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The impact of TUG1 on tumorigenesis in vivo . ( a and b ) Scramble or shTUG1 was transfected into SPC-A1 cells, which were injected in the nude mice ( n =10), respectively. Tumor volumes were calculated after injection every 2 days. Bars indicate S.D. ( c ) Tumor weights are represented as means of tumor weights ±S.D. <t>qRT-PCR</t> was performed to detect the average expression of TUG1. ( d ) Histopathology of xenograft tumors. The tumor sections were under H E staining and IHC staining using antibodies against Ki-67. Bar, 100 μ m. Error bars indicate means±S.E.M. * P
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1) Product Images from "P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression"

Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.201

The impact of TUG1 on tumorigenesis in vivo . ( a and b ) Scramble or shTUG1 was transfected into SPC-A1 cells, which were injected in the nude mice ( n =10), respectively. Tumor volumes were calculated after injection every 2 days. Bars indicate S.D. ( c ) Tumor weights are represented as means of tumor weights ±S.D. qRT-PCR was performed to detect the average expression of TUG1. ( d ) Histopathology of xenograft tumors. The tumor sections were under H E staining and IHC staining using antibodies against Ki-67. Bar, 100 μ m. Error bars indicate means±S.E.M. * P
Figure Legend Snippet: The impact of TUG1 on tumorigenesis in vivo . ( a and b ) Scramble or shTUG1 was transfected into SPC-A1 cells, which were injected in the nude mice ( n =10), respectively. Tumor volumes were calculated after injection every 2 days. Bars indicate S.D. ( c ) Tumor weights are represented as means of tumor weights ±S.D. qRT-PCR was performed to detect the average expression of TUG1. ( d ) Histopathology of xenograft tumors. The tumor sections were under H E staining and IHC staining using antibodies against Ki-67. Bar, 100 μ m. Error bars indicate means±S.E.M. * P

Techniques Used: In Vivo, Transfection, Injection, Mouse Assay, Quantitative RT-PCR, Expressing, Histopathology, Staining, Immunohistochemistry

TUG1 could epigenetically regulate HOXB7 by binding to PRC2. ( a ) qRT-PCR was performed to detect the expression of HOX genes in transfected cells, and western blot assays were used to detect the level of HOXB7 after the transfection of si-TUG1. IHC assays were used to detect the HOXB7 in tumor sections from shTUG1-transfected cells. ( b ) RIP experiments were performed in SPC-A1 and the coprecipitated RNA was subjected to qRT-PCR for TUG1. HOTAIR was used as a positive control. The fold enrichment of TUG1 in EZH2 RIP is relative to its matching IgG control RIP. TUG1 nuclear localization, as identified using qRT-PCR in fractionated SPC-A1 and A549 cells. ( c ) ChIP of H3K27me3 and EZH2 of the promoter region of HOXB7 locus after siRNA treatment targeting si-NC or si-TUG1; qPCR were performed to determine the quantitation of ChIP assays. The levels of qPCR products are expressed as a percentage of the input DNA. Error bars indicate means±S.E.M. * P
Figure Legend Snippet: TUG1 could epigenetically regulate HOXB7 by binding to PRC2. ( a ) qRT-PCR was performed to detect the expression of HOX genes in transfected cells, and western blot assays were used to detect the level of HOXB7 after the transfection of si-TUG1. IHC assays were used to detect the HOXB7 in tumor sections from shTUG1-transfected cells. ( b ) RIP experiments were performed in SPC-A1 and the coprecipitated RNA was subjected to qRT-PCR for TUG1. HOTAIR was used as a positive control. The fold enrichment of TUG1 in EZH2 RIP is relative to its matching IgG control RIP. TUG1 nuclear localization, as identified using qRT-PCR in fractionated SPC-A1 and A549 cells. ( c ) ChIP of H3K27me3 and EZH2 of the promoter region of HOXB7 locus after siRNA treatment targeting si-NC or si-TUG1; qPCR were performed to determine the quantitation of ChIP assays. The levels of qPCR products are expressed as a percentage of the input DNA. Error bars indicate means±S.E.M. * P

Techniques Used: Binding Assay, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Immunohistochemistry, Positive Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitation Assay

p53 induces TUG1 through interacting with the promoter region of TUG1. ( a ) Analysis of TUG1 expression levels in NSCLC cell lines (A549, SPC-A1, SK-MES-1, NCI-H1299 and NCI-H1650) compared with the normal bronchial epithelial cell line (16HBE) by qRT-PCR. ( b ) Description of p53RE and mutant p53RE in promoter region of TUG1. The position of ChIP primers ( e ) was indicated by arrows. ( c ) Western blotting was used to detect the p53 induction by doxo. qRT-PCR was used to detect the effect of doxo on TUG1 expression in p53-WT and p53-null cells. ( d ) Induction of TUG1 by ectopically expressed p53 (wild-type p53 or mutant p53). Overexpression was confirmed by western blotting. ( e ) Induction of TUG1 promoter activity by p53, but not mutant p53 in NCI-H1299 cell lines. ( f ) Deletion and mutation analysis of the promoter activity to determine the role of the p53RE in p53-mediated regulation of TUG1. ( g ) The p53 binding at the promoter regions of TUG1 was assessed by ChIP analysis. ChIP primers were detailed in Materials and Methods section. Shown are representative images of three independent experiments. Error bars indicate means±S.E.M. * P
Figure Legend Snippet: p53 induces TUG1 through interacting with the promoter region of TUG1. ( a ) Analysis of TUG1 expression levels in NSCLC cell lines (A549, SPC-A1, SK-MES-1, NCI-H1299 and NCI-H1650) compared with the normal bronchial epithelial cell line (16HBE) by qRT-PCR. ( b ) Description of p53RE and mutant p53RE in promoter region of TUG1. The position of ChIP primers ( e ) was indicated by arrows. ( c ) Western blotting was used to detect the p53 induction by doxo. qRT-PCR was used to detect the effect of doxo on TUG1 expression in p53-WT and p53-null cells. ( d ) Induction of TUG1 by ectopically expressed p53 (wild-type p53 or mutant p53). Overexpression was confirmed by western blotting. ( e ) Induction of TUG1 promoter activity by p53, but not mutant p53 in NCI-H1299 cell lines. ( f ) Deletion and mutation analysis of the promoter activity to determine the role of the p53RE in p53-mediated regulation of TUG1. ( g ) The p53 binding at the promoter regions of TUG1 was assessed by ChIP analysis. ChIP primers were detailed in Materials and Methods section. Shown are representative images of three independent experiments. Error bars indicate means±S.E.M. * P

Techniques Used: Expressing, Quantitative RT-PCR, Mutagenesis, Chromatin Immunoprecipitation, Western Blot, Over Expression, Activity Assay, Binding Assay

Analysis of TUG1 expression in NSCLC tissues and clinical parameters. ( a ) TUG1 was detected in 192 pairs of NSCLC tissues by qRT-PCR. The levels of TUG1 in NSCLC tissues are significantly lower than those in non-tumorous tissues. The ΔCt value was determined by subtracting the GAPDH Ct value from the TUG1 Ct value (relative to a single reference value). Smaller ΔCt value indicates higher expression. ( b and c ) Data are presented as relative expression level in tumor tissues (shown as ΔCt). TUG1 expression was significantly lower in patients with a higher pathological stage and big tumor size. ( d ) Patients with low levels of TUG1 expression showed reduced survival times compared with patients with high levels of TUG1 expression ( P
Figure Legend Snippet: Analysis of TUG1 expression in NSCLC tissues and clinical parameters. ( a ) TUG1 was detected in 192 pairs of NSCLC tissues by qRT-PCR. The levels of TUG1 in NSCLC tissues are significantly lower than those in non-tumorous tissues. The ΔCt value was determined by subtracting the GAPDH Ct value from the TUG1 Ct value (relative to a single reference value). Smaller ΔCt value indicates higher expression. ( b and c ) Data are presented as relative expression level in tumor tissues (shown as ΔCt). TUG1 expression was significantly lower in patients with a higher pathological stage and big tumor size. ( d ) Patients with low levels of TUG1 expression showed reduced survival times compared with patients with high levels of TUG1 expression ( P

