quantitative rt pcr analysis total rna  (Thermo Fisher)


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    Thermo Fisher quantitative rt pcr analysis total rna
    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger <t>RNA</t> (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time <t>(qRT)‐PCR.</t> ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P
    Quantitative Rt Pcr Analysis Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB"

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

    Journal: Thoracic Cancer

    doi: 10.1111/1759-7714.12895

    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P
    Figure Legend Snippet: Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

    Techniques Used: Expressing, Migration, In Vitro, Quantitative RT-PCR, Western Blot

    Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.
    Figure Legend Snippet: Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

    Techniques Used: Migration, In Vitro, Real-time Polymerase Chain Reaction, Expressing, shRNA, Western Blot

    2) Product Images from "Tonoplast- and Plasma Membrane-Localized Aquaporin-Family Transporters in Blue Hydrangea Sepals of Aluminum Hyperaccumulating Plant"

    Article Title: Tonoplast- and Plasma Membrane-Localized Aquaporin-Family Transporters in Blue Hydrangea Sepals of Aluminum Hyperaccumulating Plant

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043189

    Expression of HmVALT and HmPALT 1 and accumulation of their gene products in hydrangea. ( A ) The amount of HmVALT and HmPALT1 mRNA was determined by quantitative RT-PCR. mRNAs were prepared from sepal at each stage and other tissues. The data are relative to the expression of 18S ribosomal RNA and were further normalized to the level of sepal at stage 1 mRNA, which was expressed as 1.0. The error bars represent SD (n = 3). ( B ) Immunoblot analyses of HmVALT and HmPALT1 in the sepals. Hydrophobic protein fractions were extracted from sepal tissues at each stage and subjected to SDS–PAGE (20 µg of protein).
    Figure Legend Snippet: Expression of HmVALT and HmPALT 1 and accumulation of their gene products in hydrangea. ( A ) The amount of HmVALT and HmPALT1 mRNA was determined by quantitative RT-PCR. mRNAs were prepared from sepal at each stage and other tissues. The data are relative to the expression of 18S ribosomal RNA and were further normalized to the level of sepal at stage 1 mRNA, which was expressed as 1.0. The error bars represent SD (n = 3). ( B ) Immunoblot analyses of HmVALT and HmPALT1 in the sepals. Hydrophobic protein fractions were extracted from sepal tissues at each stage and subjected to SDS–PAGE (20 µg of protein).

    Techniques Used: Expressing, Quantitative RT-PCR, SDS Page

    3) Product Images from "MiR-184 regulates insulin secretion through repression of Slc25a22"

    Article Title: MiR-184 regulates insulin secretion through repression of Slc25a22

    Journal: PeerJ

    doi: 10.7717/peerj.162

    SiRNAs for Slc25a22 inhibits glucose-induced insulin secretion in the MIN6 islet β-cell line. SiRNA for Slc25a22 (Slc25a22_2, Slc25a22_4) or control siRNA was transfected into the MIN6 islet β-cell line. (A) Expression of Slc25a22 mRNA. Quantitative RT-PCR analyses of the expression levels of Slc25a22 mRNA were performed 48 h after transfection of MIN6 cells with Slc25a22 (Slc25a22_2, Slc25a22_4) or control siRNA. The 18S ribosomal RNA was used to normalize the expression levels. Data show the mean + SD for n = 3 repeats. ** p
    Figure Legend Snippet: SiRNAs for Slc25a22 inhibits glucose-induced insulin secretion in the MIN6 islet β-cell line. SiRNA for Slc25a22 (Slc25a22_2, Slc25a22_4) or control siRNA was transfected into the MIN6 islet β-cell line. (A) Expression of Slc25a22 mRNA. Quantitative RT-PCR analyses of the expression levels of Slc25a22 mRNA were performed 48 h after transfection of MIN6 cells with Slc25a22 (Slc25a22_2, Slc25a22_4) or control siRNA. The 18S ribosomal RNA was used to normalize the expression levels. Data show the mean + SD for n = 3 repeats. ** p

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR

    The effect of MiR-184 on endogenous Slc25a22. (A) Expression of Slc25a22 mRNA. Quantitative RT-PCR analyses of the expression levels of Slc25a22 mRNA were performed 48 h after transfection of MIN6 cells with miR-184 or control miRNA. The 18S ribosomal RNA was used to normalize the expression levels. Data show the mean + SD for n = 3 repeats. ** p
    Figure Legend Snippet: The effect of MiR-184 on endogenous Slc25a22. (A) Expression of Slc25a22 mRNA. Quantitative RT-PCR analyses of the expression levels of Slc25a22 mRNA were performed 48 h after transfection of MIN6 cells with miR-184 or control miRNA. The 18S ribosomal RNA was used to normalize the expression levels. Data show the mean + SD for n = 3 repeats. ** p

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection

    4) Product Images from "Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase"

    Article Title: Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-12-154

    Effect of treatment with anti-neutrophil antibody on lung inflammation and tumor promotion. (A) Total and lineage-specific leukocyte number in BALF of CC-LR mice treated or non-treated with mLy-6G Ab at the age of 14 weeks (mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR plus mLy-6G). (B) Total and lineage-specific leukocyte number in BALF of NTHi-exposed CC-LR mice treated or non-treated with mLy-6G Ab collected 1 day after last NTHi aerosol exposure at the age of 14 weeks (mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR plus mLy-6G with NTHi exposure). (C) Real-time Q-PCR expression analysis of arginase 1 on the RNA extracted from whole lung tissue (normalized to GAPDH expression level, mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR with NTHi exposure; # = P ≤ 0.05 for CC-LR with NTHi exposure vs CC-LR with NTHi exposure plus mLy-6G treatment). (D) Western blot analysis of arginase 1 on the protein extracted from whole lung tissue. (E) Lung surface tumor number after mLy-6G Ab treatment in NTHi exposed or non-exposed 14-week-old CC-LR mice. (mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR plus mLy-6G treatment; # = P ≤ 0.05 for CC-LR with NTHi exposure vs CC-LR with NTHi exposure plus mLy-6G treatment). (F) Histopathological appearance of lung tissue after treatment with mLy-6G Ab in NTHi exposed or non-exposed CC-LR mice. (4× magnification, scale bar = 50 mm, applicable to all panels).
    Figure Legend Snippet: Effect of treatment with anti-neutrophil antibody on lung inflammation and tumor promotion. (A) Total and lineage-specific leukocyte number in BALF of CC-LR mice treated or non-treated with mLy-6G Ab at the age of 14 weeks (mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR plus mLy-6G). (B) Total and lineage-specific leukocyte number in BALF of NTHi-exposed CC-LR mice treated or non-treated with mLy-6G Ab collected 1 day after last NTHi aerosol exposure at the age of 14 weeks (mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR plus mLy-6G with NTHi exposure). (C) Real-time Q-PCR expression analysis of arginase 1 on the RNA extracted from whole lung tissue (normalized to GAPDH expression level, mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR with NTHi exposure; # = P ≤ 0.05 for CC-LR with NTHi exposure vs CC-LR with NTHi exposure plus mLy-6G treatment). (D) Western blot analysis of arginase 1 on the protein extracted from whole lung tissue. (E) Lung surface tumor number after mLy-6G Ab treatment in NTHi exposed or non-exposed 14-week-old CC-LR mice. (mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR plus mLy-6G treatment; # = P ≤ 0.05 for CC-LR with NTHi exposure vs CC-LR with NTHi exposure plus mLy-6G treatment). (F) Histopathological appearance of lung tissue after treatment with mLy-6G Ab in NTHi exposed or non-exposed CC-LR mice. (4× magnification, scale bar = 50 mm, applicable to all panels).

