA (+) or control template from cDNA synthesis reactions lacking RT (-) were created from the tissue sources listed above the lanes.

    Techniques Used: Polymerase Chain Reaction, Synthesized, Marker, In Utero

    Argonaute -family genes are differentially expressed between aphid morphs. Shown are PCRs of aphid Argonaute -family genes Piwi3 , Piwi8 and Ago3a , along with GAPDH as a loading control. Products were amplified from cDNA pools synthesized with (+) or without (-) RT from RNA from asexual or sexual LSR1.G1.AC ovaries.
    Figure Legend Snippet: Argonaute -family genes are differentially expressed between aphid morphs. Shown are PCRs of aphid Argonaute -family genes Piwi3 , Piwi8 and Ago3a , along with GAPDH as a loading control. Products were amplified from cDNA pools synthesized with (+) or without (-) RT from RNA from asexual or sexual LSR1.G1.AC ovaries.

    Techniques Used: Amplification, Synthesized

    9) Product Images from "A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype"

    Article Title: A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype

    Journal: Cancer Growth and Metastasis

    doi: 10.4137/CGM.S18581

    Overexpression of recombinant cavin-3 specifically decreases cell migration and abrogates PMA-induced MMP-9 expression. ( A ) Human HT-1080 fibrosarcoma cells were transiently transfected with 2 μg of cDNA plasmids and fluorescent confocal microscopy was used to visualize the encoded recombinant GFP-tagged cavin-1, cavin-2 or cavin-3 proteins 24–48 hours post transfection. pEGFP cDNA plasmid was used as control. ( B ) Cells were then harvested and cell migration assayed as described in the Methods section. ( C ) HT-1080 cells were transiently transfected with plasmids as described above. Twenty-four hours post transfection, cells were treated with vehicle (white bars) or 100 nM PMA (Black bars) in a serum-free medium for 18 hours. Total RNA was extracted and qRT-PCR was performed to measure the levels of MMP-9 transcript. Values of PCR are normalized over the expression of housekeeping genes GAPDH and PPI γ and are the mean ± S.E.M of triplicate values from one representative experiment.
    Figure Legend Snippet: Overexpression of recombinant cavin-3 specifically decreases cell migration and abrogates PMA-induced MMP-9 expression. ( A ) Human HT-1080 fibrosarcoma cells were transiently transfected with 2 μg of cDNA plasmids and fluorescent confocal microscopy was used to visualize the encoded recombinant GFP-tagged cavin-1, cavin-2 or cavin-3 proteins 24–48 hours post transfection. pEGFP cDNA plasmid was used as control. ( B ) Cells were then harvested and cell migration assayed as described in the Methods section. ( C ) HT-1080 cells were transiently transfected with plasmids as described above. Twenty-four hours post transfection, cells were treated with vehicle (white bars) or 100 nM PMA (Black bars) in a serum-free medium for 18 hours. Total RNA was extracted and qRT-PCR was performed to measure the levels of MMP-9 transcript. Values of PCR are normalized over the expression of housekeeping genes GAPDH and PPI γ and are the mean ± S.E.M of triplicate values from one representative experiment.

    Techniques Used: Over Expression, Recombinant, Migration, Expressing, Transfection, Confocal Microscopy, Plasmid Preparation, Quantitative RT-PCR, Polymerase Chain Reaction

    10) Product Images from "Tetracycline Derivative Minocycline Inhibits Autophagy and Inflammation in Concanavalin-A-Activated Human Hepatoma Cells"

    Article Title: Tetracycline Derivative Minocycline Inhibits Autophagy and Inflammation in Concanavalin-A-Activated Human Hepatoma Cells

    Journal: Gene Regulation and Systems Biology

    doi: 10.4137/GRSB.S13946

    MT1-MMP is required for ConA-induced BNIP3 expression and formation of acidic vacuoles. Serum-starved HepG2 hepatoma cells were transiently transfected with a control siRNA (siScrambled) or an siRNA directed against MT1-MMP (siMT1-MMP); then the cells were treated with 30 μg/mL ConA for 24 hours in the presence or absence of 10 μM minocycline. ( A ) Cell lysates were isolated and electrophoresed, and immunodetection performed as described in the Methods section. ( B ) Total RNA was isolated from cells transfected with either a scrambled siRNA sequence (siScr) or a specific siRNA against MT1-MMP (siMT1-MMP), and followed by 30 μg/mL ConA treatment. cDNA was then synthesized and qRT-PCR performed as described in the Methods section. A representative qPCR profile, out of three independent experiments, is shown for the corresponding genes. Data represent mean values in triplicates. Statistical significance was assessed using Student’s unpaired t -test. Probability values of less than 0.05 were considered significant, and an asterisk (*) denotes such significance. ( C ) Cells were stained with Acridine Orange as described in the Methods section, and acidic vacuole formation was examined by fluorescence microscopy in control, ConA-, and ConA/minocycline-treated cells.
    Figure Legend Snippet: MT1-MMP is required for ConA-induced BNIP3 expression and formation of acidic vacuoles. Serum-starved HepG2 hepatoma cells were transiently transfected with a control siRNA (siScrambled) or an siRNA directed against MT1-MMP (siMT1-MMP); then the cells were treated with 30 μg/mL ConA for 24 hours in the presence or absence of 10 μM minocycline. ( A ) Cell lysates were isolated and electrophoresed, and immunodetection performed as described in the Methods section. ( B ) Total RNA was isolated from cells transfected with either a scrambled siRNA sequence (siScr) or a specific siRNA against MT1-MMP (siMT1-MMP), and followed by 30 μg/mL ConA treatment. cDNA was then synthesized and qRT-PCR performed as described in the Methods section. A representative qPCR profile, out of three independent experiments, is shown for the corresponding genes. Data represent mean values in triplicates. Statistical significance was assessed using Student’s unpaired t -test. Probability values of less than 0.05 were considered significant, and an asterisk (*) denotes such significance. ( C ) Cells were stained with Acridine Orange as described in the Methods section, and acidic vacuole formation was examined by fluorescence microscopy in control, ConA-, and ConA/minocycline-treated cells.

    Techniques Used: Expressing, Transfection, Isolation, Immunodetection, Sequencing, Synthesized, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Staining, Fluorescence, Microscopy

    STAT3 and NANOS1 gene silencing abrogate ConA-induced MT1-MMP and BNIP3 expression. HepG2 hepatoma cells were transiently transfected with a control siRNA (siScrambled) or an siRNA directed against STAT3 (siSTAT3) or NANOS1 (siNANOS1); then serum-starved cells were treated with 30 μg/mL ConA for 24 hours. ( A ) Cell lysates were isolated and electrophoresed, and immunodetection performed as described in the Methods sections. ( B ) MT1-MMP gene expression was assessed in cells that were treated with 10 μM minocycline or 30 μg/mL ConA. Total RNA was isolated, cDNA synthesized, and qRT-PCR performed as described in the Methods section. ( C ) The inter-relationship between NANOS1 and STAT3 gene expression was assessed in cells that were either silenced for NANOS1 (siNANOS1, black bars) or silenced for STAT3 (siSTAT3, gray bars). Total RNA was isolated, cDNA synthesized, and qRT-PCR performed as described in the Methods section. A representative qPCR profile, out of three independent experiments, is shown for the corresponding genes. Data represent mean values in triplicates. Statistical significance was assessed using Student’s unpaired t -test. Probability values of less than 0.05 were considered significant, and an asterisk (*) denotes such significance.
    Figure Legend Snippet: STAT3 and NANOS1 gene silencing abrogate ConA-induced MT1-MMP and BNIP3 expression. HepG2 hepatoma cells were transiently transfected with a control siRNA (siScrambled) or an siRNA directed against STAT3 (siSTAT3) or NANOS1 (siNANOS1); then serum-starved cells were treated with 30 μg/mL ConA for 24 hours. ( A ) Cell lysates were isolated and electrophoresed, and immunodetection performed as described in the Methods sections. ( B ) MT1-MMP gene expression was assessed in cells that were treated with 10 μM minocycline or 30 μg/mL ConA. Total RNA was isolated, cDNA synthesized, and qRT-PCR performed as described in the Methods section. ( C ) The inter-relationship between NANOS1 and STAT3 gene expression was assessed in cells that were either silenced for NANOS1 (siNANOS1, black bars) or silenced for STAT3 (siSTAT3, gray bars). Total RNA was isolated, cDNA synthesized, and qRT-PCR performed as described in the Methods section. A representative qPCR profile, out of three independent experiments, is shown for the corresponding genes. Data represent mean values in triplicates. Statistical significance was assessed using Student’s unpaired t -test. Probability values of less than 0.05 were considered significant, and an asterisk (*) denotes such significance.

    Techniques Used: Expressing, Transfection, Isolation, Immunodetection, Synthesized, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Minocycline inhibits ConA-induced NANOS1 expression and phosphorylation of STAT3. ( A ) Serum-starved HepG2 hepatoma cells were treated with increasing ConA concentrations. NANOS1 and MT1-MMP gene expression was expressed as the percentage of maximal ConA-induced effect for each gene. ( B ) ConA-treated HepG2 hepatoma cells were incubated with up to 10 μM minocycline for 24 hours. Total RNA was isolated, cDNA synthesized, and qPCR performed as described in the Methods section. The levels of STAT3 phosphorylation were assessed in cells that were ( C ) treated with 30 μg/mL ConA in the presence or absence of 10 μM minocycline or ( D ) transiently transfected with either a control siRNA (siScrambled) or an siRNA directed against NANOS1 (siNANOS1). A representative qPCR profile, from two independent experiments, is shown for the corresponding genes. Data represent mean values from triplicates.
    Figure Legend Snippet: Minocycline inhibits ConA-induced NANOS1 expression and phosphorylation of STAT3. ( A ) Serum-starved HepG2 hepatoma cells were treated with increasing ConA concentrations. NANOS1 and MT1-MMP gene expression was expressed as the percentage of maximal ConA-induced effect for each gene. ( B ) ConA-treated HepG2 hepatoma cells were incubated with up to 10 μM minocycline for 24 hours. Total RNA was isolated, cDNA synthesized, and qPCR performed as described in the Methods section. The levels of STAT3 phosphorylation were assessed in cells that were ( C ) treated with 30 μg/mL ConA in the presence or absence of 10 μM minocycline or ( D ) transiently transfected with either a control siRNA (siScrambled) or an siRNA directed against NANOS1 (siNANOS1). A representative qPCR profile, from two independent experiments, is shown for the corresponding genes. Data represent mean values from triplicates.

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

    11) Product Images from "Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime"

    Article Title: Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1259

    Correction of endogenous gDNA with ValidPrime. Comparison of results obtained with two assays targeting Serpine1 (left) or Chi3l3 (right) in cDNA prepared from mouse peritoneal macrophages and measured in the BioMark qPCR system. The ‘gDNA-sensitive’ assays amplify both gDNA and cDNA, while the ‘gDNA-insensitive’ assays only recognize the transcript. ( A ) Scatter plots showing the correlation between the %DNA [as defined in Equation (6) ] and Cq NA data obtained with the gDNA-sensitive assays in each of 81 independent RNA preparations (means of duplicates). The positive correlation between %DNA and Cq illustrates the increasing impact of the gDNA contamination with decreasing total signal. Mean and median values refer to %DNA levels. ( B ) Cq NA data measured with the gDNA-insensitive assays plotted against the corresponding Cq NA data (dark blue) or ValidPrime-estimated Cq RNA (light blue and orange), obtained with the gDNA-sensitive assays. Samples with a DNA contribution of 60–90 % are shown in orange and those with
    Figure Legend Snippet: Correction of endogenous gDNA with ValidPrime. Comparison of results obtained with two assays targeting Serpine1 (left) or Chi3l3 (right) in cDNA prepared from mouse peritoneal macrophages and measured in the BioMark qPCR system. The ‘gDNA-sensitive’ assays amplify both gDNA and cDNA, while the ‘gDNA-insensitive’ assays only recognize the transcript. ( A ) Scatter plots showing the correlation between the %DNA [as defined in Equation (6) ] and Cq NA data obtained with the gDNA-sensitive assays in each of 81 independent RNA preparations (means of duplicates). The positive correlation between %DNA and Cq illustrates the increasing impact of the gDNA contamination with decreasing total signal. Mean and median values refer to %DNA levels. ( B ) Cq NA data measured with the gDNA-insensitive assays plotted against the corresponding Cq NA data (dark blue) or ValidPrime-estimated Cq RNA (light blue and orange), obtained with the gDNA-sensitive assays. Samples with a DNA contribution of 60–90 % are shown in orange and those with