Techniques Used: Expressing, Quantitative RT-PCR

TUG1 could participate in AKT and MAPK pathway through modulating HOXB7. ( A ) MTT analysis of cell proliferation by co-transfection (si-NC, si-HOXB7, si-HOXB7+si-TUG1). At 48 h after transfection of si-HOXB7, the cell cycle and apoptosis were analyzed by flow cytometry. LR, early apoptotic cells. UR, terminal apoptotic cells. ( B ) qRT-PCR were performed to detect the expression of HOXB7 after overexpression of p53 and transfected with p53 followed by transfection with si-TUG1. ( C ) Western blotting analysis of the expression of p-ERK, total ERK, p-AKT, total AKT, p-GSK-3 β , total GSK-3 β proteins in indicated si-NC-transfected, si-HOXB7 and si-TUG1-transfected SPC-A1 cell lines. ( D ) Immunostaining of HOXB7 was negatively or very weakly positive in corresponding non-tumor lung tissues (a and b), but was strongly positive in squamous cell carcinoma tissues (c and d) and lung adenocarcinoma (e and f). Bar, 100 μ m. ( E ) The immunoreactivity of HOXB7 protein in NSCLC tissues showed a statistically significant inverse correlation with the relative level of TUG1 expression. Error bars indicate means±S.E.M. * P
Figure Legend Snippet: TUG1 could participate in AKT and MAPK pathway through modulating HOXB7. ( A ) MTT analysis of cell proliferation by co-transfection (si-NC, si-HOXB7, si-HOXB7+si-TUG1). At 48 h after transfection of si-HOXB7, the cell cycle and apoptosis were analyzed by flow cytometry. LR, early apoptotic cells. UR, terminal apoptotic cells. ( B ) qRT-PCR were performed to detect the expression of HOXB7 after overexpression of p53 and transfected with p53 followed by transfection with si-TUG1. ( C ) Western blotting analysis of the expression of p-ERK, total ERK, p-AKT, total AKT, p-GSK-3 β , total GSK-3 β proteins in indicated si-NC-transfected, si-HOXB7 and si-TUG1-transfected SPC-A1 cell lines. ( D ) Immunostaining of HOXB7 was negatively or very weakly positive in corresponding non-tumor lung tissues (a and b), but was strongly positive in squamous cell carcinoma tissues (c and d) and lung adenocarcinoma (e and f). Bar, 100 μ m. ( E ) The immunoreactivity of HOXB7 protein in NSCLC tissues showed a statistically significant inverse correlation with the relative level of TUG1 expression. Error bars indicate means±S.E.M. * P

Techniques Used: MTT Assay, Cotransfection, Transfection, Flow Cytometry, Cytometry, Quantitative RT-PCR, Expressing, Over Expression, Western Blot, Immunostaining

2) Product Images from "Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression"

Article Title: Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression

Journal: Molecular Cancer

doi: 10.1186/s12943-018-0836-7

AFAP1-AS1 could directly bind to EZH2. a After nuclear and cytosolic separation, RNA expression levels were measured by qRT-PCR. GAPDH was used as a cytosol marker and U6 was used as a nucleus marker. b RIPs experiments for EZH2, SUZ12 were performed and the coprecipitated RNA was subjected to qRT-PCR for AFAP1-AS1
Figure Legend Snippet: AFAP1-AS1 could directly bind to EZH2. a After nuclear and cytosolic separation, RNA expression levels were measured by qRT-PCR. GAPDH was used as a cytosol marker and U6 was used as a nucleus marker. b RIPs experiments for EZH2, SUZ12 were performed and the coprecipitated RNA was subjected to qRT-PCR for AFAP1-AS1

Techniques Used: RNA Expression, Quantitative RT-PCR, Marker

AFAP1-AS1 regulates NSCLC cell proliferation in vitro. a Analysis of AFAP1-AS1 expression levels in NSCLC cell lines compared with a normal bronchial epithelial cell line (16HBE) using qRT-PCR. b The relative expression levels of AFAP1-AS1 in A549 and SPCA1 cells transfected with si-NC or si-AFAP1-AS1 (si-AFAP1-AS1 1# and si-AFAP1-AS1 2#) were measured using qPCR. c MTT assays were performed after transfection to determine the cell viability of the transfected A549 and SPCA1 cells. d Colony-forming assays were conducted after transfection to determine the proliferation of the transfected A549 and SPCA1 cells. The colonies were counted and captured. e BrdU assays were used to detect the cell proliferation after transfection, respectively. f After transfection, the cell cycle stages of A549 and SPCA1 cells were analyzed by flow cytometry. The bar chart represents the percentages of cells in the G1/G0, S, or G2/M phases, as indicated.*, P
Figure Legend Snippet: AFAP1-AS1 regulates NSCLC cell proliferation in vitro. a Analysis of AFAP1-AS1 expression levels in NSCLC cell lines compared with a normal bronchial epithelial cell line (16HBE) using qRT-PCR. b The relative expression levels of AFAP1-AS1 in A549 and SPCA1 cells transfected with si-NC or si-AFAP1-AS1 (si-AFAP1-AS1 1# and si-AFAP1-AS1 2#) were measured using qPCR. c MTT assays were performed after transfection to determine the cell viability of the transfected A549 and SPCA1 cells. d Colony-forming assays were conducted after transfection to determine the proliferation of the transfected A549 and SPCA1 cells. The colonies were counted and captured. e BrdU assays were used to detect the cell proliferation after transfection, respectively. f After transfection, the cell cycle stages of A549 and SPCA1 cells were analyzed by flow cytometry. The bar chart represents the percentages of cells in the G1/G0, S, or G2/M phases, as indicated.*, P

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, Real-time Polymerase Chain Reaction, MTT Assay, Flow Cytometry, Cytometry

RNA-seq assay after AFAP1-AS1 knockdown in A549 cells. a Mean-centered, hierarchical clustering of 591 transcripts altered (≥2-fold change) in si-NC-treated cells and siRNA-AFAP1-AS1 treated A549 cells. b Gene Ontology analysis for all genes with altered expressions. c The altered mRNA levels of genes were selectively confirmed by qRT-PCR in knockdown AFAP1-AS1
Figure Legend Snippet: RNA-seq assay after AFAP1-AS1 knockdown in A549 cells. a Mean-centered, hierarchical clustering of 591 transcripts altered (≥2-fold change) in si-NC-treated cells and siRNA-AFAP1-AS1 treated A549 cells. b Gene Ontology analysis for all genes with altered expressions. c The altered mRNA levels of genes were selectively confirmed by qRT-PCR in knockdown AFAP1-AS1

Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR

3) Product Images from "Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma"

Article Title: Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma

Journal: BioMed Research International

doi: 10.1155/2013/251098

Degradation of matrix in situ is suppressed by knockdown of HOTAIR. (a) qRT-PCR analysis was performed to examine HOTAIR RNA levels in A375 cells transfected with siControl or siHOTAIR (I and II). (b) A375 cells were transfected with siControl or siHOTAIR (I and II) for 24 hours. In situ zymography was performed with Oregon green 488-conjugated gelatin. Cells were plated on gelatin matrix and incubated for 24 hours. The slides were observed by phase-contrast microscopy (left column), and gelatin degradation was visualized by fluorescence microscopy (right column).
Figure Legend Snippet: Degradation of matrix in situ is suppressed by knockdown of HOTAIR. (a) qRT-PCR analysis was performed to examine HOTAIR RNA levels in A375 cells transfected with siControl or siHOTAIR (I and II). (b) A375 cells were transfected with siControl or siHOTAIR (I and II) for 24 hours. In situ zymography was performed with Oregon green 488-conjugated gelatin. Cells were plated on gelatin matrix and incubated for 24 hours. The slides were observed by phase-contrast microscopy (left column), and gelatin degradation was visualized by fluorescence microscopy (right column).

Techniques Used: In Situ, Quantitative RT-PCR, Transfection, Zymography, Incubation, Microscopy, Fluorescence

Knockdown of HOTAIR decreases melanoma A375 cell motility. (a) qRT-PCR analysis was performed to examine HOTAIR RNA levels in A375 cells transfected with siControl, siHOTAIR I, or siHOTAIR II. (b) Wounds were introduced by scratching confluent monolayers of A375 cells transfected with siControl, siHOTAIR I, or siHOTAIR II. Migration was monitored by light microscopy at 0 hours and 24 hours (upper panel). The widths of the gaps from 3 experiments were measured and the results are presented in a bar graph (lower panel). * P
Figure Legend Snippet: Knockdown of HOTAIR decreases melanoma A375 cell motility. (a) qRT-PCR analysis was performed to examine HOTAIR RNA levels in A375 cells transfected with siControl, siHOTAIR I, or siHOTAIR II. (b) Wounds were introduced by scratching confluent monolayers of A375 cells transfected with siControl, siHOTAIR I, or siHOTAIR II. Migration was monitored by light microscopy at 0 hours and 24 hours (upper panel). The widths of the gaps from 3 experiments were measured and the results are presented in a bar graph (lower panel). * P

Techniques Used: Quantitative RT-PCR, Transfection, Migration, Light Microscopy

4) Product Images from "A transcriptome study on Macrobrachium nipponense hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)"

Article Title: A transcriptome study on Macrobrachium nipponense hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200222

Comparison of the expression profiles of selected genes as determined by RNA-Seq (Illumina Hiseq2500 TM sequencing) and qRT-PCR. The letter “A” and “B” in the RNA-Seq and qRT-PCR mean the comparison of expression profiles of selected genes between control and moribund samples (the correlation coefficient was 0.92), the comparison of expression profiles of selected genes between control and survived samples (the correlation coefficient was 0.95), respectively. Target gene abbreviations are as follows: TSPAN8 –TSPAN8, DPAGT1 –UDP-N-acetylglucosamine—dolichyl-phosphate N-acetylglucosaminephosphotransferase, Gag-pol –Gag-Pol polyprotein, ARSB–arylsulfatase B, MKK2 –MAP kinase-activated protein kinase 2, PDK–pyruvate dehydrogenase (acetyl-transferring) kinase, ILK–integrin-linked protein kinase, AP–acid phosphatase, ERI3 –ERI1 exoribonuclease 3, ZMYM1 –zinc finger MYM-type protein 1. Error bars indicated standard deviations of averages from three replicates.
Figure Legend Snippet: Comparison of the expression profiles of selected genes as determined by RNA-Seq (Illumina Hiseq2500 TM sequencing) and qRT-PCR. The letter “A” and “B” in the RNA-Seq and qRT-PCR mean the comparison of expression profiles of selected genes between control and moribund samples (the correlation coefficient was 0.92), the comparison of expression profiles of selected genes between control and survived samples (the correlation coefficient was 0.95), respectively. Target gene abbreviations are as follows: TSPAN8 –TSPAN8, DPAGT1 –UDP-N-acetylglucosamine—dolichyl-phosphate N-acetylglucosaminephosphotransferase, Gag-pol –Gag-Pol polyprotein, ARSB–arylsulfatase B, MKK2 –MAP kinase-activated protein kinase 2, PDK–pyruvate dehydrogenase (acetyl-transferring) kinase, ILK–integrin-linked protein kinase, AP–acid phosphatase, ERI3 –ERI1 exoribonuclease 3, ZMYM1 –zinc finger MYM-type protein 1. Error bars indicated standard deviations of averages from three replicates.