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Expressing, Western Blot

    Effect of treatment with a selective CXCR2 inhibitor on lung inflammation and tumor promotion. (A) Total and lineage-specific leukocyte number in BALF of CC-LR mice treated or non-treated with SBZ (selective CXCR2 inhibitor) at the age of 14 weeks (mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR plus SBZ). (B) Total and lineage-specific leukocyte number in BALF of NTHi-exposed CC-LR mice treated or non-treated with SBZ collected 1 day after last NTHi aerosol exposure at the age of 14 weeks. (C) Real-time Q-PCR analysis of RNA extracted from whole lung tissue for relative mRNA expression of arginase 1 (normalized to GAPDH expression level, mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR plus SBZ treatment in figure D, # = P ≤ 0.05 for CC-LR with NTHi exposure versus CC-LR with NTHi exposure plus SBZ treatment). (D) Western blot analysis of arginase 1 on the protein extracted from whole lung tissue. (E) Lung surface tumor number after SBZ treatment in NTHi exposed or unexposed CC-LR mice. (mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR plus SBZ treatment, # = P ≤ 0.05 for CC-LR with NTHi exposure vs CC-LR with NTHi exposure plus SBZ treatment). (F) Histopathological appearance of lung tissue after treatment with SBZ in NTHi exposed or unexposed CC-LR mice (4× magnification, scale bar = 50 mm, applicable to all panels).
    Figure Legend Snippet: Effect of treatment with a selective CXCR2 inhibitor on lung inflammation and tumor promotion. (A) Total and lineage-specific leukocyte number in BALF of CC-LR mice treated or non-treated with SBZ (selective CXCR2 inhibitor) at the age of 14 weeks (mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR plus SBZ). (B) Total and lineage-specific leukocyte number in BALF of NTHi-exposed CC-LR mice treated or non-treated with SBZ collected 1 day after last NTHi aerosol exposure at the age of 14 weeks. (C) Real-time Q-PCR analysis of RNA extracted from whole lung tissue for relative mRNA expression of arginase 1 (normalized to GAPDH expression level, mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR plus SBZ treatment in figure D, # = P ≤ 0.05 for CC-LR with NTHi exposure versus CC-LR with NTHi exposure plus SBZ treatment). (D) Western blot analysis of arginase 1 on the protein extracted from whole lung tissue. (E) Lung surface tumor number after SBZ treatment in NTHi exposed or unexposed CC-LR mice. (mean ± SE; * = P ≤ 0.05 for CC-LR vs CC-LR plus SBZ treatment, # = P ≤ 0.05 for CC-LR with NTHi exposure vs CC-LR with NTHi exposure plus SBZ treatment). (F) Histopathological appearance of lung tissue after treatment with SBZ in NTHi exposed or unexposed CC-LR mice (4× magnification, scale bar = 50 mm, applicable to all panels).

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Expressing, Western Blot

    5) Product Images from "Application of hairless mouse strain to bioluminescence imaging of Arc expression in mouse brain"

    Article Title: Application of hairless mouse strain to bioluminescence imaging of Arc expression in mouse brain

    Journal: BMC Neuroscience

    doi: 10.1186/s12868-017-0335-6

    Comparison of pigmentation of head between C57BL/6 and Arc - Luc Tg HL mouse strains. a Dorsal views of head of C57BL/6 ( upper panels ) and Arc - Luc Tg HL ( lower panels ) mice at 4 ( left panels ) and 8 weeks of age ( right panels ). The photographs of the C57BL/6 mouse were taken after depilation. The C57BL/6 mouse at 8 weeks of age ( upper right panel ) and the Arc - Luc Tg HL mouse at both ages ( lower left and right panels ) are examples of suitable samples for BLI because of little pigmentation. The C57BL/6 mouse at 4 weeks of age ( upper left ) is an example of an unsuitable sample for BLI with skin pigmentation enclosed in a white dotted circle. Scale bar 10 mm. b Expression of IEGs in C57BL/6 and Arc - Luc Tg HL mice. Total RNA was extracted from the hippocampus 30 min after KA injection (n = 3, respectively). The changes in Arc , Egr - 1 , and c - fos expression levels were determined by quantitative RT-PCR analysis. The data represent mean ± SD
    Figure Legend Snippet: Comparison of pigmentation of head between C57BL/6 and Arc - Luc Tg HL mouse strains. a Dorsal views of head of C57BL/6 ( upper panels ) and Arc - Luc Tg HL ( lower panels ) mice at 4 ( left panels ) and 8 weeks of age ( right panels ). The photographs of the C57BL/6 mouse were taken after depilation. The C57BL/6 mouse at 8 weeks of age ( upper right panel ) and the Arc - Luc Tg HL mouse at both ages ( lower left and right panels ) are examples of suitable samples for BLI because of little pigmentation. The C57BL/6 mouse at 4 weeks of age ( upper left ) is an example of an unsuitable sample for BLI with skin pigmentation enclosed in a white dotted circle. Scale bar 10 mm. b Expression of IEGs in C57BL/6 and Arc - Luc Tg HL mice. Total RNA was extracted from the hippocampus 30 min after KA injection (n = 3, respectively). The changes in Arc , Egr - 1 , and c - fos expression levels were determined by quantitative RT-PCR analysis. The data represent mean ± SD

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

    6) Product Images from "Overexpression of Cotton RAV1 Gene in Arabidopsis Confers Transgenic Plants High Salinity and Drought Sensitivity"

    Article Title: Overexpression of Cotton RAV1 Gene in Arabidopsis Confers Transgenic Plants High Salinity and Drought Sensitivity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0118056