    Techniques Used: Real-time Polymerase Chain Reaction

    ValidPrime flowchart. ValidPrime GOI assay validation. ValidPrime can be used as a reliable, cost-efficient alternative to RT(−) controls to survey gDNA background in RT–qPCR, and as a tool to determine the RNA-derived signal ( Cq RNA ) in RT(+)–qPCR reactions. To optimize its accuracy when Cq RNA calculation is desired, validation of GOI assays in gDNA samples is recommended, as outlined in ( A ). Asterisks indicates the efficiency evaluation and melting curve/electrophoresis-based analysis. This includes an evaluation of the gDNA sensitivity of GOI assays using dilution series with gDNA samples spanning at least three log 10 in copy number. GOI assays that do not amplify gDNA are attributed the grade A+. The amplification of gDNA by high-confidence assays should be specific and with an efficiency similar to that of the VPA (see ‘Discussion’ section and Supplementary Figure S3 ). For GOI assays with suboptimal, but confidently determined ( 17 , 21 ) efficiency, Equation (7) could be applied to adjust Cq NA data. To optimize specificity, there should also be consistency between the melting curves of PCR products in gDNA and cDNA samples. ( B ) Cq RNA calculation with ValidPrime-validated GOI assays. High confidence and A+ assays can be used with less gDNA samples for Cq RNA determination. It is recommended to confirm the absence of gDNA amplification at least once for A+ assays. Samples that do not contain sufficient gDNA to generate a signal with the VPA are attributed A*. As for gDNA insensitive A+ assays, Cq RNA equals Cq NA (i.e. output = input) in A* samples, since the DNA-derived signal is negligible [see Equations ( 2 and 4 )]. For gDNA-sensitive GOI assays, Cq RNA is calculated by a Cq DNA -based correction of Cq NA using Equations ( 4 and 5 ). To minimize the risk of jeopardizing the accuracy of the Cq RNA estimation, it is not advisable to perform correction on samples where the DNA-derived signal exceeds 60%. The calculations are facilitated using the ValidPrime software. Details on additional assay/sample grading and data output formats employed by the software are provided in Supplementary Figure S7 . The Cq RNA output data can be used for downstream data processing, such as normalization against reference genes.
    Figure Legend Snippet: ValidPrime flowchart. ValidPrime GOI assay validation. ValidPrime can be used as a reliable, cost-efficient alternative to RT(−) controls to survey gDNA background in RT–qPCR, and as a tool to determine the RNA-derived signal ( Cq RNA ) in RT(+)–qPCR reactions. To optimize its accuracy when Cq RNA calculation is desired, validation of GOI assays in gDNA samples is recommended, as outlined in ( A ). Asterisks indicates the efficiency evaluation and melting curve/electrophoresis-based analysis. This includes an evaluation of the gDNA sensitivity of GOI assays using dilution series with gDNA samples spanning at least three log 10 in copy number. GOI assays that do not amplify gDNA are attributed the grade A+. The amplification of gDNA by high-confidence assays should be specific and with an efficiency similar to that of the VPA (see ‘Discussion’ section and Supplementary Figure S3 ). For GOI assays with suboptimal, but confidently determined ( 17 , 21 ) efficiency, Equation (7) could be applied to adjust Cq NA data. To optimize specificity, there should also be consistency between the melting curves of PCR products in gDNA and cDNA samples. ( B ) Cq RNA calculation with ValidPrime-validated GOI assays. High confidence and A+ assays can be used with less gDNA samples for Cq RNA determination. It is recommended to confirm the absence of gDNA amplification at least once for A+ assays. Samples that do not contain sufficient gDNA to generate a signal with the VPA are attributed A*. As for gDNA insensitive A+ assays, Cq RNA equals Cq NA (i.e. output = input) in A* samples, since the DNA-derived signal is negligible [see Equations ( 2 and 4 )]. For gDNA-sensitive GOI assays, Cq RNA is calculated by a Cq DNA -based correction of Cq NA using Equations ( 4 and 5 ). To minimize the risk of jeopardizing the accuracy of the Cq RNA estimation, it is not advisable to perform correction on samples where the DNA-derived signal exceeds 60%. The calculations are facilitated using the ValidPrime software. Details on additional assay/sample grading and data output formats employed by the software are provided in Supplementary Figure S7 . The Cq RNA output data can be used for downstream data processing, such as normalization against reference genes.

    Techniques Used: Quantitative RT-PCR, Derivative Assay, Electrophoresis, Amplification, Polymerase Chain Reaction, Software

    12) Product Images from "Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure"

    Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

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

    doi: 10.1161/JAHA.114.001104

    Thapsigargin (TG)‐induced ER stress causes and time‐dependent corin mRNA decay in neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes were plated in 6‐well plates. Twenty‐four hours later, cells were cultured in serum‐free DMEM‐M199 (4:1) with and without 0.1 μmol/L TG for 6, 16, 24, and 30 hours, separately. Total RNA was extracted and reverse‐transcribed into cDNA and gene expression of the following genes was assessed by RT‐PCR (A through F): (A) GRP78; (B) XBP1 splicing; (C) CHOP; (D) corin; (E) GRP94; and (F) ATF4. Compared with non‐TG‐treated controls, cells treated with TG demonstrated increased ER stress as evidenced by significant increases in GRP78, GRP94, ATF4, and CHOP mRNA, with a corresponding significant increase in XBP1 splicing based on the increase in the spliced form (intron removed) XBP1s mRNA; * P
    Figure Legend Snippet: Thapsigargin (TG)‐induced ER stress causes and time‐dependent corin mRNA decay in neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes were plated in 6‐well plates. Twenty‐four hours later, cells were cultured in serum‐free DMEM‐M199 (4:1) with and without 0.1 μmol/L TG for 6, 16, 24, and 30 hours, separately. Total RNA was extracted and reverse‐transcribed into cDNA and gene expression of the following genes was assessed by RT‐PCR (A through F): (A) GRP78; (B) XBP1 splicing; (C) CHOP; (D) corin; (E) GRP94; and (F) ATF4. Compared with non‐TG‐treated controls, cells treated with TG demonstrated increased ER stress as evidenced by significant increases in GRP78, GRP94, ATF4, and CHOP mRNA, with a corresponding significant increase in XBP1 splicing based on the increase in the spliced form (intron removed) XBP1s mRNA; * P

    Techniques Used: Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction

    Thapsigargin (TG)‐induced ER stress and time‐dependent corin mRNA decay in adult mouse atrial cardiomyocytes (HL‐1) cells. HL‐1 cells were plated in 6‐well plates. Twenty‐four hours later, cells were cultured in serum‐free Claycomb medium with and without thapsigargin (TG) 0.1 μmol/L for 6, 16, 24, 30 hours, separately. Total RNA was extracted and reverse‐transcribed into cDNA and mRNA abundance of the following genes was assessed by RT‐PCR ( Figure 4 A through 4 F): (A) GRP78; (B) XBP1 splicing; (C) CHOP; (D) corin; (E) GRP94; (F) ATF4. Compared with non‐TG‐treated HL‐1 control cells, HL‐1 cells treated with TG demonstrated increased ER stress as evidenced by significant increases in GRP78, GRP94, ATF4 and CHOP mRNA, with a corresponding significant increase in XBP1 splicing based on the increase in the spliced form (intron removed) XBP1s mRNA; * P
    Figure Legend Snippet: Thapsigargin (TG)‐induced ER stress and time‐dependent corin mRNA decay in adult mouse atrial cardiomyocytes (HL‐1) cells. HL‐1 cells were plated in 6‐well plates. Twenty‐four hours later, cells were cultured in serum‐free Claycomb medium with and without thapsigargin (TG) 0.1 μmol/L for 6, 16, 24, 30 hours, separately. Total RNA was extracted and reverse‐transcribed into cDNA and mRNA abundance of the following genes was assessed by RT‐PCR ( Figure 4 A through 4 F): (A) GRP78; (B) XBP1 splicing; (C) CHOP; (D) corin; (E) GRP94; (F) ATF4. Compared with non‐TG‐treated HL‐1 control cells, HL‐1 cells treated with TG demonstrated increased ER stress as evidenced by significant increases in GRP78, GRP94, ATF4 and CHOP mRNA, with a corresponding significant increase in XBP1 splicing based on the increase in the spliced form (intron removed) XBP1s mRNA; * P

    Techniques Used: Cell Culture, Reverse Transcription Polymerase Chain Reaction

    13) Product Images from "Global Transcriptional Response of Macrophage-Like THP-1 Cells to Shiga Toxin Type 1"

    Article Title: Global Transcriptional Response of Macrophage-Like THP-1 Cells to Shiga Toxin Type 1

    Journal:

    doi: 10.1128/IAI.01341-09

    COX-2 mRNA expression by macrophage-like THP-1 cells. Cells were stimulated with Stx1 (400 ng/ml) (A), LPS (200 ng/ml) (B), or Stx1+LPS (400 and 200 ng/ml, respectively) (C) for different times. Total RNA was isolated, DNase treated, and cDNA
    Figure Legend Snippet: COX-2 mRNA expression by macrophage-like THP-1 cells. Cells were stimulated with Stx1 (400 ng/ml) (A), LPS (200 ng/ml) (B), or Stx1+LPS (400 and 200 ng/ml, respectively) (C) for different times. Total RNA was isolated, DNase treated, and cDNA

    Techniques Used: Expressing, Isolation

    Egr-1 mRNA expression by macrophage-like THP-1 cells. Cells were stimulated with Stx1 (400 ng/ml) (A), LPS (200 ng/ml) (B), or Stx1+LPS (400 and 200 ng/ml, respectively) (C) for different times. Total RNA was isolated, DNase treated, and cDNA
    Figure Legend Snippet: Egr-1 mRNA expression by macrophage-like THP-1 cells. Cells were stimulated with Stx1 (400 ng/ml) (A), LPS (200 ng/ml) (B), or Stx1+LPS (400 and 200 ng/ml, respectively) (C) for different times. Total RNA was isolated, DNase treated, and cDNA

    Techniques Used: Expressing, Isolation

    14) Product Images from "Tissue-specific and time-dependent regulation of the endothelin axis by the circadian clock protein Per1"

    Article Title: Tissue-specific and time-dependent regulation of the endothelin axis by the circadian clock protein Per1

    Journal: Life sciences

    doi: 10.1016/j.lfs.2014.03.028

    Time-dependent regulation of ET A and ET B mRNA expressions in the renal inner medulla and cortex. Wild type (light bars) or Per1 het (dark bars) mice were euthanized at noon or midnight. Total RNA was isolated from dissected inner medulla and cortex and converted to cDNA. Real time quantitative RT-PCR (qPCR)was performed to evaluate mRNA expressions of ET A (Panel A) and ET B (Panel B). Data are presented relative to the WT at noon, ±SEM. †P
    Figure Legend Snippet: Time-dependent regulation of ET A and ET B mRNA expressions in the renal inner medulla and cortex. Wild type (light bars) or Per1 het (dark bars) mice were euthanized at noon or midnight. Total RNA was isolated from dissected inner medulla and cortex and converted to cDNA. Real time quantitative RT-PCR (qPCR)was performed to evaluate mRNA expressions of ET A (Panel A) and ET B (Panel B). Data are presented relative to the WT at noon, ±SEM. †P

    Techniques Used: Mouse Assay, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Regulation of ET-1, ET A and ET B mRNA expression in the heart by time and Per1. Wild type or Per1 hetmice were euthanized at noon or midnight. Total RNA was isolated from the heart and converted to cDNA. Real time quantitative RT-PCR (qPCR)was performed to evaluate in ET-1 (Panel A), ET A (Panel B), and ET B (Panel C). Data are presented relative to the WT at noon, ±SEM. *P
    Figure Legend Snippet: Regulation of ET-1, ET A and ET B mRNA expression in the heart by time and Per1. Wild type or Per1 hetmice were euthanized at noon or midnight. Total RNA was isolated from the heart and converted to cDNA. Real time quantitative RT-PCR (qPCR)was performed to evaluate in ET-1 (Panel A), ET A (Panel B), and ET B (Panel C). Data are presented relative to the WT at noon, ±SEM. *P

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

    Regulation of ET-1, ET A , and ET B mRNA expressions in the liver by time and Per1. Wild type or Per1 hetmice were euthanized at noon or midnight. Total RNA was isolated from the liver and converted to cDNA. Real time quantitative RT-PCR (QPCR) was performed to evaluate expression of ET-1 (Panel A), ET A (Panel B), and ET B (Panel C). Data are presented relative to the WT at noon, ±SEM. *P
    Figure Legend Snippet: Regulation of ET-1, ET A , and ET B mRNA expressions in the liver by time and Per1. Wild type or Per1 hetmice were euthanized at noon or midnight. Total RNA was isolated from the liver and converted to cDNA. Real time quantitative RT-PCR (QPCR) was performed to evaluate expression of ET-1 (Panel A), ET A (Panel B), and ET B (Panel C). Data are presented relative to the WT at noon, ±SEM. *P