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

5) Product Images from "Prospective Isolation of ISL1+ Cardiac Progenitors from Human ESCs for Myocardial Infarction Therapy"

Article Title: Prospective Isolation of ISL1+ Cardiac Progenitors from Human ESCs for Myocardial Infarction Therapy

Journal: Stem Cell Reports

doi: 10.1016/j.stemcr.2018.01.037

Enrichment and Proteomic Characterization of hESC-Derived ISL1 + Progenitors (A) Schematic representation of the differentiation protocol. (B) Real-time qRT-PCR for ISL1 expression during cardiac differentiation of rH5-isl1-Hygro. n = 3. (C) Antibiotic treatment paradigm for enrichment of ISL1 + cells. (D) Immunofluorescence staining and flow cytometry of hESCs at day 8 of differentiation with or without antibiotic treatment for ISL1. (E) Membrane proteins that were ≥1.5-fold differentially expressed (n = 3 independent experiments) between antibiotic-treated cells versus untreated cells. AA, activin A. Data are mean ± SEM. ∗ p
Figure Legend Snippet: Enrichment and Proteomic Characterization of hESC-Derived ISL1 + Progenitors (A) Schematic representation of the differentiation protocol. (B) Real-time qRT-PCR for ISL1 expression during cardiac differentiation of rH5-isl1-Hygro. n = 3. (C) Antibiotic treatment paradigm for enrichment of ISL1 + cells. (D) Immunofluorescence staining and flow cytometry of hESCs at day 8 of differentiation with or without antibiotic treatment for ISL1. (E) Membrane proteins that were ≥1.5-fold differentially expressed (n = 3 independent experiments) between antibiotic-treated cells versus untreated cells. AA, activin A. Data are mean ± SEM. ∗ p

Techniques Used: Derivative Assay, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Flow Cytometry, Cytometry

Related Articles

RNA Extraction:

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RNA Sequencing Assay:

Article Title: Prospective Isolation of ISL1+ Cardiac Progenitors from Human ESCs for Myocardial Infarction Therapy
Article Snippet: For RNA sequencing, total RNA was extracted using the RNeasy Mini Kit (Qiagen, #74104) according to the manufacturer's protocol. .. For qRT-PCR assay, 2 μg of total RNA was reverse transcribed to cDNA using RevertAid First Strand cDNA Synthesis Kit (Fermentas, #K1621). qRT-PCR reactions used SYBRPremix Ex Taq II (Takara Bio, #RR081Q).

Synthesized:

Article Title: A transcriptome study on Macrobrachium nipponense hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)
Article Snippet: The first strand of cDNA was synthesized using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, China) according to the manufacturer’s instructions. .. Optimized qRT-PCR reactions were performed following the manufacturer’s instructions (SYBR Premix Ex Taq, Takara Bio, Japan.) on a real-time thermal cycler (Bio-Rad, USA), using β-actin as a reference gene.

Isolation:

Article Title: Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma
Article Snippet: Total RNA was isolated from melanoma and lymph node metastatic tissues using the RNeasy kit (Qiagen, Grand Island, NY) according to the manufacturer's instructions. .. GAPDH was used as an endogenous control for the qRT-PCR reactions (TaKaRa, Shiga, Japan).

Cell Culture:

Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression
Article Snippet: The total RNA was extracted from tissues or cultured cells with TRIzol reagent (Invitrogen, Grand Island, NY, USA), according to the manufacturer's protocol. .. After the RT reaction, 1 μ l of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) according to the manufacturer's instructions.

Article Title: Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression
Article Snippet: The total RNA was extracted from tissues or cultured cells with TRIzol reagent (Invitrogen), according to the manufacturer’s protocol. .. One microgram total RNA was reverse transcribed using PrimeScript RT Reagent Kit with gDNA Eraser (Takara). cDNA was used for subsequent qRT-PCR reactions (SYBR, TaKaRa) according to the manufacturer’s instructions.

Real-time Polymerase Chain Reaction:

Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression
Article Snippet: After the RT reaction, 1 μ l of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) according to the manufacturer's instructions. .. The results were normalized to the expression of GAPDH.

Article Title: Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression
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Article Title: Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma
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Article Title: A transcriptome study on Macrobrachium nipponense hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)
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Article Title: Long non-coding RNA ANRIL is upregulated in hepatocellular carcinoma and regulates cell proliferation by epigenetic silencing of KLF2
Article Snippet: After the RT reaction, 1 μL of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) following the manufacturer’s protocol. .. The results were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Article Title: Long non-coding RNA ANRIL is upregulated in hepatocellular carcinoma and regulates cell apoptosis by epigenetic silencing of KLF2
Article Snippet: After the RT reaction, 1 μL of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) following the manufacturer’s protocol. .. The results were normalized to the expression of GAPDH.

Polymerase Chain Reaction:

Article Title: Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma
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Random Hexamer Labeling:

Article Title: Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma
Article Snippet: Random hexamer primers were used in the RT reactions. .. GAPDH was used as an endogenous control for the qRT-PCR reactions (TaKaRa, Shiga, Japan).

Quantitative RT-PCR:

Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression
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Article Title: Long noncoding RNA AFAP1-AS1 predicts a poor prognosis and regulates non–small cell lung cancer cell proliferation by epigenetically repressing p21 expression
Article Snippet: The total RNA was extracted from tissues or cultured cells with TRIzol reagent (Invitrogen), according to the manufacturer’s protocol. .. One microgram total RNA was reverse transcribed using PrimeScript RT Reagent Kit with gDNA Eraser (Takara). cDNA was used for subsequent qRT-PCR reactions (SYBR, TaKaRa) according to the manufacturer’s instructions. .. The results were normalized to the expression of GAPDH.

Article Title: Long Noncoding RNA HOTAIR Is Associated with Motility, Invasion, and Metastatic Potential of Metastatic Melanoma
Article Snippet: Real-time qPCR was performed on a Bio-Rad CFX-96 real-time PCR system (Bio-Rad, Hercules, CA) using SYBR Premix DimerEraser kit (TaKaRa, Shiga, Japan). .. GAPDH was used as an endogenous control for the qRT-PCR reactions (TaKaRa, Shiga, Japan). .. The 2−ΔΔCt method was used to calculate the expression of each lncRNA.

Article Title: A transcriptome study on Macrobrachium nipponense hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)
Article Snippet: The first strand of cDNA was synthesized using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, China) according to the manufacturer’s instructions. .. Optimized qRT-PCR reactions were performed following the manufacturer’s instructions (SYBR Premix Ex Taq, Takara Bio, Japan.) on a real-time thermal cycler (Bio-Rad, USA), using β-actin as a reference gene. .. Primers for qRT-PCR were carefully designed using Primer Premier 3 software and listed in .

Article Title: Prospective Isolation of ISL1+ Cardiac Progenitors from Human ESCs for Myocardial Infarction Therapy
Article Snippet: For RNA sequencing, total RNA was extracted using the RNeasy Mini Kit (Qiagen, #74104) according to the manufacturer's protocol. .. For qRT-PCR assay, 2 μg of total RNA was reverse transcribed to cDNA using RevertAid First Strand cDNA Synthesis Kit (Fermentas, #K1621). qRT-PCR reactions used SYBRPremix Ex Taq II (Takara Bio, #RR081Q). .. Each data point represents three independent biological replicates.

Article Title: Long non-coding RNA ANRIL is upregulated in hepatocellular carcinoma and regulates cell proliferation by epigenetic silencing of KLF2
Article Snippet: One microgram total RNA was reverse transcribed in a final volume of 20 μL under standard conditions using PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Dalian, China; RR047A). .. After the RT reaction, 1 μL of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) following the manufacturer’s protocol. .. The results were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Article Title: Long non-coding RNA ANRIL is upregulated in hepatocellular carcinoma and regulates cell apoptosis by epigenetic silencing of KLF2
Article Snippet: One microgram total RNA was reverse transcribed in a final volume of 20 μL under standard conditions using PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Dalian, China; RR047A). .. After the RT reaction, 1 μL of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) following the manufacturer’s protocol. .. The results were normalized to the expression of GAPDH.