    Quantitative RT-PCR analysis of expressions of the stress-related genes in GhRAV1 -overexpression transgenic Arabidopsis under drought and salt stresses. Total RNA was isolated from the four-week-old transgenic plants and wild type grown under normal conditions and drought and NaCl stresses (see Methods ). Transcript levels of Arabidopsis RAB18, ABI1, ERD10, ERD15, KIN1, RD29A, RD29B and COR15a genes in the transgenic plants and wild type were determined by quantitative RT-PCR, using Arabidopsis ACTIN2 gene ( AtACT2 ) as a quantification control. Mean values and standard errors were shown from three independent experiments with three biological replicates of plant materials. Independent t-tests demonstrated that there was significant difference (* P
    Figure Legend Snippet: Quantitative RT-PCR analysis of expressions of the stress-related genes in GhRAV1 -overexpression transgenic Arabidopsis under drought and salt stresses. Total RNA was isolated from the four-week-old transgenic plants and wild type grown under normal conditions and drought and NaCl stresses (see Methods ). Transcript levels of Arabidopsis RAB18, ABI1, ERD10, ERD15, KIN1, RD29A, RD29B and COR15a genes in the transgenic plants and wild type were determined by quantitative RT-PCR, using Arabidopsis ACTIN2 gene ( AtACT2 ) as a quantification control. Mean values and standard errors were shown from three independent experiments with three biological replicates of plant materials. Independent t-tests demonstrated that there was significant difference (* P

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

    7) Product Images from "Plin4-Dependent Lipid Droplets Hamper Neuronal Mitophagy in the MPTP/p-Induced Mouse Model of Parkinson’s Disease"

    Article Title: Plin4-Dependent Lipid Droplets Hamper Neuronal Mitophagy in the MPTP/p-Induced Mouse Model of Parkinson’s Disease

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2018.00397

    Increased Plin4 is positively correlated with LD accumulation in the midbrain of MPTP/p-treated mice. (A,B) A: Quantitative RT-PCR analysis of RNA-seq indicated increased genes. B: RT-PCR analysis of Plin4 and other perilipins in mesencephalon samples from matched PD and NC mice. (C) Western blot analysis was performed to assess Plin4 expression in vivo. (D,E) For cellular location, the expression of Plin4 and TH/GFAP were analyzed by immunofluorescence of frozen brain sections. Arrows indicate neurons with coexpression of Plin4 and TH; the integrated optical density (IOD) of Plin4 and TH staining is presented on the right. (F) SH-SY5Y cells were stimulated with MPP + for 24 h followed by IF of Plin4 and BODIPY 493/503 staining (arrows marked), quantitation showed in right. Scale bar as indicated. Data are presented as the means ± SEM. Data in (A,E) : ∗ P
    Figure Legend Snippet: Increased Plin4 is positively correlated with LD accumulation in the midbrain of MPTP/p-treated mice. (A,B) A: Quantitative RT-PCR analysis of RNA-seq indicated increased genes. B: RT-PCR analysis of Plin4 and other perilipins in mesencephalon samples from matched PD and NC mice. (C) Western blot analysis was performed to assess Plin4 expression in vivo. (D,E) For cellular location, the expression of Plin4 and TH/GFAP were analyzed by immunofluorescence of frozen brain sections. Arrows indicate neurons with coexpression of Plin4 and TH; the integrated optical density (IOD) of Plin4 and TH staining is presented on the right. (F) SH-SY5Y cells were stimulated with MPP + for 24 h followed by IF of Plin4 and BODIPY 493/503 staining (arrows marked), quantitation showed in right. Scale bar as indicated. Data are presented as the means ± SEM. Data in (A,E) : ∗ P

    Techniques Used: Mouse Assay, Quantitative RT-PCR, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, In Vivo, Immunofluorescence, Staining, Quantitation Assay

    8) Product Images from "MicroRNA miR-124 Controls the Choice between Neuronal and Astrocyte Differentiation by Fine-tuning Ezh2 Expression *"

    Article Title: MicroRNA miR-124 Controls the Choice between Neuronal and Astrocyte Differentiation by Fine-tuning Ezh2 Expression *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.525493

    miR-124 down-regulates Ezh2 expression during P19 neuronal differentiation. A–E , inverse expression patterns of PRC2 members and miR-124 during P19 neuronal differentiation. Neuronal differentiation of P19 cells was induced by 1 μ m all- trans -RA treatment for 4 days. Cell aggregates were dissociated into single cell suspensions at day 4 and recultured in neurobasal medium with N2 supplement. Cells were further cultured and collected at the indicated time points. A , Ezh2 and Tuj1 protein levels were analyzed by Western blot, and Erk1/2 served as a loading control. B , expression of Ezh2 mRNA level was determined by semiquantitative RT-PCR. Hprt served as a loading control. C , increased miR-124 expression level during P19 neuronal differentiation. miR-124 expression levels were determined by Northern blot, and U6 RNA served as a loading control. A representative figure of two independent experiments is shown. This observation is shown in an earlier report ( 41 ). D , Suz12, Eed, and Tuj1 protein levels were analyzed as described above. E , expression of Suz12 and Eed mRNA levels was analyzed by semiquantitative RT-PCR. HPRT served as a loading control. F–H , miR-124 inhibitor up-regulates endogenous Ezh2. miR-124 inhibitor was transfected into differentiating P19 cells at day 6 after the start of RA treatment. The levels of miR-124 and PRC2 members were analyzed at day 10. F , down-regulation of miR-124 expression level by inhibitor was confirmed by Northern blot analysis. A representative figure of two independent experiments is shown. The miR-124-specific inhibitor has been tested and published previously ( 11 ). G , Ezh2 protein expression was up-regulated upon miR-124 inhibitor treatment. H , Suz12 protein level was not altered upon miR-124 inhibitor treatment, whereas Eed protein level was below the detection limit of the Western blot assay. Ezh2, Suz12, Eed, and Tuj1 protein expression levels were normalized to Erk1/2. Their -fold changes were calculated relative to the protein levels at day 0 ( Ezh2 and Suz12 ), day 10 ( Tuj1 ), or in non-transfected controls ( G and H ) and indicated below their respective blots. N.D. , not detectable. All data shown, unless otherwise stated, are representative of at least three independent experiments.
    Figure Legend Snippet: miR-124 down-regulates Ezh2 expression during P19 neuronal differentiation. A–E , inverse expression patterns of PRC2 members and miR-124 during P19 neuronal differentiation. Neuronal differentiation of P19 cells was induced by 1 μ m all- trans -RA treatment for 4 days. Cell aggregates were dissociated into single cell suspensions at day 4 and recultured in neurobasal medium with N2 supplement. Cells were further cultured and collected at the indicated time points. A , Ezh2 and Tuj1 protein levels were analyzed by Western blot, and Erk1/2 served as a loading control. B , expression of Ezh2 mRNA level was determined by semiquantitative RT-PCR. Hprt served as a loading control. C , increased miR-124 expression level during P19 neuronal differentiation. miR-124 expression levels were determined by Northern blot, and U6 RNA served as a loading control. A representative figure of two independent experiments is shown. This observation is shown in an earlier report ( 41 ). D , Suz12, Eed, and Tuj1 protein levels were analyzed as described above. E , expression of Suz12 and Eed mRNA levels was analyzed by semiquantitative RT-PCR. HPRT served as a loading control. F–H , miR-124 inhibitor up-regulates endogenous Ezh2. miR-124 inhibitor was transfected into differentiating P19 cells at day 6 after the start of RA treatment. The levels of miR-124 and PRC2 members were analyzed at day 10. F , down-regulation of miR-124 expression level by inhibitor was confirmed by Northern blot analysis. A representative figure of two independent experiments is shown. The miR-124-specific inhibitor has been tested and published previously ( 11 ). G , Ezh2 protein expression was up-regulated upon miR-124 inhibitor treatment. H , Suz12 protein level was not altered upon miR-124 inhibitor treatment, whereas Eed protein level was below the detection limit of the Western blot assay. Ezh2, Suz12, Eed, and Tuj1 protein expression levels were normalized to Erk1/2. Their -fold changes were calculated relative to the protein levels at day 0 ( Ezh2 and Suz12 ), day 10 ( Tuj1 ), or in non-transfected controls ( G and H ) and indicated below their respective blots. N.D. , not detectable. All data shown, unless otherwise stated, are representative of at least three independent experiments.