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

    Regulation of the endothelin axis in the lung by time and Per1. Wild type or Per1 het mice were euthanized at noon or midnight. Total RNA was isolated from the lung and converted to cDNA. Real time quantitative RT-PCR (qPCR) was performed to evaluate expressions of ET-1 (Panel A), ET A (Panel B), and ET B (Panel C). Data are presented relative to the WT at noon, ±SEM. *P
    Figure Legend Snippet: Regulation of the endothelin axis in the lung by time and Per1. Wild type or Per1 het mice were euthanized at noon or midnight. Total RNA was isolated from the lung and converted to cDNA. Real time quantitative RT-PCR (qPCR) was performed to evaluate expressions of ET-1 (Panel A), ET A (Panel B), and ET B (Panel C). Data are presented relative to the WT at noon, ±SEM. *P

    Techniques Used: Mouse Assay, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    15) Product Images from "Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin"

    Article Title: Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082346

    Changes in the percentage of EpCAM-positive cells upon control medium (DMEM containing 10% FBS) with 4 mM BCAA stimulation or 100 nM rapamycin pretreatment and 4 mM BCAA stimulation (A) or liver cirrhosis modified medium (LC) containing 10% FBS stimulation (B) for 72 h in Huh7 by using the target activation protocol of array scan. The rate change of EpCAM-positive cells in 5000 cells with overexpression of caRheb or control plasmid cDNA (pc DNA) in control medium (DMEM containing 10% FBS) with and without 4 mM BCAA stimulation for 24 h in Huh7 (C). The detection of P70S6 kinase phosphorylation, a member of downstream mTOR signaling, in the presence of DMEM, BCAA treatment, pretreatment with rapamycin and BCAA treatment, or LC stimulation for 72 h in Huh7 (A,B). Tukey’s test **p
    Figure Legend Snippet: Changes in the percentage of EpCAM-positive cells upon control medium (DMEM containing 10% FBS) with 4 mM BCAA stimulation or 100 nM rapamycin pretreatment and 4 mM BCAA stimulation (A) or liver cirrhosis modified medium (LC) containing 10% FBS stimulation (B) for 72 h in Huh7 by using the target activation protocol of array scan. The rate change of EpCAM-positive cells in 5000 cells with overexpression of caRheb or control plasmid cDNA (pc DNA) in control medium (DMEM containing 10% FBS) with and without 4 mM BCAA stimulation for 24 h in Huh7 (C). The detection of P70S6 kinase phosphorylation, a member of downstream mTOR signaling, in the presence of DMEM, BCAA treatment, pretreatment with rapamycin and BCAA treatment, or LC stimulation for 72 h in Huh7 (A,B). Tukey’s test **p

    Techniques Used: Modification, Liquid Chromatography, Activation Assay, Over Expression, Plasmid Preparation

    Protein expression and phosphorylation in Huh7 cells under Knockdown conditions for 5 days and overexpression conditions for 1 day, and detection of phosphorylation after 4 mM BCAA treatment for 30 min. Rictor or Raptor Knockdown compared to negative control (NC), caRheb compared to control plasmid cDNA (pc DNA), BCAA treatment compared to DMEM (FBS 10%) only (Ctrl) (A). The average tumor volumes and tumorigenesis ratio at the 4 th week in a xenograft model with transplanted cells with negative control, knockdown of Raptor, Rictor for 5 days, or overexpression of control plasmid DNA, caRheb for 1 day (C), and tumorigenesis rate (B). Dunnett's test, *p
    Figure Legend Snippet: Protein expression and phosphorylation in Huh7 cells under Knockdown conditions for 5 days and overexpression conditions for 1 day, and detection of phosphorylation after 4 mM BCAA treatment for 30 min. Rictor or Raptor Knockdown compared to negative control (NC), caRheb compared to control plasmid cDNA (pc DNA), BCAA treatment compared to DMEM (FBS 10%) only (Ctrl) (A). The average tumor volumes and tumorigenesis ratio at the 4 th week in a xenograft model with transplanted cells with negative control, knockdown of Raptor, Rictor for 5 days, or overexpression of control plasmid DNA, caRheb for 1 day (C), and tumorigenesis rate (B). Dunnett's test, *p

    Techniques Used: Expressing, Over Expression, Negative Control, Plasmid Preparation

    16) Product Images from "Na+/K+ ATPase activity promotes invasion of endocrine resistant breast cancer cells"

    Article Title: Na+/K+ ATPase activity promotes invasion of endocrine resistant breast cancer cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0193779

    NKP expression and activity in normal and breast cancer cell lines. Panel A: NKP mRNA expression in ER+ (YS 1.2) and ER-ve (pII and MDA-MB-231) breast cancer cells. RNA was extracted from cells, converted to cDNA and PCR amplified. Ct values were converted to ratios relative to actin levels as described in Methods. Panel B: NKP protein expression level in the membranous compartment of the tested cell lines was determined by western blotting. Membranous fraction lysate protein (3 μg) was electrophoresed on 10% SDS polyacrylamide gel, blotted onto nitrocellulose membrane and probed with antisera to T-NKP and actin (loading control). This blot represents one of 3 independent determinations. NKP activity in the membranous fraction of all cell lines (panel C), and in pII cells in response to ouabain/TTX treatment (panel D) was determined by a colorimetric NKP activity kit. Histobars represent means ± SEM of at least 3 independent determinations. *denotes significant difference from MCF10A (panel C) or pII control (panel D) with p
    Figure Legend Snippet: NKP expression and activity in normal and breast cancer cell lines. Panel A: NKP mRNA expression in ER+ (YS 1.2) and ER-ve (pII and MDA-MB-231) breast cancer cells. RNA was extracted from cells, converted to cDNA and PCR amplified. Ct values were converted to ratios relative to actin levels as described in Methods. Panel B: NKP protein expression level in the membranous compartment of the tested cell lines was determined by western blotting. Membranous fraction lysate protein (3 μg) was electrophoresed on 10% SDS polyacrylamide gel, blotted onto nitrocellulose membrane and probed with antisera to T-NKP and actin (loading control). This blot represents one of 3 independent determinations. NKP activity in the membranous fraction of all cell lines (panel C), and in pII cells in response to ouabain/TTX treatment (panel D) was determined by a colorimetric NKP activity kit. Histobars represent means ± SEM of at least 3 independent determinations. *denotes significant difference from MCF10A (panel C) or pII control (panel D) with p

    Techniques Used: Expressing, Activity Assay, Multiple Displacement Amplification, Polymerase Chain Reaction, Amplification, Western Blot

    Effect of siRNA-mediated knockdown of NKP on pII cell motility. Panel A: NKP mRNA expression in pII cells transfected with a scrambled sequence (control, open bar) or NKP siRNA (48 h, solid bar). RNA was extracted from cells, converted to cDNA and amplified by PCR. Ct values were converted to ratios relative to actin. Panel B: mean distance moved (in pixels) as a measurement of cell motility for pII cell transfected with a scrambled sequence (control, open bar, taken as 100%) or NKP siRNA (48 h, solid bar) determined after 24 h of incubation. Histobars represent means ± SEM of at least 3 independent determinations. Asterisk denotes significant difference from control with p
    Figure Legend Snippet: Effect of siRNA-mediated knockdown of NKP on pII cell motility. Panel A: NKP mRNA expression in pII cells transfected with a scrambled sequence (control, open bar) or NKP siRNA (48 h, solid bar). RNA was extracted from cells, converted to cDNA and amplified by PCR. Ct values were converted to ratios relative to actin. Panel B: mean distance moved (in pixels) as a measurement of cell motility for pII cell transfected with a scrambled sequence (control, open bar, taken as 100%) or NKP siRNA (48 h, solid bar) determined after 24 h of incubation. Histobars represent means ± SEM of at least 3 independent determinations. Asterisk denotes significant difference from control with p

    Techniques Used: Expressing, Transfection, Sequencing, Amplification, Polymerase Chain Reaction, Incubation

    17) Product Images from "HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells, et al. HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells"

    Article Title: HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells, et al. HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells

    Journal: Cancer Science

    doi: 10.1111/cas.13660

    HNRNPLL promotes cell cycle progression in colon cancer cells. A‐D, Cell cycle analysis for SW 480 (A) and HT 29 (C) cells transduced with HNRNPLL cDNA , Luc sh RNA or HNRNPLL sh RNA 2 was performed using a flow cytometer. The sum of the percentages of S and G2/M phases in SW 480 (B) and HT 29 (D) cells are shown in the bar graph. Error bars, SD . E, Immunostaining of HT 29 cells using antibodies for HNRNPLL , GMNN and CDT 1. Note that cells showing no nuclear GMNN expression and high CDT 1 expression (*) exhibit higher HNRNPLL expression compared to cells showing nuclear GMNN expression and low CDT 1 expression ( † ). Scale bar, 10 μm
    Figure Legend Snippet: HNRNPLL promotes cell cycle progression in colon cancer cells. A‐D, Cell cycle analysis for SW 480 (A) and HT 29 (C) cells transduced with HNRNPLL cDNA , Luc sh RNA or HNRNPLL sh RNA 2 was performed using a flow cytometer. The sum of the percentages of S and G2/M phases in SW 480 (B) and HT 29 (D) cells are shown in the bar graph. Error bars, SD . E, Immunostaining of HT 29 cells using antibodies for HNRNPLL , GMNN and CDT 1. Note that cells showing no nuclear GMNN expression and high CDT 1 expression (*) exhibit higher HNRNPLL expression compared to cells showing nuclear GMNN expression and low CDT 1 expression ( † ). Scale bar, 10 μm

    Techniques Used: Cell Cycle Assay, Transduction, Flow Cytometry, Cytometry, Immunostaining, Expressing

    Knockdown of PCNA , RFC 3 or FEN 1 suppresses the increased cell proliferation caused by HNRNPLL overexpression. A, Western blot analysis to confirm the overexpression of HNRNPLL and knockdown of PCNA , RFC 3 and FEN 1. Arrows and arrowheads indicate FLAG ‐tagged or endogenous HNRNPLL , respectively. B, MTT assay was performed for Mock‐ or HNRNPLL cDNA ‐transduced SW 480 cells 72 h after transfection of control si RNA or si RNA (s) targeting PCNA , RFC 3 , or FEN 1 . Error bars, SD . P = .036 (*), .20 ( † ), .32 ( †† ), .12 ( ††† ), .030 ( ‡ ), .038 ( ‡‡ ), .027 ( ‡‡‡ ), .50 ( § ), .78 ( §§ ) and .21 ( §§§ ). C, MTT assay was performed for Mock‐ or HNRNPLL cDNA ‐transduced HT 29 cells 72 h after transfection of control si RNA or si RNA (s) targeting PCNA , RFC 3 , or FEN 1 . Error bars, SD . P = .026 (*), .079 ( † ), .79 ( †† ), .12 ( ††† ), .036 ( ‡ ), .040 ( ‡‡ ), .033 ( ‡‡‡ ), .12 ( § ), .34 ( §§ ) and .18 ( §§§ ). D, Western blot analysis to confirm the overexpression of FLAG ‐ PCNA , FLAG ‐ RFC 3 and FLAG ‐ FEN 1 in SW 480 and HT 29 cells simultaneously transduced with their cDNA s. Arrows and arrowheads indicate FLAG ‐tagged or endogenous proteins, respectively. E, MTT assay was performed for SW 480 and HT 29 cells transduced with mock vector or PCNA , RFC 3 and FEN 1 cDNA . Error bars, SD . P = .76 (*) and .090 ( † )
    Figure Legend Snippet: Knockdown of PCNA , RFC 3 or FEN 1 suppresses the increased cell proliferation caused by HNRNPLL overexpression. A, Western blot analysis to confirm the overexpression of HNRNPLL and knockdown of PCNA , RFC 3 and FEN 1. Arrows and arrowheads indicate FLAG ‐tagged or endogenous HNRNPLL , respectively. B, MTT assay was performed for Mock‐ or HNRNPLL cDNA ‐transduced SW 480 cells 72 h after transfection of control si RNA or si RNA (s) targeting PCNA , RFC 3 , or FEN 1 . Error bars, SD . P = .036 (*), .20 ( † ), .32 ( †† ), .12 ( ††† ), .030 ( ‡ ), .038 ( ‡‡ ), .027 ( ‡‡‡ ), .50 ( § ), .78 ( §§ ) and .21 ( §§§ ). C, MTT assay was performed for Mock‐ or HNRNPLL cDNA ‐transduced HT 29 cells 72 h after transfection of control si RNA or si RNA (s) targeting PCNA , RFC 3 , or FEN 1 . Error bars, SD . P = .026 (*), .079 ( † ), .79 ( †† ), .12 ( ††† ), .036 ( ‡ ), .040 ( ‡‡ ), .033 ( ‡‡‡ ), .12 ( § ), .34 ( §§ ) and .18 ( §§§ ). D, Western blot analysis to confirm the overexpression of FLAG ‐ PCNA , FLAG ‐ RFC 3 and FLAG ‐ FEN 1 in SW 480 and HT 29 cells simultaneously transduced with their cDNA s. Arrows and arrowheads indicate FLAG ‐tagged or endogenous proteins, respectively. E, MTT assay was performed for SW 480 and HT 29 cells transduced with mock vector or PCNA , RFC 3 and FEN 1 cDNA . Error bars, SD . P = .76 (*) and .090 ( † )