Expressing:

Article Title: A transcriptome study on Macrobrachium nipponense hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)
Article Snippet: Optimized qRT-PCR reactions were performed following the manufacturer’s instructions (SYBR Premix Ex Taq, Takara Bio, Japan.) on a real-time thermal cycler (Bio-Rad, USA), using β-actin as a reference gene. .. The qRT-PCR program conducted as showed in Zhao et al [ ].

Article Title: Prospective Isolation of ISL1+ Cardiac Progenitors from Human ESCs for Myocardial Infarction Therapy
Article Snippet: Paragraph title: Gene Expression Analysis ... For qRT-PCR assay, 2 μg of total RNA was reverse transcribed to cDNA using RevertAid First Strand cDNA Synthesis Kit (Fermentas, #K1621). qRT-PCR reactions used SYBRPremix Ex Taq II (Takara Bio, #RR081Q).

Sequencing:

Article Title: A transcriptome study on Macrobrachium nipponense hepatopancreas experimentally challenged with white spot syndrome virus (WSSV)
Article Snippet: The tissues used for qRT-PCR were same as the samples used in transcriptome sequencing. .. Optimized qRT-PCR reactions were performed following the manufacturer’s instructions (SYBR Premix Ex Taq, Takara Bio, Japan.) on a real-time thermal cycler (Bio-Rad, USA), using β-actin as a reference gene.

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    TaKaRa qrt pcr kits
    Hcy-induced elevation of EZH2 is responsible for CFTR promoter H3K27me3. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring EZH2 small interfering RNA (si-EZH2) respectively for 24 h. a The H3K27me1, 2, 3 levels on the CFTR promoter were examined in liver of mice by <t>ChIP-PCR.</t> b The levels of H3K27me3 on the CFTR promoter were examined by ChIP-PCR in hepatocytes treated with 100μmol/L Hcy and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. c The H3K27me3 levels in the CFTR promoter of hepatocytes treated with different concentration of agonist of H3K27me3 (1, 5, 10 μmol/L EPZ005687). d , e <t>qRT-PCR</t> and western blot were used to detect the mRNA and protein expression of CFTR in the hepatic cells treated with 10 μmol/L EPZ005687 and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. f , g The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in HL-7702 cells exposed to 100μmol/L Hcy for 24 h. ( H - I ) The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-EZH2. ( J ) The level of H3K27me3 on CFTR promoter were detected by ChIP-PCR in hepatic cells infected with adenovirus si-EZH2. ( K ) The mRNA and protein expression of CFTR were detected by western blot in hepatic cells infected with adenovirus si-EZH2. ( L ) The protein expression of autophagy related protein factors p62, BECN1, LC3 and Atg12 were detected by western blot in hepatocytes infected with adenovirus (Ad-CFTR, si-EZH2, Ad-CFTR and si-EZH2). Mean ± SD of three experiments performed in triplicate. * P
    Qrt Pcr Kits, supplied by TaKaRa, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Propagation of HCV 122KO in 751-122KO cells. (A) HCV was inoculated into Huh7-122KO (#2), Huh7-122KO-cured (#3 or #5), or 751-122KO (#1, #2 or #3) cells, and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined by <t>qRT-PCR</t> and focus formation assay, respectively, at 72 hpi. (B) Infectious titer in the culture medium on serial passage of each 751-122KO cell clone. (C) HCV and HCV 122KO were inoculated into 751-122KO and Huh7.5.1 cells and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined at 72 hpi. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P
    Lenti X Qrt Pcr Titration Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    IFNα-Mediated Inhibition of Knockdown following H1 Promoter-Driven Expression of shRNA (A and B) H1299 and A549 cells were transfected with pHMH1-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by <t>qRT-PCR.</t> All data are expressed as the means ± SD (n = 4). *p
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    Hcy-induced elevation of EZH2 is responsible for CFTR promoter H3K27me3. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring EZH2 small interfering RNA (si-EZH2) respectively for 24 h. a The H3K27me1, 2, 3 levels on the CFTR promoter were examined in liver of mice by ChIP-PCR. b The levels of H3K27me3 on the CFTR promoter were examined by ChIP-PCR in hepatocytes treated with 100μmol/L Hcy and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. c The H3K27me3 levels in the CFTR promoter of hepatocytes treated with different concentration of agonist of H3K27me3 (1, 5, 10 μmol/L EPZ005687). d , e qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in the hepatic cells treated with 10 μmol/L EPZ005687 and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. f , g The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in HL-7702 cells exposed to 100μmol/L Hcy for 24 h. ( H - I ) The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-EZH2. ( J ) The level of H3K27me3 on CFTR promoter were detected by ChIP-PCR in hepatic cells infected with adenovirus si-EZH2. ( K ) The mRNA and protein expression of CFTR were detected by western blot in hepatic cells infected with adenovirus si-EZH2. ( L ) The protein expression of autophagy related protein factors p62, BECN1, LC3 and Atg12 were detected by western blot in hepatocytes infected with adenovirus (Ad-CFTR, si-EZH2, Ad-CFTR and si-EZH2). Mean ± SD of three experiments performed in triplicate. * P

    Journal: Cell Death & Disease

    Article Title: Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver

    doi: 10.1038/s41419-017-0216-z

    Figure Lengend Snippet: Hcy-induced elevation of EZH2 is responsible for CFTR promoter H3K27me3. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring EZH2 small interfering RNA (si-EZH2) respectively for 24 h. a The H3K27me1, 2, 3 levels on the CFTR promoter were examined in liver of mice by ChIP-PCR. b The levels of H3K27me3 on the CFTR promoter were examined by ChIP-PCR in hepatocytes treated with 100μmol/L Hcy and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. c The H3K27me3 levels in the CFTR promoter of hepatocytes treated with different concentration of agonist of H3K27me3 (1, 5, 10 μmol/L EPZ005687). d , e qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in the hepatic cells treated with 10 μmol/L EPZ005687 and 100 μmol/L Hcy plus 10 μmol/L EPZ005687. f , g The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in HL-7702 cells exposed to 100μmol/L Hcy for 24 h. ( H - I ) The mRNA and protein expression of EZH2 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-EZH2. ( J ) The level of H3K27me3 on CFTR promoter were detected by ChIP-PCR in hepatic cells infected with adenovirus si-EZH2. ( K ) The mRNA and protein expression of CFTR were detected by western blot in hepatic cells infected with adenovirus si-EZH2. ( L ) The protein expression of autophagy related protein factors p62, BECN1, LC3 and Atg12 were detected by western blot in hepatocytes infected with adenovirus (Ad-CFTR, si-EZH2, Ad-CFTR and si-EZH2). Mean ± SD of three experiments performed in triplicate. * P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) for CFTR , DNMT1 and EZH2 genes was performed using an FTC3000 qRT-PCR detection system as described previously , . qRT-PCR kits were from Takara Biotechnology Co., Ltd. (Dalian, China).

    Techniques: Knock-Out, Mouse Assay, Small Interfering RNA, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Concentration Assay, Quantitative RT-PCR, Western Blot, Expressing, Infection

    Hcy induces hepatic autophagy in CBS +/- mice. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy (100 μmol/L) for 24 h. a The concentration of Hcy in plasma of mice was measured by automatic biochemical analyzer. b Contents of ALT and AST in serum of CBS +/- mice were analyzed using automatic biochemical analyzer. c Hematoxylin and eosin (H E) and Oil Red O staining of CBS +/- mice liver. d Transmission electron microscope (TEM) was used to analyse cell autophagy in liver tissues of CBS +/− mice. The arrows indicate the double-membrane vacuoles digesting organelles or cytosolic contents. e and f mRNA and protein expression of p62, BECN1, LC3 and Atg12 in the liver tissue of CBS +/− mice by qRT-PCR and western blot, respectively. g Hepatocytes were treated with different concentrations of Hcy (50–500 μmol/L) for 24 h before MTT assay. h The activities of AST and ALT in hepatocytes after treatment with Hcy were detected by ELISA. ( I ) Confocal fluorescent microscopy analysis of hepatocytes overexpressing mRFP-GFP-LC3, treated with 100 µmol/L Hcy for 24 h. Quantification of mean red and green fluorescent puncta of at least 10 cells per condition is shown. The efficiency of transfection was also shown. ( J ) and ( K ) mRNA and protein expression of p62, BECN1, LC3 and Atg12 in hepatic cells treated with 100 µmol/L L-Hcy by qRT-PCR and western blot. Densitometry analysis of the proteins was performed for each sample (mean ± s.d.). * P

    Journal: Cell Death & Disease

    Article Title: Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver

    doi: 10.1038/s41419-017-0216-z

    Figure Lengend Snippet: Hcy induces hepatic autophagy in CBS +/- mice. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy (100 μmol/L) for 24 h. a The concentration of Hcy in plasma of mice was measured by automatic biochemical analyzer. b Contents of ALT and AST in serum of CBS +/- mice were analyzed using automatic biochemical analyzer. c Hematoxylin and eosin (H E) and Oil Red O staining of CBS +/- mice liver. d Transmission electron microscope (TEM) was used to analyse cell autophagy in liver tissues of CBS +/− mice. The arrows indicate the double-membrane vacuoles digesting organelles or cytosolic contents. e and f mRNA and protein expression of p62, BECN1, LC3 and Atg12 in the liver tissue of CBS +/− mice by qRT-PCR and western blot, respectively. g Hepatocytes were treated with different concentrations of Hcy (50–500 μmol/L) for 24 h before MTT assay. h The activities of AST and ALT in hepatocytes after treatment with Hcy were detected by ELISA. ( I ) Confocal fluorescent microscopy analysis of hepatocytes overexpressing mRFP-GFP-LC3, treated with 100 µmol/L Hcy for 24 h. Quantification of mean red and green fluorescent puncta of at least 10 cells per condition is shown. The efficiency of transfection was also shown. ( J ) and ( K ) mRNA and protein expression of p62, BECN1, LC3 and Atg12 in hepatic cells treated with 100 µmol/L L-Hcy by qRT-PCR and western blot. Densitometry analysis of the proteins was performed for each sample (mean ± s.d.). * P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) for CFTR , DNMT1 and EZH2 genes was performed using an FTC3000 qRT-PCR detection system as described previously , . qRT-PCR kits were from Takara Biotechnology Co., Ltd. (Dalian, China).