    Techniques Used: Expressing, Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Transfection

    9) Product Images from "CRL3IBTK Regulates the Tumor Suppressor Pdcd4 through Ubiquitylation Coupled to Proteasomal Degradation *"

    Article Title: CRL3IBTK Regulates the Tumor Suppressor Pdcd4 through Ubiquitylation Coupled to Proteasomal Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.634535

    IBtkα promotes the Pdcd4 polyubiquitylation and degradation. A , IBtkα promotes the polyubiquitylation of exogenously expressed Pdcd4. HEK293T cells (3 × 10 6 ) were transfected with Pdcd4 (4 μg) with or without IBtkα-FLAG, IBtkαΔBTB-FLAG, HA-tagged ubiquitin, or empty vector (4 μg). Forty-eight hours later, cells were treated with MG132 (20 μ m ) for 4 h before lysis. Cell extracts were subjected to IP with anti-HA antibody, and immunocomplexes were resolved by SDS-PAGE on a 6% gel, followed by WB with the indicated antibodies. B , IBtkα promotes the polyubiquitylation of endogenous Pdcd4 in vivo. C , IBtkα RNA interference increases the Pdcd4 protein content. HeLa cells (3 × 10 6 ) were transduced with control shRNA ( CNT-shRNA ), or IBtk shRNA for 48 h and then incubated with CHX (100 μg/ml) for up to 5 h. Cell lysates (30 μg) were analyzed by WB with the indicated antibodies. D , densitometric analysis of WB protein bands relative to the experiment described in C . Optical density of WB protein bands was expressed as arbitrary units normalized to control shRNA taken as a value of 100. Mean values ± S.D. ( error bars ) of three independent experiments are shown. E , IBtkα RNA interference does not affect the Pdcd4 mRNA level. HeLa cells were transduced as described in C , total RNA was extracted 48 h later, and IBtkα and Pdcd4 transcripts were measured by quantitative RT-PCR. Relative mRNA levels were expressed as arbitrary units normalized to control shRNA taken as 1.0. Mean values ± S.D. of three independent experiments are shown.
    Figure Legend Snippet: IBtkα promotes the Pdcd4 polyubiquitylation and degradation. A , IBtkα promotes the polyubiquitylation of exogenously expressed Pdcd4. HEK293T cells (3 × 10 6 ) were transfected with Pdcd4 (4 μg) with or without IBtkα-FLAG, IBtkαΔBTB-FLAG, HA-tagged ubiquitin, or empty vector (4 μg). Forty-eight hours later, cells were treated with MG132 (20 μ m ) for 4 h before lysis. Cell extracts were subjected to IP with anti-HA antibody, and immunocomplexes were resolved by SDS-PAGE on a 6% gel, followed by WB with the indicated antibodies. B , IBtkα promotes the polyubiquitylation of endogenous Pdcd4 in vivo. C , IBtkα RNA interference increases the Pdcd4 protein content. HeLa cells (3 × 10 6 ) were transduced with control shRNA ( CNT-shRNA ), or IBtk shRNA for 48 h and then incubated with CHX (100 μg/ml) for up to 5 h. Cell lysates (30 μg) were analyzed by WB with the indicated antibodies. D , densitometric analysis of WB protein bands relative to the experiment described in C . Optical density of WB protein bands was expressed as arbitrary units normalized to control shRNA taken as a value of 100. Mean values ± S.D. ( error bars ) of three independent experiments are shown. E , IBtkα RNA interference does not affect the Pdcd4 mRNA level. HeLa cells were transduced as described in C , total RNA was extracted 48 h later, and IBtkα and Pdcd4 transcripts were measured by quantitative RT-PCR. Relative mRNA levels were expressed as arbitrary units normalized to control shRNA taken as 1.0. Mean values ± S.D. of three independent experiments are shown.

    Techniques Used: Transfection, Plasmid Preparation, Lysis, SDS Page, Western Blot, In Vivo, Transduction, shRNA, Incubation, Quantitative RT-PCR