    Techniques Used: Over Expression, Western Blot, MTT Assay, Transfection, Transduction, Plasmid Preparation

    18) Product Images from "GLUT3 is induced during epithelial-mesenchymal transition and promotes tumor cell proliferation in non-small cell lung cancer"

    Article Title: GLUT3 is induced during epithelial-mesenchymal transition and promotes tumor cell proliferation in non-small cell lung cancer

    Journal: Cancer & Metabolism

    doi: 10.1186/2049-3002-2-11

    GLUT3 is induced upon EMT. (A) H2122 cells were stimulated with 10 ng/ml TGF-β for the indicated time points, after which the cells were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 4) of mRNA expression ratios between TGF-β treated and non-treated conditions. (B) H727 or H2009 cell populations stably expressing a control plasmid (ctl), SNAIL, or ZEB1 were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 3) of mRNA expression relative to the cell line expressing the least amount of the same gene (set to 1). (C) H727 or H2009 stable cell populations were lysed with RIPA buffer to prepare protein extracts and to analyze the expression of the indicated proteins by Western blot.
    Figure Legend Snippet: GLUT3 is induced upon EMT. (A) H2122 cells were stimulated with 10 ng/ml TGF-β for the indicated time points, after which the cells were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 4) of mRNA expression ratios between TGF-β treated and non-treated conditions. (B) H727 or H2009 cell populations stably expressing a control plasmid (ctl), SNAIL, or ZEB1 were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 3) of mRNA expression relative to the cell line expressing the least amount of the same gene (set to 1). (C) H727 or H2009 stable cell populations were lysed with RIPA buffer to prepare protein extracts and to analyze the expression of the indicated proteins by Western blot.

    Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Expressing, Stable Transfection, Plasmid Preparation, CTL Assay, Western Blot

    GLUT3 is strongly expressed in mesenchymal liver tumor cells. (A) The indicated cell lines were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 3) of mRNA expression relative to the cell line expressing the least amount of the same gene (set to 1). N.D. , not detected. (B) Hep3B cells were stimulated with 10 ng/ml TGF-β for 72 h, after which the cells were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 3) of mRNA expression relative to the untreated conditions (set to 1).
    Figure Legend Snippet: GLUT3 is strongly expressed in mesenchymal liver tumor cells. (A) The indicated cell lines were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 3) of mRNA expression relative to the cell line expressing the least amount of the same gene (set to 1). N.D. , not detected. (B) Hep3B cells were stimulated with 10 ng/ml TGF-β for 72 h, after which the cells were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 3) of mRNA expression relative to the untreated conditions (set to 1).

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

    GLUT3 is strongly expressed in mesenchymal lung tumor cells. (A) The indicated cell lines were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 3) of mRNA or mature microRNA expression relative to the cell line expressing the least amount of the same gene (set to 1). (B) The indicated cells were stained to analyze the expression of GLUT3 or E-cadherin by immunocytochemistry. Scale bar, 100 μm. In (A) and (B) , the dashed line indicates the separation between the mesenchymal and the epithelial groups of cells.
    Figure Legend Snippet: GLUT3 is strongly expressed in mesenchymal lung tumor cells. (A) The indicated cell lines were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 3) of mRNA or mature microRNA expression relative to the cell line expressing the least amount of the same gene (set to 1). (B) The indicated cells were stained to analyze the expression of GLUT3 or E-cadherin by immunocytochemistry. Scale bar, 100 μm. In (A) and (B) , the dashed line indicates the separation between the mesenchymal and the epithelial groups of cells.

    Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Expressing, Staining, Immunocytochemistry

    19) Product Images from "A MT1-MMP/NF-?B signaling axis as a checkpoint controller of COX-2 expression in CD133(+) U87 glioblastoma cells"

    Article Title: A MT1-MMP/NF-?B signaling axis as a checkpoint controller of COX-2 expression in CD133(+) U87 glioblastoma cells

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-6-8

    Cell-based evidence that MT1-MMP directly regulates COX-2 expression in U87 glioma cell lines . Monolayers or neurospheres from glioblastoma-derived cells were either Mock-transfected, transfected with a cDNA plasmid encoding MT1-MMP (Wt), or transfected with an siRNA (Si) against MT1-MMP as described in the Methods section. (A) Gelatin zymography was performed to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells. Cell lysates were isolated from U87 glioblastoma-derived cells and SDS-PAGE performed (20 μg protein/well), followed by Western-blotting and COX-2 or GAPDH immunodetection. (B) Total RNA was isolated from monolayers (white bars) or neurospheres (black bars) of U87 Mock-transfected cells, or from U87 cells transfected with MT1-MMP cDNA or siRNA against MT1-MMP, and reverse-transcribed as described in the Methods section. Quantitative PCR was performed in order to monitor COX-2 gene expression levels.
    Figure Legend Snippet: Cell-based evidence that MT1-MMP directly regulates COX-2 expression in U87 glioma cell lines . Monolayers or neurospheres from glioblastoma-derived cells were either Mock-transfected, transfected with a cDNA plasmid encoding MT1-MMP (Wt), or transfected with an siRNA (Si) against MT1-MMP as described in the Methods section. (A) Gelatin zymography was performed to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells. Cell lysates were isolated from U87 glioblastoma-derived cells and SDS-PAGE performed (20 μg protein/well), followed by Western-blotting and COX-2 or GAPDH immunodetection. (B) Total RNA was isolated from monolayers (white bars) or neurospheres (black bars) of U87 Mock-transfected cells, or from U87 cells transfected with MT1-MMP cDNA or siRNA against MT1-MMP, and reverse-transcribed as described in the Methods section. Quantitative PCR was performed in order to monitor COX-2 gene expression levels.

    Techniques Used: Expressing, Derivative Assay, Transfection, Plasmid Preparation, Zymography, Isolation, SDS Page, Western Blot, Immunodetection, Real-time Polymerase Chain Reaction

    MT1-MMP-mediated regulation of COX-2 gene and protein expression is independent from MT1-MMP's catalytic functions . (A) Cell lysates were isolated from Mock-transfected, or U87 glioma cells that had been transiently transfected with a cDNA plasmid encoding MT1-MMP, which were subsequently treated (or not) with 10 μM Ilomastat (Ilo). SDS-PAGE was performed (20 μg protein/well), followed by Western-blotting and COX-2 or MT1-MMP immunodetection. Gelatin zymography was also used to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells. (B) Total RNA was extracted from the above described cell conditions and gene expression levels assessed by qRT-PCR for COX-2 in the absence (white bars) or the presence of Ilomastat (black bars).
    Figure Legend Snippet: MT1-MMP-mediated regulation of COX-2 gene and protein expression is independent from MT1-MMP's catalytic functions . (A) Cell lysates were isolated from Mock-transfected, or U87 glioma cells that had been transiently transfected with a cDNA plasmid encoding MT1-MMP, which were subsequently treated (or not) with 10 μM Ilomastat (Ilo). SDS-PAGE was performed (20 μg protein/well), followed by Western-blotting and COX-2 or MT1-MMP immunodetection. Gelatin zymography was also used to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells. (B) Total RNA was extracted from the above described cell conditions and gene expression levels assessed by qRT-PCR for COX-2 in the absence (white bars) or the presence of Ilomastat (black bars).

    Techniques Used: Expressing, Isolation, Transfection, Plasmid Preparation, SDS Page, Western Blot, Immunodetection, Zymography, Quantitative RT-PCR

    20) Product Images from "Endoplasmic reticulum stress in adipose tissue augments lipolysis"

    Article Title: Endoplasmic reticulum stress in adipose tissue augments lipolysis

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12384

    Male Balb/c mice were injected with either control buffer (Con) or tunicamycin (Tun). After 24 hrs, the epididymal fat pads were dissected and ( A ) total RNA was extracted and transcribed to cDNA. Relative mRNA levels of the indicated genes were quantified using real-time PCR and normalized to 18S rRNA. The value obtained for the control for each gene was assigned a value of 1. The value obtained following injection with tunicamycin was expressed relative to the control value. Results represent the mean ± SD of n = 3–8 experiments. ▪ Control, □ Tunicamycin. ( B ) Equal amounts of protein were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing the indicated proteins. For IRE1α, GRP78 and GAPDH intervening lanes have been removed. ( C – E ) The intensities of the bands in ( B ) were quantified from n = 3–6 experiments. The control values were set to 1. The values obtained from tunicamycin treated mice were expressed relative to the control. * Indicates P
    Figure Legend Snippet: Male Balb/c mice were injected with either control buffer (Con) or tunicamycin (Tun). After 24 hrs, the epididymal fat pads were dissected and ( A ) total RNA was extracted and transcribed to cDNA. Relative mRNA levels of the indicated genes were quantified using real-time PCR and normalized to 18S rRNA. The value obtained for the control for each gene was assigned a value of 1. The value obtained following injection with tunicamycin was expressed relative to the control value. Results represent the mean ± SD of n = 3–8 experiments. ▪ Control, □ Tunicamycin. ( B ) Equal amounts of protein were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing the indicated proteins. For IRE1α, GRP78 and GAPDH intervening lanes have been removed. ( C – E ) The intensities of the bands in ( B ) were quantified from n = 3–6 experiments. The control values were set to 1. The values obtained from tunicamycin treated mice were expressed relative to the control. * Indicates P

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

    Subcutaneous white adipose tissue was obtained during surgery from burned patients (burn) or non-burned patients undergoing elective surgery (non-burn). ( A ) Total RNA was extracted from adipose tissue obtained from non-burned ( n = 3–4) and burned patients ( n = 5–8) and transcribed to cDNA. Real-time quantitative PCR was performed to evaluate the relative mRNA levels of the indicated genes that were normalized to IDH1 mRNA. The value obtained from one non-burn sample was set to 1. The values obtained from all other samples were expressed relative to the non-burn sample. ▪ Non-burn, □ Burn. ( B , Top and Bottom Panels ) Adipose tissue was homogenized and equal amounts of total protein in the tissue homogenates were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing GRP78 and alpha/beta tubulin. ( C ) The intensities of the bands corresponding to GRP78 in ( B ) were quantified. ( D ) Human adipocytes (non-burn) were isolated and cultured either in the presence or absence of tunicamycin (5 μg/ml) at 37°C for the indicated times. The culture media was collected and the concentration of free glycerol determined. To account for the variability between patients in basal lipolysis rates, the amount of glycerol released from control cells from each patient and at each time-point was given a value of 100%. The amount of glycerol released from adipocytes in the presence of tunicamycin is expressed as a percentage of the value obtained from the control cells. Results represent the mean ± SD from n = 3 female patients ages 48 and 61 years, ▪ Control, □ Tunicamycin. * indicates P
    Figure Legend Snippet: Subcutaneous white adipose tissue was obtained during surgery from burned patients (burn) or non-burned patients undergoing elective surgery (non-burn). ( A ) Total RNA was extracted from adipose tissue obtained from non-burned ( n = 3–4) and burned patients ( n = 5–8) and transcribed to cDNA. Real-time quantitative PCR was performed to evaluate the relative mRNA levels of the indicated genes that were normalized to IDH1 mRNA. The value obtained from one non-burn sample was set to 1. The values obtained from all other samples were expressed relative to the non-burn sample. ▪ Non-burn, □ Burn. ( B , Top and Bottom Panels ) Adipose tissue was homogenized and equal amounts of total protein in the tissue homogenates were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing GRP78 and alpha/beta tubulin. ( C ) The intensities of the bands corresponding to GRP78 in ( B ) were quantified. ( D ) Human adipocytes (non-burn) were isolated and cultured either in the presence or absence of tunicamycin (5 μg/ml) at 37°C for the indicated times. The culture media was collected and the concentration of free glycerol determined. To account for the variability between patients in basal lipolysis rates, the amount of glycerol released from control cells from each patient and at each time-point was given a value of 100%. The amount of glycerol released from adipocytes in the presence of tunicamycin is expressed as a percentage of the value obtained from the control cells. Results represent the mean ± SD from n = 3 female patients ages 48 and 61 years, ▪ Control, □ Tunicamycin. * indicates P

    Techniques Used: Real-time Polymerase Chain Reaction, SDS Page, Isolation, Cell Culture, Concentration Assay

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    Positive Control:

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    Synthesized:

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    Pyrolysis Gas Chromatography:

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    Immunostaining:

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    Real-time Polymerase Chain Reaction:

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    Microarray:

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    Quantitation Assay:

    Article Title:
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    Expressing:

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    Article Title:
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    Cell Fractionation:

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    Western Blot:

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    Activated Clotting Time Assay:

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    Countercurrent Chromatography:

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    Transfection:

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    Dissection:

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    Northern Blot:

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    Generated:

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    Inhibition:

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    Polymerase Chain Reaction:

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    Quantitative RT-PCR:

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    Article Title: CALCIUM OXALATE STONE FRAGMENT AND CRYSTAL PHAGOCYTOSIS BY HUMAN MACROPHAGES
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    TaqMan Assay:

    Article Title: p130Cas/Cyclooxygenase-2 axis in the control of mesenchymal plasticity of breast cancer cells
    Article Snippet: 1 μg of DNAse-treated RNA (RQ1 RNase-Free DNase kit, Promega, Madison, WI, USA) was retrotranscribed with High Capacity cDNA Reverse Transcription Kit (Invitrogen Life Technologies, Carlsbad, CA, USA). .. Quantitative PCR was performed on an Applied Biosystems, 7900HT Fast Real-Time PCR System (standard settings) using the Universal Probe Library system (Roche Italia, Monza, Italy) and Platinum™ Quantitative PCR SuperMix-UDG (Invitrogen Life Technologies, Carlsbad, CA, USA).