    Techniques: Mouse Assay, Knock-Out, Concentration Assay, AST Assay, Staining, Transmission Assay, Microscopy, Transmission Electron Microscopy, Expressing, Quantitative RT-PCR, Western Blot, MTT Assay, Enzyme-linked Immunosorbent Assay, Transfection

    Hcy promotes the autophagy of hepatocytes by downregulation of cystic fibrosis trans membrane conductance regulator ( CFTR ). Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy, VX-770 and CFTR(inh)-172, respectively, for 24 h. a , b qRT-PCR and western blot detected the mRNA and protein expression of CFTR in the liver of CBS +/- mice. c , d qRT-PCR and western blot detected the mRNA and protein expression of CFTR in HL-7702 cells treated with 100 μmol/L Hcy. e , f Effect of CFTR activation on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with VX-770. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. g , h Effect of CFTR inhibition on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with CFTR(inh)-172. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. i , j, k An adenoviral vector carrying either a scrambled sequence or CFTR plasmid infected hepatocytes and protein expression of CFTR was detected by western blot, and the effect of CFTR overexpression and activation on the ratio of LC3-II/I and the expression of autophagy related proteins p62, BECN1 and Atg12 was examined in hepatocytes infected with Ad-CFTR. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. Results represent the means ± s.d. of independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver

    doi: 10.1038/s41419-017-0216-z

    Figure Lengend Snippet: Hcy promotes the autophagy of hepatocytes by downregulation of cystic fibrosis trans membrane conductance regulator ( CFTR ). Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy, VX-770 and CFTR(inh)-172, respectively, for 24 h. a , b qRT-PCR and western blot detected the mRNA and protein expression of CFTR in the liver of CBS +/- mice. c , d qRT-PCR and western blot detected the mRNA and protein expression of CFTR in HL-7702 cells treated with 100 μmol/L Hcy. e , f Effect of CFTR activation on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with VX-770. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. g , h Effect of CFTR inhibition on the expression of autophagy related proteins p62, BECN1, LC3-II/I and Atg12 in hepatocytes treated with CFTR(inh)-172. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. i , j, k An adenoviral vector carrying either a scrambled sequence or CFTR plasmid infected hepatocytes and protein expression of CFTR was detected by western blot, and the effect of CFTR overexpression and activation on the ratio of LC3-II/I and the expression of autophagy related proteins p62, BECN1 and Atg12 was examined in hepatocytes infected with Ad-CFTR. The mRNA and protein changes were detected by qRT-PCR and western blot respectively. Results represent the means ± s.d. of independent experiments. * P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) for CFTR , DNMT1 and EZH2 genes was performed using an FTC3000 qRT-PCR detection system as described previously , . qRT-PCR kits were from Takara Biotechnology Co., Ltd. (Dalian, China).

    Techniques: Knock-Out, Mouse Assay, Quantitative RT-PCR, Western Blot, Expressing, Activation Assay, Inhibition, Plasmid Preparation, Sequencing, Infection, Over Expression

    Hcy induces liver autophagy by downregulation of CFTR expression via DNA methylation. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring DNMT1 small interfering RNA (si-DNMT1) respectively for 24 h. a A schematic drawing of the putative CpG islands in the 5’-flank region of CFTR . (B, C) The total methylation rate of CpG rich region (−572 bp/−262 bp) within the proximal promoter of the CFTR gene was detected by bisulfite-sequencing PCR (BSP) method in liver of CBS +/- mice and ( d , E ) HL-7702 cells. Black and white circles represent methylated and unmethylated CpGs respectively. f qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in HL-7702 cells treated with Hcy (100 μmol/L) or Hcy (100 μmol/L) plus 5-azacytidine (AZC) (10 μmol/L). g , h The mRNA and protein expression of DNMTs in liver tissues of mice were detected by qRT-PCR and western blot. i The mRNA and protein expression of DNMT1 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-DNMT1. j Bisulfite sequencing detected the DNA methylation of CFTR in HL-7702 cells infected with the adenovirus si-DNMT1. k Western blot was performed to detect the protein expression of autophagy related proteins p62, BECN1, LC3 and Atg12 in hepatocytes infected with adenovirus CFTR and adenovirus si-DNMT1. Mean ± s.d. of three experiments performed in triplicate. * P

    Journal: Cell Death & Disease

    Article Title: Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver

    doi: 10.1038/s41419-017-0216-z

    Figure Lengend Snippet: Hcy induces liver autophagy by downregulation of CFTR expression via DNA methylation. Eight to 10 weeks old cystathionine b-synthase ( CBS ) heterozygous knockout mice ( CBS +/− ) were fed with regular mice chow and water ad libitum. Human hepatocytes HL-7702 were treated with L-Hcy and adenovirus harboring DNMT1 small interfering RNA (si-DNMT1) respectively for 24 h. a A schematic drawing of the putative CpG islands in the 5’-flank region of CFTR . (B, C) The total methylation rate of CpG rich region (−572 bp/−262 bp) within the proximal promoter of the CFTR gene was detected by bisulfite-sequencing PCR (BSP) method in liver of CBS +/- mice and ( d , E ) HL-7702 cells. Black and white circles represent methylated and unmethylated CpGs respectively. f qRT-PCR and western blot were used to detect the mRNA and protein expression of CFTR in HL-7702 cells treated with Hcy (100 μmol/L) or Hcy (100 μmol/L) plus 5-azacytidine (AZC) (10 μmol/L). g , h The mRNA and protein expression of DNMTs in liver tissues of mice were detected by qRT-PCR and western blot. i The mRNA and protein expression of DNMT1 were detected by qRT-PCR and western blot in hepatic cells infected with adenovirus si-DNMT1. j Bisulfite sequencing detected the DNA methylation of CFTR in HL-7702 cells infected with the adenovirus si-DNMT1. k Western blot was performed to detect the protein expression of autophagy related proteins p62, BECN1, LC3 and Atg12 in hepatocytes infected with adenovirus CFTR and adenovirus si-DNMT1. Mean ± s.d. of three experiments performed in triplicate. * P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) for CFTR , DNMT1 and EZH2 genes was performed using an FTC3000 qRT-PCR detection system as described previously , . qRT-PCR kits were from Takara Biotechnology Co., Ltd. (Dalian, China).

    Techniques: Expressing, DNA Methylation Assay, Knock-Out, Mouse Assay, Small Interfering RNA, Methylation, Methylation Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Infection

    CFTR alters the levels of liver injury biomarker and autophagy in CBS +/ − mice. CBS +/− mice injected with lentivirus Lv-CFTR or Lv-GFP through the tail vein (see Materials and Methods). a CFTR mRNA and protein levels were determined by qRT-PCR and western blot respectively. Data are presented as mean ± s.d. relative to Lv-GFP-injected mice. b Circulating levels of ALT and AST were analyzed. Results are means ± s.d. ( n = 6). * P

    Journal: Cell Death & Disease

    Article Title: Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver

    doi: 10.1038/s41419-017-0216-z

    Figure Lengend Snippet: CFTR alters the levels of liver injury biomarker and autophagy in CBS +/ − mice. CBS +/− mice injected with lentivirus Lv-CFTR or Lv-GFP through the tail vein (see Materials and Methods). a CFTR mRNA and protein levels were determined by qRT-PCR and western blot respectively. Data are presented as mean ± s.d. relative to Lv-GFP-injected mice. b Circulating levels of ALT and AST were analyzed. Results are means ± s.d. ( n = 6). * P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) for CFTR , DNMT1 and EZH2 genes was performed using an FTC3000 qRT-PCR detection system as described previously , . qRT-PCR kits were from Takara Biotechnology Co., Ltd. (Dalian, China).