    IBtkα enhances translation by counteracting the Pdcd4 repression of target mRNAs. A , schematic representation of luciferase reporter mRNAs. B , depletion of IBtkα by RNA interference decreases the translation of reporter mRNAs with stem-loop structured or unstructured 5′-UTR. HeLa cells (3 × 10 6 ) cells were transfected with IBtk siRNA, Pdcd4 siRNA, or control siRNA or left untransfected ( mock ). After 24 h, cells were transfected with pCMV-LUC (0.2 μg) or pCMV-SL-LUC (0.2 μg) and serum-starved for 12 h, followed by growth in complete medium (10% FBS) for additional 24 h. The luciferase activity measured in untransfected cells was designated as 100%. Mean values ± S.D. ( error bars ) of five independent experiments are shown. C , overexpression of IBtkα enhances the translation of reporter mRNAs with stem-loop structured or unstructured 5′-UTR. HeLa cells (3 × 10 6 ) were transfected with IBtkα-FLAG, IBtkα-FLAG mutants, or empty vector (4 μg). Subsequent steps were performed as described in B. D , IBtkα RNA interference does not affect the global protein synthesis. HeLa cells (3 × 10 6 ) were transfected with IBtk siRNA or control siRNA or left untransfected ( mock ). The rate of protein synthesis was measured by incorporation of 35 S-labeled methionine and cysteine into translated protein and normalized to total protein concentration. E , IBtkα depletion by RNA interference decreases the intracellular amount of Bcl-xL. HeLa cells (3 × 10 6 ) were transfected with IBtk siRNA or control siRNA or left untransfected ( mock ), and 48 h later, cell lysates were analyzed by WB with the indicated antibodies. F , HeLa cells were transfected as described in E , and total RNA was analyzed by real-time PCR to measure the level of the indicated transcripts. Mean values ± S.D. of three independent experiments are shown.
    Figure Legend Snippet: IBtkα enhances translation by counteracting the Pdcd4 repression of target mRNAs. A , schematic representation of luciferase reporter mRNAs. B , depletion of IBtkα by RNA interference decreases the translation of reporter mRNAs with stem-loop structured or unstructured 5′-UTR. HeLa cells (3 × 10 6 ) cells were transfected with IBtk siRNA, Pdcd4 siRNA, or control siRNA or left untransfected ( mock ). After 24 h, cells were transfected with pCMV-LUC (0.2 μg) or pCMV-SL-LUC (0.2 μg) and serum-starved for 12 h, followed by growth in complete medium (10% FBS) for additional 24 h. The luciferase activity measured in untransfected cells was designated as 100%. Mean values ± S.D. ( error bars ) of five independent experiments are shown. C , overexpression of IBtkα enhances the translation of reporter mRNAs with stem-loop structured or unstructured 5′-UTR. HeLa cells (3 × 10 6 ) were transfected with IBtkα-FLAG, IBtkα-FLAG mutants, or empty vector (4 μg). Subsequent steps were performed as described in B. D , IBtkα RNA interference does not affect the global protein synthesis. HeLa cells (3 × 10 6 ) were transfected with IBtk siRNA or control siRNA or left untransfected ( mock ). The rate of protein synthesis was measured by incorporation of 35 S-labeled methionine and cysteine into translated protein and normalized to total protein concentration. E , IBtkα depletion by RNA interference decreases the intracellular amount of Bcl-xL. HeLa cells (3 × 10 6 ) were transfected with IBtk siRNA or control siRNA or left untransfected ( mock ), and 48 h later, cell lysates were analyzed by WB with the indicated antibodies. F , HeLa cells were transfected as described in E , and total RNA was analyzed by real-time PCR to measure the level of the indicated transcripts. Mean values ± S.D. of three independent experiments are shown.

    Techniques Used: Luciferase, Transfection, Activity Assay, Over Expression, Plasmid Preparation, Labeling, Protein Concentration, Western Blot, Real-time Polymerase Chain Reaction

    10) Product Images from "AMPK Activation Reduces Hepatic Lipid Content by Increasing Fat Oxidation In Vivo"

    Article Title: AMPK Activation Reduces Hepatic Lipid Content by Increasing Fat Oxidation In Vivo

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19092826

    Effects of the expression of an active form of AMP-activated protein kinase (AMPK) in the liver on body weight and food intake. Ten-week-old male C57BL/6J mice received injections of adenovirus (Ad) expressing the green fluorescent protein (GFP) or a constitutively active form of AMPKα2 (AMPK-CA) and were studied for the indicated times after adenovirus injection and in the indicated nutritional state. ( A ) Western blot analysis of liver lysates with antibodies raised against pan-AMPKα and myc-tagged AMPK-CA was performed on days 2 and day 8 after adenovirus administration; ( B ) Western blot analysis of liver lysates from fed mice 48 h after the injection of Ad GFP or Ad AMPK-CA, with the antibodies indicated. Each lane represents a liver sample from an individual mouse. The panel on the right shows Ser79 phosphorylated acetyl CoA carboxylase/ total acetyl CoA carboxylase (P-ACC/ACC) ratios from the quantification of immunoblot images ( n = 5); ( C ) Hepatic malonyl-CoA levels in 8 h-fasted mice 48 h after the injection of Ad GFP or Ad AMPK-CA ( n = 5); ( D ) Effect of AMPK activation in the liver on the expression of Cpt1a and Cpt 2 genes. Total RNA was isolated from the liver of 24 h-fasted mice 48 h after the injection of Ad GFP or Ad AMPK-CA ( n = 6). The expression of Cpt1a and Cpt2 genes was assessed by real-time quantitative RT-PCR. Relative mRNA levels are expressed as fold-activation relative to levels in Ad GFP livers; ( E ) Body weight changes and ( F ) cumulative food intake measured for 8 days after adenovirus administration ( n = 11–12 per group). Data are means ± standard error of mean (SEM). * p
    Figure Legend Snippet: Effects of the expression of an active form of AMP-activated protein kinase (AMPK) in the liver on body weight and food intake. Ten-week-old male C57BL/6J mice received injections of adenovirus (Ad) expressing the green fluorescent protein (GFP) or a constitutively active form of AMPKα2 (AMPK-CA) and were studied for the indicated times after adenovirus injection and in the indicated nutritional state. ( A ) Western blot analysis of liver lysates with antibodies raised against pan-AMPKα and myc-tagged AMPK-CA was performed on days 2 and day 8 after adenovirus administration; ( B ) Western blot analysis of liver lysates from fed mice 48 h after the injection of Ad GFP or Ad AMPK-CA, with the antibodies indicated. Each lane represents a liver sample from an individual mouse. The panel on the right shows Ser79 phosphorylated acetyl CoA carboxylase/ total acetyl CoA carboxylase (P-ACC/ACC) ratios from the quantification of immunoblot images ( n = 5); ( C ) Hepatic malonyl-CoA levels in 8 h-fasted mice 48 h after the injection of Ad GFP or Ad AMPK-CA ( n = 5); ( D ) Effect of AMPK activation in the liver on the expression of Cpt1a and Cpt 2 genes. Total RNA was isolated from the liver of 24 h-fasted mice 48 h after the injection of Ad GFP or Ad AMPK-CA ( n = 6). The expression of Cpt1a and Cpt2 genes was assessed by real-time quantitative RT-PCR. Relative mRNA levels are expressed as fold-activation relative to levels in Ad GFP livers; ( E ) Body weight changes and ( F ) cumulative food intake measured for 8 days after adenovirus administration ( n = 11–12 per group). Data are means ± standard error of mean (SEM). * p

    Techniques Used: Expressing, Mouse Assay, Injection, Western Blot, Activation Assay, Cycling Probe Technology, Isolation, Quantitative RT-PCR