    In Vivo:

    Article Title: Discovery and pharmacological characterization of a new class of prolyl-tRNA synthetase inhibitor for anti-fibrosis therapy
    Article Snippet: Total RNA was isolated from in vivo samples using ISOGEN II (Nippongene) according to the manufacturer's instructions. .. High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA) was used for cDNA synthesis.

    Fluorescence:

    Article Title: CALCIUM OXALATE STONE FRAGMENT AND CRYSTAL PHAGOCYTOSIS BY HUMAN MACROPHAGES
    Article Snippet: CCL2, CCL3, CCL4, CCL22, IL-1Rα, IL-6, IL-8, S100a9, and GAPDH levels were analyzed by qRT-PCR. mRNA qRT-PCR was performed using the TaqMan High-Capacity cDNA Reverse Transcription Kit, TaqMan Fast Advance PCR Master Mix, and TaqMan mRNA assay primers (Applied Biosystems, ?city state?). .. All reactions were analyzed using 7900HT Fast Real-Time PCR System (Applied Biosystems).

    Isolation:

    Article Title: Role of Chronic Inflammation in Myopia Progression: Clinical Evidence and Experimental Validation
    Article Snippet: 2.6 Total RNA of sclera tissues was isolated using an RNeasy Mini kit (Qiagen) and used for PCR array analysis. .. Total RNA (1 μg) in a 20-μl final volume was reverse-transcribed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Article Title: Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation
    Article Snippet: RNAs isolated from primary oligodendrocytes ( > 93% GalC+ ) of wild type B6 mice served as IL-17R positive control [ ], and of IL-17R-deficient mice (the Jackson Laboratory) as negative control. .. High Capacity cDNA Reverse Transcription Kit (Invitrogen), RNeasy Mini Kit and QuantiFast SYBR Green PCR Kit (QIAGEN) were used for real-time PCR according to the manufacturer’s instructions.

    Article Title: The Deacetylase Sirt6 Activates the Acetyltransferase GCN5 and Suppresses Hepatic Gluconeogenesis
    Article Snippet: Total RNA was isolated from cells or pulverized liver using Trizol (Invitrogen). .. For Q-RT-PCR analysis, cDNA was synthesized from 2 μg RNA using random primers and High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).

    Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits Interferon-Gamma Production in B Cells and Suppresses Colitis in Mice
    Article Snippet: Analysis of Abs in sera collected from 12- to 18-month-old mice was performed by ELISA using IL-4, IL-10, IL-17, and IFN-γ ELISA Kits (Alpha Diagnostic International) according to the manufacturer’s instructions. .. RNA was isolated using an RNeasy Plus Micro Kit (Qiagen) and reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was performed using the 7300 Real-Time PCR System (Applied Biosystems) and TaqMan Gene Expression Master Mix (Applied Biosystems). .. The qPCR TaqMan probes (Applied Biosystems) were as follows: IFN-γ, Mm01168134_m1; IL-4, Mm99999154_m1; IL-10, Mm01288386_m1; IL-17, Mm00439618; Actb, 4352341E.

    Article Title: Hydrogen Sulfide and Carbon Monoxide Protect Gastric Mucosa Compromised by Mild Stress Against Alendronate Injury
    Article Snippet: Expression of mRNA in gastric mucosa was determined by real-time PCR as described previously [ ]. .. Briefly, RNA was isolated from gastric mucosal biopsies stored at −80 °C using GeneMATRIX Universal RNA Purification Kit (EURx, Gdansk, Poland) and reversed transcription to cDNA was performed using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Life Technologies, MA, USA). .. Expression for HO-1, HO-2, HIF-1α, NF-κB, COX-2, iNOS, TNF-α, IL-1β, SOD-2, and GPx-1 was determined by real-time PCR using specific primers, SG qPCR Master Mix (2x) including SYBR-Green (EURx, Gdansk, Poland) and appropriate thermal cycler (7900HT Fast Real-Time PCR System, Thermo Fisher Scientific, Life Technologies, MA, USA).

    Article Title:
    Article Snippet: Phosphorylation of VEGFR-2 and VEGFR-3 on adult LECs (AdLECs, Lonza) treated with VEGF-C or VEGF-D variants at 200 ng/ml was analyzed as described previously ( ). .. AdLECs were serum-starved overnight and then exposed to VEGF-C or VEGF-D variants (at 100 ng/ml, unless stated otherwise) before isolation of total RNA using an RNeasy minikit (Qiagen, Valencia, CA) and preparation of cDNA with a high capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA) using 1 μg of total RNA. .. Quantitation of cDNA for COX-2 and the internal reference gene (β-actin) was carried out with TaqMan Fast Universal PCR Master Mix using an Applied Biosystems 7500 fast real-time PCR machine (both from Thermo Fisher Scientific).

    Article Title: p130Cas/Cyclooxygenase-2 axis in the control of mesenchymal plasticity of breast cancer cells
    Article Snippet: Paragraph title: RNA isolation and qRT-PCR for mRNA detection ... 1 μg of DNAse-treated RNA (RQ1 RNase-Free DNase kit, Promega, Madison, WI, USA) was retrotranscribed with High Capacity cDNA Reverse Transcription Kit (Invitrogen Life Technologies, Carlsbad, CA, USA).

    Article Title: Optimizing multi-dimensional high throughput screening using zebrafish
    Article Snippet: Briefly, total RNA was isolated from groups of 120 hpf larval zebrafish using RNAzol® RT (Molecular Research Center, Inc., Cincinnati, OH). .. One microgram of total RNA was converted to cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA).

    Article Title: Discovery and pharmacological characterization of a new class of prolyl-tRNA synthetase inhibitor for anti-fibrosis therapy
    Article Snippet: Total RNA was isolated from in vivo samples using ISOGEN II (Nippongene) according to the manufacturer's instructions. .. High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA) was used for cDNA synthesis.

    Negative Control:

    Article Title: Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation
    Article Snippet: RNAs isolated from primary oligodendrocytes ( > 93% GalC+ ) of wild type B6 mice served as IL-17R positive control [ ], and of IL-17R-deficient mice (the Jackson Laboratory) as negative control. .. High Capacity cDNA Reverse Transcription Kit (Invitrogen), RNeasy Mini Kit and QuantiFast SYBR Green PCR Kit (QIAGEN) were used for real-time PCR according to the manufacturer’s instructions.

    Article Title: Cell-autonomous regulation of fast troponin T pre-mRNA alternative splicing in response to mechanical stretch
    Article Snippet: The RNA and nontemplate (i.e., H2 O instead of RNA) samples were reverse transcribed using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). .. PCR was performed using HotStart GoTaq polymerase (Promega) under the following cycling conditions: 2 min at 95°C, followed by 4 cycles of 30 s at 94°C, 30 s at 65°C (−1.0°C/cycle), followed by 1 min and 15 s at 72°C.

    Article Title: miRNA in the Regulation of Skeletal Muscle Adaptation to Acute Endurance Exercise in C57Bl/6J Male Mice
    Article Snippet: First-strand cDNA synthesis from 1 µg of total RNA was performed with random primers using a high capacity cDNA reverse transcription kit (Applied Biosystems Inc., Foster City, CA) according to manufacturer's directions. .. Forward and reverse primers ( ) for the aforementioned genes were designed based on sequences available in GenBank ( http://www.ncbi.nlm.nih.gov/entrez/query.fcgi ) using the MIT Primer 3 designer software ( http://wi.mit.edu/cgi-bin/primer3/primer3_www.cgi ), and were confirmed for specificity using the basic local alignment search tool ( www.ncbi.nlm.nih.gov/BLAST/ ). β-2 microglobulin was used as a control house-keeping gene.

    Mouse Assay:

    Article Title: Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation
    Article Snippet: RNAs isolated from primary oligodendrocytes ( > 93% GalC+ ) of wild type B6 mice served as IL-17R positive control [ ], and of IL-17R-deficient mice (the Jackson Laboratory) as negative control. .. High Capacity cDNA Reverse Transcription Kit (Invitrogen), RNeasy Mini Kit and QuantiFast SYBR Green PCR Kit (QIAGEN) were used for real-time PCR according to the manufacturer’s instructions.

    Article Title: Systematic evaluation of medium-throughput mRNA abundance platforms
    Article Snippet: RNA was reverse-transcribed using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems) following the manufacturer's instructions. .. Assuming a 100% reaction efficiency, cDNA was diluted to a concentration of 5 ng/μL in RNase-free H2 O and stored at −20°C.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response
    Article Snippet: Inhibition of de novo RNA transcription by ActD was validated by quantitative PCR. .. 1 μg total RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer’s instructions. cDNAs were quantified on a Eppendorf Masterplex thermocycler using QuantiTect SYBR Green RT-PCR Kit (QIAGEN). .. Primer sequences are detailed in the .

    Blocking Assay:

    Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response
    Article Snippet: For detection of DIG-labeled probes, the membrane was briefly washed in washing buffer (0.1M Maleic Acid, 0.15M NaCl, pH 7,5 and 0.3% Tween, pH7.5) and blocked with 0.1M Maleic Acid, 0.15M NaCl, 1% Blocking Reagent (Roche) at room temperature. .. 1 μg total RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer’s instructions. cDNAs were quantified on a Eppendorf Masterplex thermocycler using QuantiTect SYBR Green RT-PCR Kit (QIAGEN).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Optimizing multi-dimensional high throughput screening using zebrafish
    Article Snippet: One microgram of total RNA was converted to cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). .. One microgram of total RNA was converted to cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA).

    Purification:

    Article Title: Expression of SV2 isoforms during rodent brain development
    Article Snippet: Paragraph title: RNA purification and quantification ... One μg of total RNA was used to synthesize cDNA with the Applied Biosystems high capacity cDNA reverse transcription kit in a total volume of 100 μl following the manufacturer’s protocol (Life Technologies Corporation®, Carlsbad, California).

    Article Title: Hydrogen Sulfide and Carbon Monoxide Protect Gastric Mucosa Compromised by Mild Stress Against Alendronate Injury
    Article Snippet: Expression of mRNA in gastric mucosa was determined by real-time PCR as described previously [ ]. .. Briefly, RNA was isolated from gastric mucosal biopsies stored at −80 °C using GeneMATRIX Universal RNA Purification Kit (EURx, Gdansk, Poland) and reversed transcription to cDNA was performed using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Life Technologies, MA, USA). .. Expression for HO-1, HO-2, HIF-1α, NF-κB, COX-2, iNOS, TNF-α, IL-1β, SOD-2, and GPx-1 was determined by real-time PCR using specific primers, SG qPCR Master Mix (2x) including SYBR-Green (EURx, Gdansk, Poland) and appropriate thermal cycler (7900HT Fast Real-Time PCR System, Thermo Fisher Scientific, Life Technologies, MA, USA).

    Article Title: Antioxidant activity and cytotoxicity of Jerusalem artichoke tubers and leaves extract on HaCaT and BJ fibroblast cells
    Article Snippet: After 24 h of pre-incubation, cells were treated with obtained leaves and tuber extracts and incubated for 24 h. Then, samples were collected, and total RNA was extracted from HaCaT and fibroblast cells using a EURx Universal RNA Purification Kit according to the manufacturer’s protocol. .. The reverse transcription (RT) reaction was performed at a final volume 20 μL with 1 μg of RNA (as a cDNA template) using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem™) according to the manufacturer’s protocol.