    Techniques: Biomarker Assay, Mouse Assay, Injection, Quantitative RT-PCR, Western Blot, AST Assay

    The impact of TUG1 on tumorigenesis in vivo . ( a and b ) Scramble or shTUG1 was transfected into SPC-A1 cells, which were injected in the nude mice ( n =10), respectively. Tumor volumes were calculated after injection every 2 days. Bars indicate S.D. ( c ) Tumor weights are represented as means of tumor weights ±S.D. qRT-PCR was performed to detect the average expression of TUG1. ( d ) Histopathology of xenograft tumors. The tumor sections were under H E staining and IHC staining using antibodies against Ki-67. Bar, 100 μ m. Error bars indicate means±S.E.M. * P

    Journal: Cell Death & Disease

    Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

    doi: 10.1038/cddis.2014.201

    Figure Lengend Snippet: The impact of TUG1 on tumorigenesis in vivo . ( a and b ) Scramble or shTUG1 was transfected into SPC-A1 cells, which were injected in the nude mice ( n =10), respectively. Tumor volumes were calculated after injection every 2 days. Bars indicate S.D. ( c ) Tumor weights are represented as means of tumor weights ±S.D. qRT-PCR was performed to detect the average expression of TUG1. ( d ) Histopathology of xenograft tumors. The tumor sections were under H E staining and IHC staining using antibodies against Ki-67. Bar, 100 μ m. Error bars indicate means±S.E.M. * P

    Article Snippet: After the RT reaction, 1 μ l of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) according to the manufacturer's instructions.

    Techniques: In Vivo, Transfection, Injection, Mouse Assay, Quantitative RT-PCR, Expressing, Histopathology, Staining, Immunohistochemistry

    TUG1 could epigenetically regulate HOXB7 by binding to PRC2. ( a ) qRT-PCR was performed to detect the expression of HOX genes in transfected cells, and western blot assays were used to detect the level of HOXB7 after the transfection of si-TUG1. IHC assays were used to detect the HOXB7 in tumor sections from shTUG1-transfected cells. ( b ) RIP experiments were performed in SPC-A1 and the coprecipitated RNA was subjected to qRT-PCR for TUG1. HOTAIR was used as a positive control. The fold enrichment of TUG1 in EZH2 RIP is relative to its matching IgG control RIP. TUG1 nuclear localization, as identified using qRT-PCR in fractionated SPC-A1 and A549 cells. ( c ) ChIP of H3K27me3 and EZH2 of the promoter region of HOXB7 locus after siRNA treatment targeting si-NC or si-TUG1; qPCR were performed to determine the quantitation of ChIP assays. The levels of qPCR products are expressed as a percentage of the input DNA. Error bars indicate means±S.E.M. * P

    Journal: Cell Death & Disease

    Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

    doi: 10.1038/cddis.2014.201

    Figure Lengend Snippet: TUG1 could epigenetically regulate HOXB7 by binding to PRC2. ( a ) qRT-PCR was performed to detect the expression of HOX genes in transfected cells, and western blot assays were used to detect the level of HOXB7 after the transfection of si-TUG1. IHC assays were used to detect the HOXB7 in tumor sections from shTUG1-transfected cells. ( b ) RIP experiments were performed in SPC-A1 and the coprecipitated RNA was subjected to qRT-PCR for TUG1. HOTAIR was used as a positive control. The fold enrichment of TUG1 in EZH2 RIP is relative to its matching IgG control RIP. TUG1 nuclear localization, as identified using qRT-PCR in fractionated SPC-A1 and A549 cells. ( c ) ChIP of H3K27me3 and EZH2 of the promoter region of HOXB7 locus after siRNA treatment targeting si-NC or si-TUG1; qPCR were performed to determine the quantitation of ChIP assays. The levels of qPCR products are expressed as a percentage of the input DNA. Error bars indicate means±S.E.M. * P

    Article Snippet: After the RT reaction, 1 μ l of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) according to the manufacturer's instructions.

    Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Immunohistochemistry, Positive Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitation Assay

    p53 induces TUG1 through interacting with the promoter region of TUG1. ( a ) Analysis of TUG1 expression levels in NSCLC cell lines (A549, SPC-A1, SK-MES-1, NCI-H1299 and NCI-H1650) compared with the normal bronchial epithelial cell line (16HBE) by qRT-PCR. ( b ) Description of p53RE and mutant p53RE in promoter region of TUG1. The position of ChIP primers ( e ) was indicated by arrows. ( c ) Western blotting was used to detect the p53 induction by doxo. qRT-PCR was used to detect the effect of doxo on TUG1 expression in p53-WT and p53-null cells. ( d ) Induction of TUG1 by ectopically expressed p53 (wild-type p53 or mutant p53). Overexpression was confirmed by western blotting. ( e ) Induction of TUG1 promoter activity by p53, but not mutant p53 in NCI-H1299 cell lines. ( f ) Deletion and mutation analysis of the promoter activity to determine the role of the p53RE in p53-mediated regulation of TUG1. ( g ) The p53 binding at the promoter regions of TUG1 was assessed by ChIP analysis. ChIP primers were detailed in Materials and Methods section. Shown are representative images of three independent experiments. Error bars indicate means±S.E.M. * P

    Journal: Cell Death & Disease

    Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

    doi: 10.1038/cddis.2014.201

    Figure Lengend Snippet: p53 induces TUG1 through interacting with the promoter region of TUG1. ( a ) Analysis of TUG1 expression levels in NSCLC cell lines (A549, SPC-A1, SK-MES-1, NCI-H1299 and NCI-H1650) compared with the normal bronchial epithelial cell line (16HBE) by qRT-PCR. ( b ) Description of p53RE and mutant p53RE in promoter region of TUG1. The position of ChIP primers ( e ) was indicated by arrows. ( c ) Western blotting was used to detect the p53 induction by doxo. qRT-PCR was used to detect the effect of doxo on TUG1 expression in p53-WT and p53-null cells. ( d ) Induction of TUG1 by ectopically expressed p53 (wild-type p53 or mutant p53). Overexpression was confirmed by western blotting. ( e ) Induction of TUG1 promoter activity by p53, but not mutant p53 in NCI-H1299 cell lines. ( f ) Deletion and mutation analysis of the promoter activity to determine the role of the p53RE in p53-mediated regulation of TUG1. ( g ) The p53 binding at the promoter regions of TUG1 was assessed by ChIP analysis. ChIP primers were detailed in Materials and Methods section. Shown are representative images of three independent experiments. Error bars indicate means±S.E.M. * P

    Article Snippet: After the RT reaction, 1 μ l of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) according to the manufacturer's instructions.

    Techniques: Expressing, Quantitative RT-PCR, Mutagenesis, Chromatin Immunoprecipitation, Western Blot, Over Expression, Activity Assay, Binding Assay

    Analysis of TUG1 expression in NSCLC tissues and clinical parameters. ( a ) TUG1 was detected in 192 pairs of NSCLC tissues by qRT-PCR. The levels of TUG1 in NSCLC tissues are significantly lower than those in non-tumorous tissues. The ΔCt value was determined by subtracting the GAPDH Ct value from the TUG1 Ct value (relative to a single reference value). Smaller ΔCt value indicates higher expression. ( b and c ) Data are presented as relative expression level in tumor tissues (shown as ΔCt). TUG1 expression was significantly lower in patients with a higher pathological stage and big tumor size. ( d ) Patients with low levels of TUG1 expression showed reduced survival times compared with patients with high levels of TUG1 expression ( P

    Journal: Cell Death & Disease

    Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

    doi: 10.1038/cddis.2014.201

    Figure Lengend Snippet: Analysis of TUG1 expression in NSCLC tissues and clinical parameters. ( a ) TUG1 was detected in 192 pairs of NSCLC tissues by qRT-PCR. The levels of TUG1 in NSCLC tissues are significantly lower than those in non-tumorous tissues. The ΔCt value was determined by subtracting the GAPDH Ct value from the TUG1 Ct value (relative to a single reference value). Smaller ΔCt value indicates higher expression. ( b and c ) Data are presented as relative expression level in tumor tissues (shown as ΔCt). TUG1 expression was significantly lower in patients with a higher pathological stage and big tumor size. ( d ) Patients with low levels of TUG1 expression showed reduced survival times compared with patients with high levels of TUG1 expression ( P

    Article Snippet: After the RT reaction, 1 μ l of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) according to the manufacturer's instructions.