    Long-term adenovirus-mediated expression of an active form of AMPK in the liver increases hepatic lipid oxidation and fatty acid uptake. Ten-week-old male C57BL/6J mice received injections of Ad GFP or Ad AMPK CA and were studied at the indicated times after adenovirus injection and in the indicated nutritional state. ( A ) Hepatic [1- 14 C]-palmitate oxidation in fed mice 48 h after the injection of Ad GFP or Ad AMPK-CA ( n = 4); ( B ) Plasma β-hydroxybutyrate levels in 24 h-fasted mice 48 h after the injection of Ad GFP or Ad AMPK-CA ( n = 6); ( C ) Plasma triglyceride (TG) and ( D ) plasma free fatty acid (FFA) levels in overnight-fasted mice 8 days after the injection of Ad GFP or Ad AMPK-CA ( n = 12); ( E ) Hepatic [1- 14 C]-palmitate uptake in 24 h-fasted mice 48 h after the injection of Ad GFP or Ad AMPK-CA ( n = 5); ( F ) Effect of AMPK activation in the liver on the expression of the fatty acid transporters. Total RNA was isolated from the liver of 24 h-fasted mice 48 h after the injection of Ad GFP or Ad AMPK-CA ( n = 5). The expression of Slc27a4 ( Fatp4 ), Cd36 , and Fabp4 genes was assessed by real-time quantitative RT-PCR. Relative mRNA levels are expressed as fold-activation relative to levels in Ad GFP livers. Data are means ± SEM. * p
    Figure Legend Snippet: Long-term adenovirus-mediated expression of an active form of AMPK in the liver increases hepatic lipid oxidation and fatty acid uptake. Ten-week-old male C57BL/6J mice received injections of Ad GFP or Ad AMPK CA and were studied at the indicated times after adenovirus injection and in the indicated nutritional state. ( A ) Hepatic [1- 14 C]-palmitate oxidation in fed mice 48 h after the injection of Ad GFP or Ad AMPK-CA ( n = 4); ( B ) Plasma β-hydroxybutyrate levels in 24 h-fasted mice 48 h after the injection of Ad GFP or Ad AMPK-CA ( n = 6); ( C ) Plasma triglyceride (TG) and ( D ) plasma free fatty acid (FFA) levels in overnight-fasted mice 8 days after the injection of Ad GFP or Ad AMPK-CA ( n = 12); ( E ) Hepatic [1- 14 C]-palmitate uptake in 24 h-fasted mice 48 h after the injection of Ad GFP or Ad AMPK-CA ( n = 5); ( F ) Effect of AMPK activation in the liver on the expression of the fatty acid transporters. Total RNA was isolated from the liver of 24 h-fasted mice 48 h after the injection of Ad GFP or Ad AMPK-CA ( n = 5). The expression of Slc27a4 ( Fatp4 ), Cd36 , and Fabp4 genes was assessed by real-time quantitative RT-PCR. Relative mRNA levels are expressed as fold-activation relative to levels in Ad GFP livers. Data are means ± SEM. * p

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

    11) Product Images from "The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri"

    Article Title: The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri

    Journal: Open Biology

    doi: 10.1098/rsob.160073

    Comparison of S. flexneri WT and pldA knockout strain transcriptomes. ( a ) Volcano plot showing a differentially expressed genes after pldA abrogation ( ΔpldA /WT). The data are expressed as a log2-fold change in gene expression levels ( x -axis) plotted against the –log2 p -value ( y -axis). The red dots represent the T3SS-related genes downregulated in the ΔpldA mutant. The pldA gene is marked by a green circle in the bottom left of the figure. ( b ) Heatmap of RNA sequencing comparing protein expression in the supernatants from WT and ΔpldA strains. Gene expression counts were log2 transformed to identify consistent changes in expression profiles between strains. The samples were sequenced in duplicate. ( c ) Quantitative real-time PCR verification of differentially expressed genes in the RNA-seq assay. Total RNA was prepared from the S. flexneri WT and ΔpldA strains, and expression levels of ipaC , ipaA , ipgC and phoN2 were analysed. The 16S rRNA expression level was evaluated as an internal control. RNA-seq was performed in duplicate on RNA from exponential phase cultures. qRT-PCR was repeated three times, and the expression level for each gene was normalized to the WT strain. The error bars represent ±s.e.m. ( n = 3). * p
    Figure Legend Snippet: Comparison of S. flexneri WT and pldA knockout strain transcriptomes. ( a ) Volcano plot showing a differentially expressed genes after pldA abrogation ( ΔpldA /WT). The data are expressed as a log2-fold change in gene expression levels ( x -axis) plotted against the –log2 p -value ( y -axis). The red dots represent the T3SS-related genes downregulated in the ΔpldA mutant. The pldA gene is marked by a green circle in the bottom left of the figure. ( b ) Heatmap of RNA sequencing comparing protein expression in the supernatants from WT and ΔpldA strains. Gene expression counts were log2 transformed to identify consistent changes in expression profiles between strains. The samples were sequenced in duplicate. ( c ) Quantitative real-time PCR verification of differentially expressed genes in the RNA-seq assay. Total RNA was prepared from the S. flexneri WT and ΔpldA strains, and expression levels of ipaC , ipaA , ipgC and phoN2 were analysed. The 16S rRNA expression level was evaluated as an internal control. RNA-seq was performed in duplicate on RNA from exponential phase cultures. qRT-PCR was repeated three times, and the expression level for each gene was normalized to the WT strain. The error bars represent ±s.e.m. ( n = 3). * p

    Techniques Used: Knock-Out, Expressing, Mutagenesis, RNA Sequencing Assay, Transformation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    12) Product Images from "Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome"

    Article Title: Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030414

    Regulation of IRAK3 expression in THP-1 monocytes. ( A ) Gene expression was analyzed by measuring relative RNA levels using qRT-PCR, protein expression and ROS production were determined by flow cytometry in THP-1 cells exposed to 1 or 10 µg/ml gADIPOQ (n = 6) or in IRAK3 -depleted THP-1 cells exposed to 10 µg/ml gADIPOQ (n = 4) for 6 h and 24 h. Data shown are means ± SEM of 24 h exposed cells normalized to 6 h exposed cells. * P
    Figure Legend Snippet: Regulation of IRAK3 expression in THP-1 monocytes. ( A ) Gene expression was analyzed by measuring relative RNA levels using qRT-PCR, protein expression and ROS production were determined by flow cytometry in THP-1 cells exposed to 1 or 10 µg/ml gADIPOQ (n = 6) or in IRAK3 -depleted THP-1 cells exposed to 10 µg/ml gADIPOQ (n = 4) for 6 h and 24 h. Data shown are means ± SEM of 24 h exposed cells normalized to 6 h exposed cells. * P