    SDS Page:

    Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response
    Article Snippet: For western blot, 1/50 of total lysates volume was loaded onto a 10% SDS-PAGE and transferred to an Immobilon-P PVDF membrane (Millipore) followed by incubation with primary and HRP-conjugated secondary antibodies and ECL detection. .. 1 μg total RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer’s instructions. cDNAs were quantified on a Eppendorf Masterplex thermocycler using QuantiTect SYBR Green RT-PCR Kit (QIAGEN).

    Software:

    Article Title: Expression of SV2 isoforms during rodent brain development
    Article Snippet: One μg of total RNA was used to synthesize cDNA with the Applied Biosystems high capacity cDNA reverse transcription kit in a total volume of 100 μl following the manufacturer’s protocol (Life Technologies Corporation®, Carlsbad, California). .. Undiluted, 10× and 100× diluted cDNA were analyzed in duplicate for SV2A, SV2B and SV2C expression, using inventoried (SV2A and SV2B) and made to order (SV2C) Applied Biosystems TaqMan gene expression assays.

    Article Title:
    Article Snippet: AdLECs were serum-starved overnight and then exposed to VEGF-C or VEGF-D variants (at 100 ng/ml, unless stated otherwise) before isolation of total RNA using an RNeasy minikit (Qiagen, Valencia, CA) and preparation of cDNA with a high capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA) using 1 μg of total RNA. .. TaqMan gene expression assays for COX-2 (HS00153133-M1) and β-actin (HS99999903-M1) were from Applied Biosystems (Thermo Fisher Scientific).

    Article Title: A Novel Predictive Equation for Potential Diagnosis of Cholangiocarcinoma
    Article Snippet: Approximately 2 µg of total RNA was reverse transcribed to cDNA by the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. .. Real-time RT-PCR was performed using the LightCycler 480 II (Roche Diagnostics, Rotkreuz, Switzerland).

    SYBR Green Assay:

    Article Title: Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation
    Article Snippet: For quantitative real-time PCR of IL-17R, specific primers were generated as follows: IL-17RrealF: 5′-AGGTCCAGCCCTTCTTCAGCA-3′, IL-17RrealR: 5′-GCTTGGGAACTGTGGTATTTGA- -GATTA-3′. .. High Capacity cDNA Reverse Transcription Kit (Invitrogen), RNeasy Mini Kit and QuantiFast SYBR Green PCR Kit (QIAGEN) were used for real-time PCR according to the manufacturer’s instructions. .. To determine the neurosphere volume of stimulated NSCs, these cells were cultured at 200 cells/ml in 96-well plates.

    Article Title: A Novel Predictive Equation for Potential Diagnosis of Cholangiocarcinoma
    Article Snippet: Approximately 2 µg of total RNA was reverse transcribed to cDNA by the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. .. An in-house PCR array was prepared using a Biomek® NXP Laboratory Automate Workstation (Beckman Coulter, Fullerton, CA).

    Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response
    Article Snippet: Inhibition of de novo RNA transcription by ActD was validated by quantitative PCR. .. 1 μg total RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer’s instructions. cDNAs were quantified on a Eppendorf Masterplex thermocycler using QuantiTect SYBR Green RT-PCR Kit (QIAGEN). .. Primer sequences are detailed in the .

    Article Title: miRNA in the Regulation of Skeletal Muscle Adaptation to Acute Endurance Exercise in C57Bl/6J Male Mice
    Article Snippet: The mRNA expression of PPARGC1 alpha (PGC-1α), citrate synthase (CS), 5-aminolevulinate synthase, (ALAS), cytochrome c , (cyt. c ), pyruvate dehydrogenase kinase 4 (PDK4), Drosha, DiGeorge syndrome critical region gene 8 (DGCR8) and Dicer were quantified using 7300 Real-time PCR System (Applied Biosystems Inc., Foster City, CA) and SYBR® Green chemistry (PerfeCT a SYBR® Green Supermix, ROX, Quanta BioSciences, Gaithersburg, MD) as previously described . .. First-strand cDNA synthesis from 1 µg of total RNA was performed with random primers using a high capacity cDNA reverse transcription kit (Applied Biosystems Inc., Foster City, CA) according to manufacturer's directions.

    RNA Extraction:

    Article Title: Expression of SV2 isoforms during rodent brain development
    Article Snippet: RNA extraction was performed with the RNeasy (Qiagen®, Venlo, Netherlands). .. One μg of total RNA was used to synthesize cDNA with the Applied Biosystems high capacity cDNA reverse transcription kit in a total volume of 100 μl following the manufacturer’s protocol (Life Technologies Corporation®, Carlsbad, California).

    RNA Expression:

    Article Title: Optimizing multi-dimensional high throughput screening using zebrafish
    Article Snippet: One microgram of total RNA was converted to cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). .. One microgram of total RNA was converted to cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA).

    In Vitro:

    Article Title: Discovery and pharmacological characterization of a new class of prolyl-tRNA synthetase inhibitor for anti-fibrosis therapy
    Article Snippet: In vitro RNA samples were prepared as mentioned above. .. High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA) was used for cDNA synthesis.

    Homogenization:

    Article Title: Improved detection of gene-microbe interactions in the mouse skin microbiota using high-resolution QTL mapping of 16S rRNA transcripts
    Article Snippet: An additional 2-h room temperature incubation step was included after homogenization in order to increase the nucleic acids’ dissolution in the RLT buffer. .. RNA was treated with DNase (RNase-Free DNase Qiagen, stock solution concentration) for 15 min, twice. cDNA synthesis was performed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems).

    Incubation:

    Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response
    Article Snippet: For western blot, 1/50 of total lysates volume was loaded onto a 10% SDS-PAGE and transferred to an Immobilon-P PVDF membrane (Millipore) followed by incubation with primary and HRP-conjugated secondary antibodies and ECL detection. .. 1 μg total RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer’s instructions. cDNAs were quantified on a Eppendorf Masterplex thermocycler using QuantiTect SYBR Green RT-PCR Kit (QIAGEN).

    Article Title: Antioxidant activity and cytotoxicity of Jerusalem artichoke tubers and leaves extract on HaCaT and BJ fibroblast cells
    Article Snippet: After 24 h of pre-incubation, cells were treated with obtained leaves and tuber extracts and incubated for 24 h. Then, samples were collected, and total RNA was extracted from HaCaT and fibroblast cells using a EURx Universal RNA Purification Kit according to the manufacturer’s protocol. .. The reverse transcription (RT) reaction was performed at a final volume 20 μL with 1 μg of RNA (as a cDNA template) using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem™) according to the manufacturer’s protocol.

    Article Title: Improved detection of gene-microbe interactions in the mouse skin microbiota using high-resolution QTL mapping of 16S rRNA transcripts
    Article Snippet: An additional 2-h room temperature incubation step was included after homogenization in order to increase the nucleic acids’ dissolution in the RLT buffer. .. RNA was treated with DNase (RNase-Free DNase Qiagen, stock solution concentration) for 15 min, twice. cDNA synthesis was performed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems).

    Concentration Assay:

    Article Title: Improved detection of gene-microbe interactions in the mouse skin microbiota using high-resolution QTL mapping of 16S rRNA transcripts
    Article Snippet: An additional 2-h room temperature incubation step was included after homogenization in order to increase the nucleic acids’ dissolution in the RLT buffer. .. RNA was treated with DNase (RNase-Free DNase Qiagen, stock solution concentration) for 15 min, twice. cDNA synthesis was performed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems). .. In addition, RNA purity was checked by a negative reverse transcriptase (without transcriptase) PCR and agarose gel electrophoresis.

    Article Title: Systematic evaluation of medium-throughput mRNA abundance platforms
    Article Snippet: RNA was reverse-transcribed using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems) following the manufacturer's instructions. .. RNA was reverse-transcribed using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems) following the manufacturer's instructions.

    CTG Assay:

    Article Title: Optimizing multi-dimensional high throughput screening using zebrafish
    Article Snippet: One microgram of total RNA was converted to cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). .. Primers for B-actin2 (ENSDART00000141737) were F: 5′-AAG CAG GAG TAC GAT GAG TC-3′ and 5′-TGG AGT CCT CAG ATG CAT TG -3′.

    Variant Assay:

    Article Title: A Novel Predictive Equation for Potential Diagnosis of Cholangiocarcinoma
    Article Snippet: Approximately 2 µg of total RNA was reverse transcribed to cDNA by the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. .. Approximately 2 µg of total RNA was reverse transcribed to cDNA by the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol.

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    Thermo Fisher high capacity cdna reverse transcription kit
    Reactivation of LINE-1 by BaP is Effected via Canonical TGF-β1 Signaling Total <t>RNA</t> was isolated from BEAS-2B cells treated with 0.5 uM BaP for 8 hours, and 1 µg of RNA subjected to <t>cDNA</t> synthesis. Samples were analyzed by RT-qPCR using specific primers for ( A ) human LINE-1 (ORF1 and ORF2) or ( B ) TGF-β1. ( C ) cells pre-treated with TGF-β1 receptor inhibitor (LY2157299) or vehicle (DMSO) for 30 min before BaP challenge or ( D ) transfected with target-specific siRNAs to SMAD2, SMAD3, or scramble siRNA or no siRNA (mock) controls. Expression levels are shown as the mean of triplicates with SEM relative to controls. ( E ) Whole cell extracts from transfected cells were analyzed by immunoblotting for SMAD2, SMAD3, SMAD2/3 or GAPDH antibodies (loading control) to confirm target knockdown. Data are representative of two or more independent experiments. Points represent mean of triple samples with SE.
    High Capacity Cdna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2431 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Reactivation of LINE-1 by BaP is Effected via Canonical TGF-β1 Signaling Total RNA was isolated from BEAS-2B cells treated with 0.5 uM BaP for 8 hours, and 1 µg of RNA subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for ( A ) human LINE-1 (ORF1 and ORF2) or ( B ) TGF-β1. ( C ) cells pre-treated with TGF-β1 receptor inhibitor (LY2157299) or vehicle (DMSO) for 30 min before BaP challenge or ( D ) transfected with target-specific siRNAs to SMAD2, SMAD3, or scramble siRNA or no siRNA (mock) controls. Expression levels are shown as the mean of triplicates with SEM relative to controls. ( E ) Whole cell extracts from transfected cells were analyzed by immunoblotting for SMAD2, SMAD3, SMAD2/3 or GAPDH antibodies (loading control) to confirm target knockdown. Data are representative of two or more independent experiments. Points represent mean of triple samples with SE.

    Journal: Oncotarget

    Article Title: LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells

    doi: 10.18632/oncotarget.21953

    Figure Lengend Snippet: Reactivation of LINE-1 by BaP is Effected via Canonical TGF-β1 Signaling Total RNA was isolated from BEAS-2B cells treated with 0.5 uM BaP for 8 hours, and 1 µg of RNA subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for ( A ) human LINE-1 (ORF1 and ORF2) or ( B ) TGF-β1. ( C ) cells pre-treated with TGF-β1 receptor inhibitor (LY2157299) or vehicle (DMSO) for 30 min before BaP challenge or ( D ) transfected with target-specific siRNAs to SMAD2, SMAD3, or scramble siRNA or no siRNA (mock) controls. Expression levels are shown as the mean of triplicates with SEM relative to controls. ( E ) Whole cell extracts from transfected cells were analyzed by immunoblotting for SMAD2, SMAD3, SMAD2/3 or GAPDH antibodies (loading control) to confirm target knockdown. Data are representative of two or more independent experiments. Points represent mean of triple samples with SE.

    Article Snippet: DNAse digested RNA (1 μg) was employed for cDNA synthesis using high-capacity cDNA reverse transcription Kit (Thermo Fisher Scientific).

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

    Specificity of TGF-β1/LINE-1 Interactions in Transformed Lung Epithelial Cell Lines NCI-H460, NCI-H520, or NCI-H1993 cell lines were challenged with BaP (0.5–2 uM) or 0.5% DMSO vehicle for 24 hours. Total RNA was isolated and 1 µg subjected to cDNA synthesis. Samples were analyzed by RT-PCR using specific primers for human TGF-β1 ( A ), LINE-1 (L1) ORF 1 ( B ) or GAPDH. Expression levels are presented relative to untreated cells. Each point represents the mean and SE of triplicate samples. The data are representative at two or more independent experiments. ( C ) Cells transfected with target-specific siRNAs to SNAIL or scramble siRNA or no siRNA (mock) were challenged with 3 ng/ml TGF-β1. Whole cell extracts were analyzed by immunoblotting for ORF1p, SNAIL, phospho(p)-SMAD2, or total SMAD2. Data are representative of two or more independent experiments.