    Techniques: Expressing, Quantitative RT-PCR

    TUG1 could participate in AKT and MAPK pathway through modulating HOXB7. ( A ) MTT analysis of cell proliferation by co-transfection (si-NC, si-HOXB7, si-HOXB7+si-TUG1). At 48 h after transfection of si-HOXB7, the cell cycle and apoptosis were analyzed by flow cytometry. LR, early apoptotic cells. UR, terminal apoptotic cells. ( B ) qRT-PCR were performed to detect the expression of HOXB7 after overexpression of p53 and transfected with p53 followed by transfection with si-TUG1. ( C ) Western blotting analysis of the expression of p-ERK, total ERK, p-AKT, total AKT, p-GSK-3 β , total GSK-3 β proteins in indicated si-NC-transfected, si-HOXB7 and si-TUG1-transfected SPC-A1 cell lines. ( D ) Immunostaining of HOXB7 was negatively or very weakly positive in corresponding non-tumor lung tissues (a and b), but was strongly positive in squamous cell carcinoma tissues (c and d) and lung adenocarcinoma (e and f). Bar, 100 μ m. ( E ) The immunoreactivity of HOXB7 protein in NSCLC tissues showed a statistically significant inverse correlation with the relative level of TUG1 expression. Error bars indicate means±S.E.M. * P

    Journal: Cell Death & Disease

    Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

    doi: 10.1038/cddis.2014.201

    Figure Lengend Snippet: TUG1 could participate in AKT and MAPK pathway through modulating HOXB7. ( A ) MTT analysis of cell proliferation by co-transfection (si-NC, si-HOXB7, si-HOXB7+si-TUG1). At 48 h after transfection of si-HOXB7, the cell cycle and apoptosis were analyzed by flow cytometry. LR, early apoptotic cells. UR, terminal apoptotic cells. ( B ) qRT-PCR were performed to detect the expression of HOXB7 after overexpression of p53 and transfected with p53 followed by transfection with si-TUG1. ( C ) Western blotting analysis of the expression of p-ERK, total ERK, p-AKT, total AKT, p-GSK-3 β , total GSK-3 β proteins in indicated si-NC-transfected, si-HOXB7 and si-TUG1-transfected SPC-A1 cell lines. ( D ) Immunostaining of HOXB7 was negatively or very weakly positive in corresponding non-tumor lung tissues (a and b), but was strongly positive in squamous cell carcinoma tissues (c and d) and lung adenocarcinoma (e and f). Bar, 100 μ m. ( E ) The immunoreactivity of HOXB7 protein in NSCLC tissues showed a statistically significant inverse correlation with the relative level of TUG1 expression. Error bars indicate means±S.E.M. * P

    Article Snippet: After the RT reaction, 1 μ l of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) according to the manufacturer's instructions.

    Techniques: MTT Assay, Cotransfection, Transfection, Flow Cytometry, Cytometry, Quantitative RT-PCR, Expressing, Over Expression, Western Blot, Immunostaining

    Propagation of HCV 122KO in 751-122KO cells. (A) HCV was inoculated into Huh7-122KO (#2), Huh7-122KO-cured (#3 or #5), or 751-122KO (#1, #2 or #3) cells, and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined by qRT-PCR and focus formation assay, respectively, at 72 hpi. (B) Infectious titer in the culture medium on serial passage of each 751-122KO cell clone. (C) HCV and HCV 122KO were inoculated into 751-122KO and Huh7.5.1 cells and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined at 72 hpi. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Journal: PLoS Pathogens

    Article Title: Characterization of miR-122-independent propagation of HCV

    doi: 10.1371/journal.ppat.1006374

    Figure Lengend Snippet: Propagation of HCV 122KO in 751-122KO cells. (A) HCV was inoculated into Huh7-122KO (#2), Huh7-122KO-cured (#3 or #5), or 751-122KO (#1, #2 or #3) cells, and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined by qRT-PCR and focus formation assay, respectively, at 72 hpi. (B) Infectious titer in the culture medium on serial passage of each 751-122KO cell clone. (C) HCV and HCV 122KO were inoculated into 751-122KO and Huh7.5.1 cells and the levels of intracellular HCV-RNA replication (top) and infectious titers in the culture supernatants (bottom) were determined at 72 hpi. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Article Snippet: The infectious titer of lentivirus was determined by a Lenti-X qRT-PCR Titration Kit (Clontech, Mountain View, CA).

    Techniques: Quantitative RT-PCR, Tube Formation Assay, Standard Deviation

    G28A mutants can replicate efficiently in an Ago2-independent manner. (A) Intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) of Huh7-122KO and Huh7-122KOR cells infected with either HCV or HCV 122KO in the presence of either control-LNA or LNA-miR-122 were determined at 72 hpi. (B) Ago2 complexes in 751-122KO and Huh7.5.1 cells infected with HCV were immunoprecipitated by either anti-IgG or anti-Ago2 mouse antibody at 12 dpi. Levels of Ago2 and HCV-RNA in the precipitates were determined by immunoblotting and qRT-PCR, respectively. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Journal: PLoS Pathogens

    Article Title: Characterization of miR-122-independent propagation of HCV

    doi: 10.1371/journal.ppat.1006374

    Figure Lengend Snippet: G28A mutants can replicate efficiently in an Ago2-independent manner. (A) Intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) of Huh7-122KO and Huh7-122KOR cells infected with either HCV or HCV 122KO in the presence of either control-LNA or LNA-miR-122 were determined at 72 hpi. (B) Ago2 complexes in 751-122KO and Huh7.5.1 cells infected with HCV were immunoprecipitated by either anti-IgG or anti-Ago2 mouse antibody at 12 dpi. Levels of Ago2 and HCV-RNA in the precipitates were determined by immunoblotting and qRT-PCR, respectively. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Article Snippet: The infectious titer of lentivirus was determined by a Lenti-X qRT-PCR Titration Kit (Clontech, Mountain View, CA).

    Techniques: Infection, Immunoprecipitation, Quantitative RT-PCR, Standard Deviation

    Establishment of replicon cells derived from Huh7-122KO cells. (A) Subgenomic HCV replicon RNA was electroporated into Huh7-122KO and Huh7-122KOR cells, or into Huh7-122KO cells together with control- or miR-122-mimic, and G418-resistant colonies were stained with crystal violet at 21 days post-transduction. (B) Expression of miR-122 in Huh7-122KO cells electroporated with either control-mimic or miR-122-mimic at 72 h post-electroporation. Relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control. (C) Each of the three clones derived from each type of replicon cells was subjected to qRT-PCR after extraction of total RNA (top) and to immunoblotting by using anti-NS5A and β-actin (middle). The relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control (bottom). (D) Intracellular HCV-RNA replication in Huh7-122KO-SGR cells (#1, #3, #5) and Huh7-122KOR-SGR cells (#1, #5, #6) in the presence of 20 nM of either LNA-control or LNA-miR122 was determined by qRT-PCR. (E) The Ago2 complex was immunoprecipitated from Huh7-122KO-SGR#1 and Huh7-122KOR-SGR#1 cells by using either anti-IgG or anti-Ago2 mouse antibody. The HCV-RNA associated with Ago2 was determined by qRT-PCR and Ago2 was detected by immunoblotting. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Journal: PLoS Pathogens

    Article Title: Characterization of miR-122-independent propagation of HCV

    doi: 10.1371/journal.ppat.1006374

    Figure Lengend Snippet: Establishment of replicon cells derived from Huh7-122KO cells. (A) Subgenomic HCV replicon RNA was electroporated into Huh7-122KO and Huh7-122KOR cells, or into Huh7-122KO cells together with control- or miR-122-mimic, and G418-resistant colonies were stained with crystal violet at 21 days post-transduction. (B) Expression of miR-122 in Huh7-122KO cells electroporated with either control-mimic or miR-122-mimic at 72 h post-electroporation. Relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control. (C) Each of the three clones derived from each type of replicon cells was subjected to qRT-PCR after extraction of total RNA (top) and to immunoblotting by using anti-NS5A and β-actin (middle). The relative expression of miR-122 was determined by qRT-PCR by using U6 snRNA as an internal control (bottom). (D) Intracellular HCV-RNA replication in Huh7-122KO-SGR cells (#1, #3, #5) and Huh7-122KOR-SGR cells (#1, #5, #6) in the presence of 20 nM of either LNA-control or LNA-miR122 was determined by qRT-PCR. (E) The Ago2 complex was immunoprecipitated from Huh7-122KO-SGR#1 and Huh7-122KOR-SGR#1 cells by using either anti-IgG or anti-Ago2 mouse antibody. The HCV-RNA associated with Ago2 was determined by qRT-PCR and Ago2 was detected by immunoblotting. Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Article Snippet: The infectious titer of lentivirus was determined by a Lenti-X qRT-PCR Titration Kit (Clontech, Mountain View, CA).

    Techniques: Derivative Assay, Staining, Transduction, Expressing, Electroporation, Quantitative RT-PCR, Clone Assay, Immunoprecipitation, Standard Deviation

    miR-122-independent HCV replication in Huh7-122KO cells. (A) HCV was inoculated into Huh7-122KO and Huh7-122KOR cells at an MOI of 3, and intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) were determined by qRT-PCR and focus formation assay, respectively. (B) HCV-RNA replication was inhibited by the treatment with IFNα, BILN, BMS790052, PSI7977 and anti-CD81 antibody. (C) HCV replication in Huh7-122KO cells was resistant to treatment with an miR-122 inhibitor, LNA (HCV-RNA replication: left; infectious titer: right). Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Journal: PLoS Pathogens

    Article Title: Characterization of miR-122-independent propagation of HCV

    doi: 10.1371/journal.ppat.1006374

    Figure Lengend Snippet: miR-122-independent HCV replication in Huh7-122KO cells. (A) HCV was inoculated into Huh7-122KO and Huh7-122KOR cells at an MOI of 3, and intracellular HCV-RNA levels (left panel) and infectious titers in the culture supernatants (right panel) were determined by qRT-PCR and focus formation assay, respectively. (B) HCV-RNA replication was inhibited by the treatment with IFNα, BILN, BMS790052, PSI7977 and anti-CD81 antibody. (C) HCV replication in Huh7-122KO cells was resistant to treatment with an miR-122 inhibitor, LNA (HCV-RNA replication: left; infectious titer: right). Error bars indicate the standard deviation of the mean and asterisks indicate significant differences (*P

    Article Snippet: The infectious titer of lentivirus was determined by a Lenti-X qRT-PCR Titration Kit (Clontech, Mountain View, CA).