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

    Gene expressions of the IRAK3 -related pathway and adipocyte differentiation markers in visceral adipose tissue. ( A ) Gene expression in visceral adipose tissue was analyzed by measuring relative RNA levels using qRT-PCR for key molecules in the TLR2/NFκB inflammatory pathway. The adipose tissue specific antioxidant gene SOD3 instead of SOD2 was used as oxidative stress marker in visceral adipose tissue. ( B ) Relative RNA levels of markers of adipocyte differentiation ( PPARs and ADIPOQ ), insulin signaling ( INSR ) and glucose uptake ( GLUT4 ) in visceral adipose tissue as determined by qRT-PCR. Data shown are means. * P
    Figure Legend Snippet: Gene expressions of the IRAK3 -related pathway and adipocyte differentiation markers in visceral adipose tissue. ( A ) Gene expression in visceral adipose tissue was analyzed by measuring relative RNA levels using qRT-PCR for key molecules in the TLR2/NFκB inflammatory pathway. The adipose tissue specific antioxidant gene SOD3 instead of SOD2 was used as oxidative stress marker in visceral adipose tissue. ( B ) Relative RNA levels of markers of adipocyte differentiation ( PPARs and ADIPOQ ), insulin signaling ( INSR ) and glucose uptake ( GLUT4 ) in visceral adipose tissue as determined by qRT-PCR. Data shown are means. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Marker

    13) Product Images from "Loss of Ezh2 synergizes with JAK2-V617F in initiating myeloproliferative neoplasms and promoting myelofibrosis"

    Article Title: Loss of Ezh2 synergizes with JAK2-V617F in initiating myeloproliferative neoplasms and promoting myelofibrosis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20151136

    Gene expression analysis by RNA sequencing of LT-HSCs and MEPs. (A) Unsupervised clustering of differentially expressed genes (P ≤ 0.05; log2-fold changes > 1.5). Each column represents data from one individual mouse. The color code for the genotypes of the individual mice is the same as in B. (B) PCA. The data for LT-HSCs and MEPs were derived from two independent experiments and combined ( n = 4 or 5 for SclCre , n = 7 or 4 for SclCre;Ezh2 +/Δ , n = 4 for SclCre;Ezh2 Δ/Δ , n = 6 or 4 for SclCre;V617F , n = 8 or 6 SclCre;V617F;Ezh2 +/Δ , and n = 6 for SclCre;V617F;Ezh2 Δ/Δ for LT-HSCs or MEP analysis). Each dot represents data from one individual mouse. (C) Competitive gene set enrichment analysis for gene expression signatures of interferon-γ, interferon-α, and fetal liver HSCs in LT-HSCs of SclCre;V617F;Ezh2 Δ/Δ compared with SclCre;V617F . (D) Plot showing the number of differentially expressed genes with cutoff of P ≤ 0.05. (E) Gene list of top 10 significant gene expression differences according to the absolute fold change. (F) Expression levels of Lin28b and Hmga2 . Each dot represents data from one individual mouse. (G) Relative expression of HMGA2 , IGF2BP3 , and Pcolce2 determined by qPCR in granulocyte RNA from patients with MPN that carry mutations in EZH2 or CALR . Each dot represents data from one individual patient. The mutations in patient granulocyte were determined by allele-specific PCR ( n = 4 for CALR mutation with WT EZH2 , n = 4 for CALR mutation with heterozygous mutation of EZH2 , n = 1 for CALR mutation with homozygous mutation of EZH2 , n = 8 for JAK2V617F mutation with WT EZH2 , n = 12 for JAK2V617F mutation with heterozygous mutation of EZH2 , and n = 4 for JAK2V617F mutation with h homozygous mutation of EZH2 ). (F and G) Horizontal lines indicate the mean of the values. *, P
    Figure Legend Snippet: Gene expression analysis by RNA sequencing of LT-HSCs and MEPs. (A) Unsupervised clustering of differentially expressed genes (P ≤ 0.05; log2-fold changes > 1.5). Each column represents data from one individual mouse. The color code for the genotypes of the individual mice is the same as in B. (B) PCA. The data for LT-HSCs and MEPs were derived from two independent experiments and combined ( n = 4 or 5 for SclCre , n = 7 or 4 for SclCre;Ezh2 +/Δ , n = 4 for SclCre;Ezh2 Δ/Δ , n = 6 or 4 for SclCre;V617F , n = 8 or 6 SclCre;V617F;Ezh2 +/Δ , and n = 6 for SclCre;V617F;Ezh2 Δ/Δ for LT-HSCs or MEP analysis). Each dot represents data from one individual mouse. (C) Competitive gene set enrichment analysis for gene expression signatures of interferon-γ, interferon-α, and fetal liver HSCs in LT-HSCs of SclCre;V617F;Ezh2 Δ/Δ compared with SclCre;V617F . (D) Plot showing the number of differentially expressed genes with cutoff of P ≤ 0.05. (E) Gene list of top 10 significant gene expression differences according to the absolute fold change. (F) Expression levels of Lin28b and Hmga2 . Each dot represents data from one individual mouse. (G) Relative expression of HMGA2 , IGF2BP3 , and Pcolce2 determined by qPCR in granulocyte RNA from patients with MPN that carry mutations in EZH2 or CALR . Each dot represents data from one individual patient. The mutations in patient granulocyte were determined by allele-specific PCR ( n = 4 for CALR mutation with WT EZH2 , n = 4 for CALR mutation with heterozygous mutation of EZH2 , n = 1 for CALR mutation with homozygous mutation of EZH2 , n = 8 for JAK2V617F mutation with WT EZH2 , n = 12 for JAK2V617F mutation with heterozygous mutation of EZH2 , and n = 4 for JAK2V617F mutation with h homozygous mutation of EZH2 ). (F and G) Horizontal lines indicate the mean of the values. *, P

    Techniques Used: Expressing, RNA Sequencing Assay, Mouse Assay, Derivative Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Mutagenesis

    14) Product Images from "SIRT1 Suppresses the Senescence-Associated Secretory Phenotype through Epigenetic Gene Regulation"

    Article Title: SIRT1 Suppresses the Senescence-Associated Secretory Phenotype through Epigenetic Gene Regulation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116480

    Effects of stable SIRT1 knockdown on mRNA expression of SASP components. A , B , and C , The indicated cells were exposed (or not) to 10Gy of X-irradiation. Total RNA was extracted on the indicated days after irradiation. I IL-8, IL-6, IL-1β, and GRO-α mRNA levels were then determined by quantitative RT-PCR analysis. The levels of the indicated mRNA were normalized to that of GAPDH mRNA and shown as fold induction by 0h. Data are means ± s.d. of triplicates from experiments that were repeated at least twice with similar results.
    Figure Legend Snippet: Effects of stable SIRT1 knockdown on mRNA expression of SASP components. A , B , and C , The indicated cells were exposed (or not) to 10Gy of X-irradiation. Total RNA was extracted on the indicated days after irradiation. I IL-8, IL-6, IL-1β, and GRO-α mRNA levels were then determined by quantitative RT-PCR analysis. The levels of the indicated mRNA were normalized to that of GAPDH mRNA and shown as fold induction by 0h. Data are means ± s.d. of triplicates from experiments that were repeated at least twice with similar results.