    Journal: Oncotarget

    Article Title: LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells

    doi: 10.18632/oncotarget.21953

    Figure Lengend Snippet: Specificity of TGF-β1/LINE-1 Interactions in Transformed Lung Epithelial Cell Lines NCI-H460, NCI-H520, or NCI-H1993 cell lines were challenged with BaP (0.5–2 uM) or 0.5% DMSO vehicle for 24 hours. Total RNA was isolated and 1 µg subjected to cDNA synthesis. Samples were analyzed by RT-PCR using specific primers for human TGF-β1 ( A ), LINE-1 (L1) ORF 1 ( B ) or GAPDH. Expression levels are presented relative to untreated cells. Each point represents the mean and SE of triplicate samples. The data are representative at two or more independent experiments. ( C ) Cells transfected with target-specific siRNAs to SNAIL or scramble siRNA or no siRNA (mock) were challenged with 3 ng/ml TGF-β1. Whole cell extracts were analyzed by immunoblotting for ORF1p, SNAIL, phospho(p)-SMAD2, or total SMAD2. Data are representative of two or more independent experiments.

    Article Snippet: DNAse digested RNA (1 μg) was employed for cDNA synthesis using high-capacity cDNA reverse transcription Kit (Thermo Fisher Scientific).

    Techniques: Transformation Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection

    Activation of EMT Programming by TGF-β1 is Associated with LINE-1 Expression in Human Bronchial Epithelial Cells ( A ) BEAS-2B whole cell lysates isolated from cells stimulated with 3 ng/mL TGF-β1 for 48 hours or control were analyzed by immunoblotting using antibodies against LINE1 (L1) ORF1 protein (ORF1p), E-cadherin, vimentin or GAPDH. ( B ) Total RNA from untreated or treated with 3 ng/ml TGF-β1 for 8 hours, and 1 µg of RNA was subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for human L1 (ORF1 and ORF2). Expression levels are shown as the mean of triplicates with SEM relative to controls. ( C ) Whole cell lysates from cells stimulated with different concentrations of TGF-β1 for 48 hours or control were analyzed for expression of L1 ORF1p by immunoblotting. Data are representative of two or more independent experiments.

    Journal: Oncotarget

    Article Title: LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells

    doi: 10.18632/oncotarget.21953

    Figure Lengend Snippet: Activation of EMT Programming by TGF-β1 is Associated with LINE-1 Expression in Human Bronchial Epithelial Cells ( A ) BEAS-2B whole cell lysates isolated from cells stimulated with 3 ng/mL TGF-β1 for 48 hours or control were analyzed by immunoblotting using antibodies against LINE1 (L1) ORF1 protein (ORF1p), E-cadherin, vimentin or GAPDH. ( B ) Total RNA from untreated or treated with 3 ng/ml TGF-β1 for 8 hours, and 1 µg of RNA was subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for human L1 (ORF1 and ORF2). Expression levels are shown as the mean of triplicates with SEM relative to controls. ( C ) Whole cell lysates from cells stimulated with different concentrations of TGF-β1 for 48 hours or control were analyzed for expression of L1 ORF1p by immunoblotting. Data are representative of two or more independent experiments.

    Article Snippet: DNAse digested RNA (1 μg) was employed for cDNA synthesis using high-capacity cDNA reverse transcription Kit (Thermo Fisher Scientific).

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

    Compound 1 treatment of human lung fibroblasts cell cultures increases intracellular cAMP and decreases TGF ‐ β ‐mediated induction of fibrotic gene expression. WI ‐38 human lung fibroblasts were seeded on 24‐well plates and treated with increasing concentrations of Compound 1 (1 × 10 −10 mol/L to 1 × 10 −5 mol/L). After 30 min, cells were stimulated by 1 μ mol/L forskolin for 30 min and cell lysates were analyzed for cAMP concentration (A). To analyze fibroblast gene expression, WI ‐38 human lung fibroblasts were cultured in media containing 0.5% FBS for 24 h and treated with increasing concentrations of Compound 1 (1 × 10 −10 mol/L to 1 × 10 −5 mol/L) for 1 h followed by TGF ‐ β (3 ng/mL) and forskolin (1 μ mol/L) for 24 h. Total RNA was extracted from cell lysates, cDNA was amplified, and target gene mRNA was measured. The target gene expression levels of Col1a1 (Type‐1 collagen), Fn (Fibronectin), CTGF (connective tissue growth factor), and PAI ‐1 (plasminogen activator inhibitor‐1) was normalized to Glyceraldehyde 3‐phosphate dehydrogenase ( GAPDH ). Data are reported as mean ± SD with individual data points shown ( n = 3 per group). # P ≤ 0.05 versus forskolin group by two‐tailed Williams’ test.

    Journal: Physiological Reports

    Article Title: Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury. Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury

    doi: 10.14814/phy2.13753

    Figure Lengend Snippet: Compound 1 treatment of human lung fibroblasts cell cultures increases intracellular cAMP and decreases TGF ‐ β ‐mediated induction of fibrotic gene expression. WI ‐38 human lung fibroblasts were seeded on 24‐well plates and treated with increasing concentrations of Compound 1 (1 × 10 −10 mol/L to 1 × 10 −5 mol/L). After 30 min, cells were stimulated by 1 μ mol/L forskolin for 30 min and cell lysates were analyzed for cAMP concentration (A). To analyze fibroblast gene expression, WI ‐38 human lung fibroblasts were cultured in media containing 0.5% FBS for 24 h and treated with increasing concentrations of Compound 1 (1 × 10 −10 mol/L to 1 × 10 −5 mol/L) for 1 h followed by TGF ‐ β (3 ng/mL) and forskolin (1 μ mol/L) for 24 h. Total RNA was extracted from cell lysates, cDNA was amplified, and target gene mRNA was measured. The target gene expression levels of Col1a1 (Type‐1 collagen), Fn (Fibronectin), CTGF (connective tissue growth factor), and PAI ‐1 (plasminogen activator inhibitor‐1) was normalized to Glyceraldehyde 3‐phosphate dehydrogenase ( GAPDH ). Data are reported as mean ± SD with individual data points shown ( n = 3 per group). # P ≤ 0.05 versus forskolin group by two‐tailed Williams’ test.

    Article Snippet: After seeding, cells were cultured in E‐MEM containing 0.5% FBS for 24 h. The cultures were then treated with Compound 1 (1 × 10‐10 mol/L to 1 × 10‐5 mol/L) for 1 h followed by TGF‐β (3 ng/mL) and forskolin (1 μ mol/L) for 24 h. Total RNA was extracted from cell lysate using RNeasy 96 Kit (QIAGEN, Germany). cDNA was amplified using High‐Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), and target gene mRNA was measured using TaqMan PCR (7900HT Thermo Fisher Scientific).

    Techniques: Expressing, Concentration Assay, Cell Culture, Amplification, Two Tailed Test

    Therapeutic administration of Compound 1 to targeted type II AEC ‐injured mice reduces the expression of TNF α within the lung. DTR ‐expressing mice ( DTR +) were administered daily I.P. PBS or DT from day 0 through Day 14. Subsets of the DTR +: DT ‐treated animals were treated by oral gavage once daily beginning on day 11 with vehicle or Compound 1 at 5.0 mg/kg. On day 21, the left lung was harvested and homogenized, and total RNA was extracted. First‐strand cDNA was synthesized and mRNA levels for Col1a1, Fibronectin, CTGF TNF α and PAI ‐1 (plasminogen activator inhibitor‐1) were assessed using SYBR Green‐based detection. The expression levels were normalized to GAPDH using the following formula: % GAPDH expression = 100/2‐ΔΔ CT . Data are presented as an average ± SEM ( n = 9 per DTR +: DT vehicle‐ and drug‐treated groups).

    Journal: Physiological Reports

    Article Title: Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury. Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury

    doi: 10.14814/phy2.13753

    Figure Lengend Snippet: Therapeutic administration of Compound 1 to targeted type II AEC ‐injured mice reduces the expression of TNF α within the lung. DTR ‐expressing mice ( DTR +) were administered daily I.P. PBS or DT from day 0 through Day 14. Subsets of the DTR +: DT ‐treated animals were treated by oral gavage once daily beginning on day 11 with vehicle or Compound 1 at 5.0 mg/kg. On day 21, the left lung was harvested and homogenized, and total RNA was extracted. First‐strand cDNA was synthesized and mRNA levels for Col1a1, Fibronectin, CTGF TNF α and PAI ‐1 (plasminogen activator inhibitor‐1) were assessed using SYBR Green‐based detection. The expression levels were normalized to GAPDH using the following formula: % GAPDH expression = 100/2‐ΔΔ CT . Data are presented as an average ± SEM ( n = 9 per DTR +: DT vehicle‐ and drug‐treated groups).

    Article Snippet: After seeding, cells were cultured in E‐MEM containing 0.5% FBS for 24 h. The cultures were then treated with Compound 1 (1 × 10‐10 mol/L to 1 × 10‐5 mol/L) for 1 h followed by TGF‐β (3 ng/mL) and forskolin (1 μ mol/L) for 24 h. Total RNA was extracted from cell lysate using RNeasy 96 Kit (QIAGEN, Germany). cDNA was amplified using High‐Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), and target gene mRNA was measured using TaqMan PCR (7900HT Thermo Fisher Scientific).

    Techniques: Mouse Assay, Expressing, Synthesized, SYBR Green Assay

    PMO Uptake into H2K- mdx 52 Myotubes Is Mediated by Endocytosis H2K-WT and mdx 52 myoblasts were differentiated for 4 days and treated with 5 or 10 μM PMO for 48 h (A and B) or 5 or 10 μM Cy5-conjugated PMO for 1, 4 (D and E), or 48 h (C). (A) RT-PCR analysis of exon 51 skipping with cDNA from the myotubes. A representative image is shown. M, 100-bp ladder marker. The dotted line shows the division of the gel. Exon-skipping efficiency was calculated using the densitometric value of the Δexon 51 plus 52 band divided by that of all bands (Δexon 51 plus 52 and Δexon 52) (n = 3 independent experiments). (B) H2K myotubes were preincubated with 1 mM NaN3 for 1 h and then supplemented with 5 μM PMO for 48 h in the presence of NaN3. The exon-skipping efficiency was calculated from the results of RT-PCR (n = 4 independent experiments). The unpaired t test was used for statistical analysis. (C) Representative image of RT-PCR after a 48-h treatment of H2K myotubes with 10 μM Cy5-PMO. The dotted line shows the division of the gel. (D) A representative image of Cy5 fluorescence in myotubes after 5 μM Cy5-PMO treatment for 1 or 4 h. The fluorescence intensities were quantified at each time point. Two-way ANOVA was used for comparisons between multiple groups. Scale bars, 200 nm (n = 7 independent experiments). (E) Representative fluorescence images of myotubes after 5 μM Cy5-PMO treatment for 4 h, merged with nuclear staining by Hoechst (blue). Cy5 fluorescence intensity in the nuclear area (μm) was quantified using the BZ-H3CM software. The unpaired t test was used for statistical analysis. Scale bars, 20 nm (n = 4 independent experiments). (F) H2K- mdx 52 myotubes were pre-incubated with 10 mM NaN3, 30 μM CPZ, 4 μg/mL filipin III, and 10 μM EIPA for 30 min and then supplemented with 5 μM Cy5-PMO for 4 h in the presence of each inhibitor. Representative fluorescence images of the myotubes are shown. The fluorescence intensities were quantified for each condition (n = 6 independent experiments). One-way ANOVA was used for comparisons between multiple groups. Results are presented as mean ± SE. *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Scavenger Receptor Class A1 Mediates Uptake of Morpholino Antisense Oligonucleotide into Dystrophic Skeletal Muscle

    doi: 10.1016/j.omtn.2019.01.008

    Figure Lengend Snippet: PMO Uptake into H2K- mdx 52 Myotubes Is Mediated by Endocytosis H2K-WT and mdx 52 myoblasts were differentiated for 4 days and treated with 5 or 10 μM PMO for 48 h (A and B) or 5 or 10 μM Cy5-conjugated PMO for 1, 4 (D and E), or 48 h (C). (A) RT-PCR analysis of exon 51 skipping with cDNA from the myotubes. A representative image is shown. M, 100-bp ladder marker. The dotted line shows the division of the gel. Exon-skipping efficiency was calculated using the densitometric value of the Δexon 51 plus 52 band divided by that of all bands (Δexon 51 plus 52 and Δexon 52) (n = 3 independent experiments). (B) H2K myotubes were preincubated with 1 mM NaN3 for 1 h and then supplemented with 5 μM PMO for 48 h in the presence of NaN3. The exon-skipping efficiency was calculated from the results of RT-PCR (n = 4 independent experiments). The unpaired t test was used for statistical analysis. (C) Representative image of RT-PCR after a 48-h treatment of H2K myotubes with 10 μM Cy5-PMO. The dotted line shows the division of the gel. (D) A representative image of Cy5 fluorescence in myotubes after 5 μM Cy5-PMO treatment for 1 or 4 h. The fluorescence intensities were quantified at each time point. Two-way ANOVA was used for comparisons between multiple groups. Scale bars, 200 nm (n = 7 independent experiments). (E) Representative fluorescence images of myotubes after 5 μM Cy5-PMO treatment for 4 h, merged with nuclear staining by Hoechst (blue). Cy5 fluorescence intensity in the nuclear area (μm) was quantified using the BZ-H3CM software. The unpaired t test was used for statistical analysis. Scale bars, 20 nm (n = 4 independent experiments). (F) H2K- mdx 52 myotubes were pre-incubated with 10 mM NaN3, 30 μM CPZ, 4 μg/mL filipin III, and 10 μM EIPA for 30 min and then supplemented with 5 μM Cy5-PMO for 4 h in the presence of each inhibitor. Representative fluorescence images of the myotubes are shown. The fluorescence intensities were quantified for each condition (n = 6 independent experiments). One-way ANOVA was used for comparisons between multiple groups. Results are presented as mean ± SE. *p