    Techniques: Quantitative RT-PCR, Tube Formation Assay, Standard Deviation

    IFNα-Mediated Inhibition of Knockdown following H1 Promoter-Driven Expression of shRNA (A and B) H1299 and A549 cells were transfected with pHMH1-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by qRT-PCR. All data are expressed as the means ± SD (n = 4). *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: IFNα-Mediated Inhibition of Knockdown following H1 Promoter-Driven Expression of shRNA (A and B) H1299 and A549 cells were transfected with pHMH1-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by qRT-PCR. All data are expressed as the means ± SD (n = 4). *p

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: Inhibition, Expressing, shRNA, Transfection, Recombinant, Incubation, Quantitative RT-PCR

    Knockdown Efficiencies of Chemically Synthetic siRNAs following IFN-α Stimulation (A and B) A549 cells were transfected with siControl, sip53 (A), or simyc (B), followed by treatment with recombinant IFNα at 10 4 U/mL. After 48-hr incubation, c- myc and p53 mRNA levels in the cells were determined by qRT-PCR. All data are expressed as the means ± SD (n = 4).

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Knockdown Efficiencies of Chemically Synthetic siRNAs following IFN-α Stimulation (A and B) A549 cells were transfected with siControl, sip53 (A), or simyc (B), followed by treatment with recombinant IFNα at 10 4 U/mL. After 48-hr incubation, c- myc and p53 mRNA levels in the cells were determined by qRT-PCR. All data are expressed as the means ± SD (n = 4).

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: Transfection, Recombinant, Incubation, Quantitative RT-PCR

    Inhibition of shRNA-Mediated Knockdown by IFN-α Stimulation (A and B) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA (A) and protein (B) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. (C) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 2 , 10 3 , or 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were similarly determined. (D and E) A549 cells were transfected with pHMU6-shLuc, -shmyc (D), or -shp53 (E), followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc (D) and p53 (E) mRNA levels in the cells were similarly determined. These experiments were repeated at least three times, and representative data are shown. All data are expressed as the means ± SD (n = 4). *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Inhibition of shRNA-Mediated Knockdown by IFN-α Stimulation (A and B) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc mRNA (A) and protein (B) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. (C) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-α at 10 2 , 10 3 , or 10 4 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were similarly determined. (D and E) A549 cells were transfected with pHMU6-shLuc, -shmyc (D), or -shp53 (E), followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, c- myc (D) and p53 (E) mRNA levels in the cells were similarly determined. These experiments were repeated at least three times, and representative data are shown. All data are expressed as the means ± SD (n = 4). *p

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: Inhibition, shRNA, Transfection, Recombinant, Incubation, Quantitative RT-PCR, Western Blot

    Dicer and Ago2 Expression Levels following IFN-α Stimulation H1299 and A549 cells were treated with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, dicer and Ago2 mRNA (A) and protein (B) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. (C) TRBP protein levels in the cells were determined by western blotting analysis. The qRT-PCR data are expressed as the means ± SD (n = 4).

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Dicer and Ago2 Expression Levels following IFN-α Stimulation H1299 and A549 cells were treated with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, dicer and Ago2 mRNA (A) and protein (B) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. (C) TRBP protein levels in the cells were determined by western blotting analysis. The qRT-PCR data are expressed as the means ± SD (n = 4).

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: Expressing, Recombinant, Incubation, Quantitative RT-PCR, Western Blot

    Inhibition of Ad Vector-Expressing shRNA-Mediated Knockdown by IFN-α Stimulation (A) 4 × 10 8 IFU Ad-shLuc was intravenously administered to C57BL/6 mice. 3 hr after the administration, IFN-β mRNA levels in the spleen were determined by qRT-PCR. Data are expressed as the means ± SE (n = 3–4). (B–D) A549 cells were transduced with Ad-shp53, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, the copy numbers of sip53 in the cells were determined by qRT-PCR (B). After 48-hr incubation, p53 mRNA (C) and protein (D) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. Data are expressed as the means ± SD (n = 4). **p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Inhibition of Ad Vector-Expressing shRNA-Mediated Knockdown by IFN-α Stimulation (A) 4 × 10 8 IFU Ad-shLuc was intravenously administered to C57BL/6 mice. 3 hr after the administration, IFN-β mRNA levels in the spleen were determined by qRT-PCR. Data are expressed as the means ± SE (n = 3–4). (B–D) A549 cells were transduced with Ad-shp53, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, the copy numbers of sip53 in the cells were determined by qRT-PCR (B). After 48-hr incubation, p53 mRNA (C) and protein (D) levels in the cells were determined by qRT-PCR and western blotting analysis, respectively. Data are expressed as the means ± SD (n = 4). **p

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: Inhibition, Plasmid Preparation, Expressing, shRNA, Mouse Assay, Quantitative RT-PCR, Transduction, Recombinant, Incubation, Western Blot

    Efficiencies of shRNA-Mediated Knockdown following Exposure to IFN-β, IFN-γ, and TNF-α (A) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-β at 10 2 or 10 3 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by qRT-PCR. (B) H1299 cells were similarly transfected, followed by treatment with recombinant IFN-γ and TNF-α at 5 × 10 3 U/mL and 100 ng/mL, respectively. After 48-hr incubation, c- myc mRNA levels in the cells were similarly determined. All data are expressed as the means ± SD (n = 4). *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Efficiencies of shRNA-Mediated Knockdown following Exposure to IFN-β, IFN-γ, and TNF-α (A) H1299 cells were transfected with pHMU6-shLuc or -shmyc, followed by treatment with recombinant IFN-β at 10 2 or 10 3 U/mL. After 48-hr incubation, c- myc mRNA levels in the cells were determined by qRT-PCR. (B) H1299 cells were similarly transfected, followed by treatment with recombinant IFN-γ and TNF-α at 5 × 10 3 U/mL and 100 ng/mL, respectively. After 48-hr incubation, c- myc mRNA levels in the cells were similarly determined. All data are expressed as the means ± SD (n = 4). *p

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: shRNA, Transfection, Recombinant, Incubation, Quantitative RT-PCR

    Type I IFN Responses Following Introduction of shRNA-Expressing Vectors (A–D) A549 (A and C) and H1299 (B and D) cells were transfected with pHMU6-shLuc (A and B) or siControl (indicated as siRNA) (C and D). After 24-hr incubation, IFNβ, ISG54, and ISG56 mRNA levels in the cells were determined by qRT-PCR. The data are expressed as the means ± SD (n = 3). (E) 100 μg pHMU6-shLuc was intramuscularly administered to C57BL/6 mice. 3 hr after administration, the IFN-β mRNA levels in the mouse muscle were determined by qRT-PCR. Data are expressed as the means ± SE (n = 3–4). *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Type I IFN Responses Following Introduction of shRNA-Expressing Vectors (A–D) A549 (A and C) and H1299 (B and D) cells were transfected with pHMU6-shLuc (A and B) or siControl (indicated as siRNA) (C and D). After 24-hr incubation, IFNβ, ISG54, and ISG56 mRNA levels in the cells were determined by qRT-PCR. The data are expressed as the means ± SD (n = 3). (E) 100 μg pHMU6-shLuc was intramuscularly administered to C57BL/6 mice. 3 hr after administration, the IFN-β mRNA levels in the mouse muscle were determined by qRT-PCR. Data are expressed as the means ± SE (n = 3–4). *p

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: shRNA, Expressing, Transfection, Incubation, Quantitative RT-PCR, Mouse Assay

    Processing of shRNA to siRNA in IFN-α-Treated Cells (A and B) H1299 cells were transfected with pHMU6-shp53 or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, shRNA and siRNA copy numbers in the cells were determined by northern blotting analysis (A) and qRT-PCR (B). Data are expressed as the means ± SD (n = 4). *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Type I Interferons Impede Short Hairpin RNA-Mediated RNAi via Inhibition of Dicer-Mediated Processing to Small Interfering RNA

    doi: 10.1016/j.omtn.2016.12.007

    Figure Lengend Snippet: Processing of shRNA to siRNA in IFN-α-Treated Cells (A and B) H1299 cells were transfected with pHMU6-shp53 or -shmyc, followed by treatment with recombinant IFN-α at 10 4 U/mL. After 48-hr incubation, shRNA and siRNA copy numbers in the cells were determined by northern blotting analysis (A) and qRT-PCR (B). Data are expressed as the means ± SD (n = 4). *p

    Article Snippet: Sequences of the primers used in this study are described in . siRNA copy numbers were determined by qRT-PCR using Mir-X miRNA First-Strand Synthesis and SYBR qRT-PCR (Clontech-Takara) and primers specific for the sip53 and simyc.

    Techniques: shRNA, Transfection, Recombinant, Incubation, Northern Blot, Quantitative RT-PCR