    Article Snippet: One hundred nanograms of total RNA template was used for RT-PCR with a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Marker, Fluorescence, Staining, Software, Incubation

    Role of SR-A1 in PMO Uptake into H2K- mdx 52 Myotubes (A) Gene expression of SR class A isoforms by qPCR using cDNA from H2K-WT and H2K- mdx 52 myotubes 6 days after differentiation. The value of 2 ΔCT is shown. Abt1 was used as a housekeeping gene. The Mann-Whitney U test was used for statistical analysis (n ≥ 3 independent experiments). (B) Western blotting of SR-A1 in the lysates of H2K- mdx52 myotubes on day 6 separated into cytosolic and membrane fractions. GAPDH was used as the control for the cytosolic fraction, and Na + /K + ATPase and caveolin-3 were used as controls for the membrane fraction. Blots of each protein were derived from the same membrane. (C) Representative orthogonal sectioning of confocal images of caveolin-3 and SR-A1 in H2K- mdx 52 myotubes on day 4 by the HyVolution method. Images of the xy, xz, and yz sections are shown. Scale bars, 10 μm. (D) H2K- mdx 52 myotubes were pre-treated with SR inhibitors for 1 h and then supplemented with 5 μM PMO for 48 h in the presence of inhibitors. Ten micrograms per microliter poly I was used as an SR class A, C, E, and F inhibitor. Ten micrograms per microliter poly C was used as a control for poly I. Ten micrograms per microliter fucoidan was used as an SR class A1/2 and C1 inhibitor. The paired t test was used for statistical analysis (n ≥ 3 independent experiments). (E) H2K- mdx 52 myoblasts were supplemented with 5 nM siRNA for each SR-isoform for 24 h, differentiated for 4 days, and supplemented with 5 μM PMO for 48 h, followed by total RNA collection for RT-PCR detection of exon 51 skipping. The Mann-Whitney U test was used for comparisons between the control and each siRNA-treated group (n ≥ 3 independent experiments). (F) Primary satellite cell-derived myoblasts from mdx 52 and DKO mice were supplemented with 5 μM Cy5-PMO for 4 h, and the fluorescence intensities were quantified. The unpaired t test was used for statistical analysis (n = 3 independent experiments). (G) GFP- or GFP-SR-A1/2-transfected HEK293T cells were supplemented with 5 μM Cy5-PMO for 4 h. Representative fluorescence images of the cells are shown. The fluorescence intensity of Cy5 divided by cell area was quantified in each condition. White arrowheads show co-localization of Cy5-PMO and GFP-SR-A1/2. The unpaired t test was used for statistical analysis (n = 4 independent experiments). Results are presented as mean ± SE. *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Scavenger Receptor Class A1 Mediates Uptake of Morpholino Antisense Oligonucleotide into Dystrophic Skeletal Muscle

    doi: 10.1016/j.omtn.2019.01.008

    Figure Lengend Snippet: Role of SR-A1 in PMO Uptake into H2K- mdx 52 Myotubes (A) Gene expression of SR class A isoforms by qPCR using cDNA from H2K-WT and H2K- mdx 52 myotubes 6 days after differentiation. The value of 2 ΔCT is shown. Abt1 was used as a housekeeping gene. The Mann-Whitney U test was used for statistical analysis (n ≥ 3 independent experiments). (B) Western blotting of SR-A1 in the lysates of H2K- mdx52 myotubes on day 6 separated into cytosolic and membrane fractions. GAPDH was used as the control for the cytosolic fraction, and Na + /K + ATPase and caveolin-3 were used as controls for the membrane fraction. Blots of each protein were derived from the same membrane. (C) Representative orthogonal sectioning of confocal images of caveolin-3 and SR-A1 in H2K- mdx 52 myotubes on day 4 by the HyVolution method. Images of the xy, xz, and yz sections are shown. Scale bars, 10 μm. (D) H2K- mdx 52 myotubes were pre-treated with SR inhibitors for 1 h and then supplemented with 5 μM PMO for 48 h in the presence of inhibitors. Ten micrograms per microliter poly I was used as an SR class A, C, E, and F inhibitor. Ten micrograms per microliter poly C was used as a control for poly I. Ten micrograms per microliter fucoidan was used as an SR class A1/2 and C1 inhibitor. The paired t test was used for statistical analysis (n ≥ 3 independent experiments). (E) H2K- mdx 52 myoblasts were supplemented with 5 nM siRNA for each SR-isoform for 24 h, differentiated for 4 days, and supplemented with 5 μM PMO for 48 h, followed by total RNA collection for RT-PCR detection of exon 51 skipping. The Mann-Whitney U test was used for comparisons between the control and each siRNA-treated group (n ≥ 3 independent experiments). (F) Primary satellite cell-derived myoblasts from mdx 52 and DKO mice were supplemented with 5 μM Cy5-PMO for 4 h, and the fluorescence intensities were quantified. The unpaired t test was used for statistical analysis (n = 3 independent experiments). (G) GFP- or GFP-SR-A1/2-transfected HEK293T cells were supplemented with 5 μM Cy5-PMO for 4 h. Representative fluorescence images of the cells are shown. The fluorescence intensity of Cy5 divided by cell area was quantified in each condition. White arrowheads show co-localization of Cy5-PMO and GFP-SR-A1/2. The unpaired t test was used for statistical analysis (n = 4 independent experiments). Results are presented as mean ± SE. *p

    Article Snippet: One hundred nanograms of total RNA template was used for RT-PCR with a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Western Blot, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Fluorescence, Transfection

    HNRNPLL promotes cell cycle progression in colon cancer cells. A‐D, Cell cycle analysis for SW 480 (A) and HT 29 (C) cells transduced with HNRNPLL cDNA , Luc sh RNA or HNRNPLL sh RNA 2 was performed using a flow cytometer. The sum of the percentages of S and G2/M phases in SW 480 (B) and HT 29 (D) cells are shown in the bar graph. Error bars, SD . E, Immunostaining of HT 29 cells using antibodies for HNRNPLL , GMNN and CDT 1. Note that cells showing no nuclear GMNN expression and high CDT 1 expression (*) exhibit higher HNRNPLL expression compared to cells showing nuclear GMNN expression and low CDT 1 expression ( † ). Scale bar, 10 μm

    Journal: Cancer Science

    Article Title: HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells, et al. HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells

    doi: 10.1111/cas.13660

    Figure Lengend Snippet: HNRNPLL promotes cell cycle progression in colon cancer cells. A‐D, Cell cycle analysis for SW 480 (A) and HT 29 (C) cells transduced with HNRNPLL cDNA , Luc sh RNA or HNRNPLL sh RNA 2 was performed using a flow cytometer. The sum of the percentages of S and G2/M phases in SW 480 (B) and HT 29 (D) cells are shown in the bar graph. Error bars, SD . E, Immunostaining of HT 29 cells using antibodies for HNRNPLL , GMNN and CDT 1. Note that cells showing no nuclear GMNN expression and high CDT 1 expression (*) exhibit higher HNRNPLL expression compared to cells showing nuclear GMNN expression and low CDT 1 expression ( † ). Scale bar, 10 μm

    Article Snippet: Collected RNA was subjected to reverse transcription using a High‐Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) with random or oligo(dT) primers and was analyzed by 35 cycles of conventional PCR using KOD‐Plus Neo (Toyobo, Osaka, Japan).

    Techniques: Cell Cycle Assay, Transduction, Flow Cytometry, Cytometry, Immunostaining, Expressing

    Knockdown of PCNA , RFC 3 or FEN 1 suppresses the increased cell proliferation caused by HNRNPLL overexpression. A, Western blot analysis to confirm the overexpression of HNRNPLL and knockdown of PCNA , RFC 3 and FEN 1. Arrows and arrowheads indicate FLAG ‐tagged or endogenous HNRNPLL , respectively. B, MTT assay was performed for Mock‐ or HNRNPLL cDNA ‐transduced SW 480 cells 72 h after transfection of control si RNA or si RNA (s) targeting PCNA , RFC 3 , or FEN 1 . Error bars, SD . P = .036 (*), .20 ( † ), .32 ( †† ), .12 ( ††† ), .030 ( ‡ ), .038 ( ‡‡ ), .027 ( ‡‡‡ ), .50 ( § ), .78 ( §§ ) and .21 ( §§§ ). C, MTT assay was performed for Mock‐ or HNRNPLL cDNA ‐transduced HT 29 cells 72 h after transfection of control si RNA or si RNA (s) targeting PCNA , RFC 3 , or FEN 1 . Error bars, SD . P = .026 (*), .079 ( † ), .79 ( †† ), .12 ( ††† ), .036 ( ‡ ), .040 ( ‡‡ ), .033 ( ‡‡‡ ), .12 ( § ), .34 ( §§ ) and .18 ( §§§ ). D, Western blot analysis to confirm the overexpression of FLAG ‐ PCNA , FLAG ‐ RFC 3 and FLAG ‐ FEN 1 in SW 480 and HT 29 cells simultaneously transduced with their cDNA s. Arrows and arrowheads indicate FLAG ‐tagged or endogenous proteins, respectively. E, MTT assay was performed for SW 480 and HT 29 cells transduced with mock vector or PCNA , RFC 3 and FEN 1 cDNA . Error bars, SD . P = .76 (*) and .090 ( † )

    Journal: Cancer Science

    Article Title: HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells, et al. HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells

    doi: 10.1111/cas.13660

    Figure Lengend Snippet: Knockdown of PCNA , RFC 3 or FEN 1 suppresses the increased cell proliferation caused by HNRNPLL overexpression. A, Western blot analysis to confirm the overexpression of HNRNPLL and knockdown of PCNA , RFC 3 and FEN 1. Arrows and arrowheads indicate FLAG ‐tagged or endogenous HNRNPLL , respectively. B, MTT assay was performed for Mock‐ or HNRNPLL cDNA ‐transduced SW 480 cells 72 h after transfection of control si RNA or si RNA (s) targeting PCNA , RFC 3 , or FEN 1 . Error bars, SD . P = .036 (*), .20 ( † ), .32 ( †† ), .12 ( ††† ), .030 ( ‡ ), .038 ( ‡‡ ), .027 ( ‡‡‡ ), .50 ( § ), .78 ( §§ ) and .21 ( §§§ ). C, MTT assay was performed for Mock‐ or HNRNPLL cDNA ‐transduced HT 29 cells 72 h after transfection of control si RNA or si RNA (s) targeting PCNA , RFC 3 , or FEN 1 . Error bars, SD . P = .026 (*), .079 ( † ), .79 ( †† ), .12 ( ††† ), .036 ( ‡ ), .040 ( ‡‡ ), .033 ( ‡‡‡ ), .12 ( § ), .34 ( §§ ) and .18 ( §§§ ). D, Western blot analysis to confirm the overexpression of FLAG ‐ PCNA , FLAG ‐ RFC 3 and FLAG ‐ FEN 1 in SW 480 and HT 29 cells simultaneously transduced with their cDNA s. Arrows and arrowheads indicate FLAG ‐tagged or endogenous proteins, respectively. E, MTT assay was performed for SW 480 and HT 29 cells transduced with mock vector or PCNA , RFC 3 and FEN 1 cDNA . Error bars, SD . P = .76 (*) and .090 ( † )

    Article Snippet: Collected RNA was subjected to reverse transcription using a High‐Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) with random or oligo(dT) primers and was analyzed by 35 cycles of conventional PCR using KOD‐Plus Neo (Toyobo, Osaka, Japan).

    Techniques: Over Expression, Western Blot, MTT Assay, Transfection, Transduction, Plasmid Preparation