    Techniques Used: Expressing, Irradiation, Quantitative RT-PCR

    Effects of SIRT1 overexpression on expression of SASP components. A , BJ/TERT cells were infected with a control retrovirus or the retrovirus expressing SIRT1 and then selected for 4 days. The resulting cells were lysed and then subjected to immunoblot analysis with antibodies to SIRT1 and γ -tubulin (loading control). B , SIRT1-overexpressing and control cells were exposed (or not) to 10Gy of X-irradiation. Cell were lysed on the indicated days after irradiation, and then subjected to immunoblot analysis using antibodies to IL-8, IL-6, SIRT1, and γ -tubulin. γ -Tubulin was used as a loading control. C , SIRT1-overexpressing and control cells were exposed (or not) to 10Gy of X-irradiation. Total RNA was extracted on the indicated days after irradiation. IL-8, IL-6, IL-1β, and GRO-α mRNA levels were then determined by quantitative RT-PCR analysis. The levels of the indicated mRNA were normalized to that of GAPDH mRNA and shown as fold induction by 0h. Data are means ± s.d. of triplicates from experiments that were repeated at least twice with similar results.
    Figure Legend Snippet: Effects of SIRT1 overexpression on expression of SASP components. A , BJ/TERT cells were infected with a control retrovirus or the retrovirus expressing SIRT1 and then selected for 4 days. The resulting cells were lysed and then subjected to immunoblot analysis with antibodies to SIRT1 and γ -tubulin (loading control). B , SIRT1-overexpressing and control cells were exposed (or not) to 10Gy of X-irradiation. Cell were lysed on the indicated days after irradiation, and then subjected to immunoblot analysis using antibodies to IL-8, IL-6, SIRT1, and γ -tubulin. γ -Tubulin was used as a loading control. C , SIRT1-overexpressing and control cells were exposed (or not) to 10Gy of X-irradiation. Total RNA was extracted on the indicated days after irradiation. IL-8, IL-6, IL-1β, and GRO-α mRNA levels were then determined by quantitative RT-PCR analysis. The levels of the indicated mRNA were normalized to that of GAPDH mRNA and shown as fold induction by 0h. Data are means ± s.d. of triplicates from experiments that were repeated at least twice with similar results.

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

    Effects of transient SIRT1 knockdown on expression of SASP components. A , MRC-5 cells were transfected with siRNA against SIRT1 (siSIRT1–8 and siSIRT1–9) or control (siControl), and then lysed 2 days after transfection. Cell lysates were subjected to immunoblot analysis using antibodies to SIRT1 and γ -tubulin (loading control). B , MRC-5 cells were transfected as in A and cultured for 2 days. The resulting cells were exposed (or not) to 10Gy of X-radiation. Cells were lysed on the indicated days after irradiation and subjected to immunoblot analysis using antibodies to IL-8, IL-6, and γ-tubulin. γ -Tubulin was used as a loading control. C , SIRT1 knockdown and control cells were exposed (or not) to 10Gy of X-irradiation. Total RNA was extracted on the indicated days after irradiation. IL-8, IL-6, IL-1β, and GRO-α mRNA levels were then determined by quantitative RT-PCR analysis. The levels of the indicated mRNA were normalized to that of GAPDH mRNA and shown as fold induction by 0h. Data are means ± s.d. of triplicates from experiments that were repeated at least twice with similar results.
    Figure Legend Snippet: Effects of transient SIRT1 knockdown on expression of SASP components. A , MRC-5 cells were transfected with siRNA against SIRT1 (siSIRT1–8 and siSIRT1–9) or control (siControl), and then lysed 2 days after transfection. Cell lysates were subjected to immunoblot analysis using antibodies to SIRT1 and γ -tubulin (loading control). B , MRC-5 cells were transfected as in A and cultured for 2 days. The resulting cells were exposed (or not) to 10Gy of X-radiation. Cells were lysed on the indicated days after irradiation and subjected to immunoblot analysis using antibodies to IL-8, IL-6, and γ-tubulin. γ -Tubulin was used as a loading control. C , SIRT1 knockdown and control cells were exposed (or not) to 10Gy of X-irradiation. Total RNA was extracted on the indicated days after irradiation. IL-8, IL-6, IL-1β, and GRO-α mRNA levels were then determined by quantitative RT-PCR analysis. The levels of the indicated mRNA were normalized to that of GAPDH mRNA and shown as fold induction by 0h. Data are means ± s.d. of triplicates from experiments that were repeated at least twice with similar results.

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

    The expression of SASP components and SIRT1 during cellular senescence. A , The indicated cells were exposed (or not) to 10 Gy of X-radiation, cultured for 10 days, and then total RNA and cell lysates were extracted. IL-8 and IL-6 mRNA levels were determined by quantitative RT-PCR analysis with normalization to GAPHD mRNA levels and expressed as fold induction by x-radiation. Data are means ± s.d. of triplicates from experiments that were repeated at least twice with similar results. Cell lysates were subjected to immunoblot analysis using antibodies to SIRT1 and γ -tubulin. B and C , MRC-5 human fibroblast cells were treated as in A , Total RNA and cell lysates were extracted at indicated days. IL-8 and IL-6 mRNA levels were then determined by quantitative RT-PCR analysis (B). The levels of the indicated mRNA were normalized to that of GAPDH mRNA and shown as fold induction by 0h. Data are means ± s.d. of triplicates from experiments that were repeated at least twice with similar results. Cell lysates were subjected to immunoblot analysis using antibodies to IL-8, IL-6, SIRT1, and γ -tubulin. γ -Tubulin was used as a loading control (C).
    Figure Legend Snippet: The expression of SASP components and SIRT1 during cellular senescence. A , The indicated cells were exposed (or not) to 10 Gy of X-radiation, cultured for 10 days, and then total RNA and cell lysates were extracted. IL-8 and IL-6 mRNA levels were determined by quantitative RT-PCR analysis with normalization to GAPHD mRNA levels and expressed as fold induction by x-radiation. Data are means ± s.d. of triplicates from experiments that were repeated at least twice with similar results. Cell lysates were subjected to immunoblot analysis using antibodies to SIRT1 and γ -tubulin. B and C , MRC-5 human fibroblast cells were treated as in A , Total RNA and cell lysates were extracted at indicated days. IL-8 and IL-6 mRNA levels were then determined by quantitative RT-PCR analysis (B). The levels of the indicated mRNA were normalized to that of GAPDH mRNA and shown as fold induction by 0h. Data are means ± s.d. of triplicates from experiments that were repeated at least twice with similar results. Cell lysates were subjected to immunoblot analysis using antibodies to IL-8, IL-6, SIRT1, and γ -tubulin. γ -Tubulin was used as a loading control (C).

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

    15) Product Images from "Comparative Leaf and Root Transcriptomic Analysis of two Rice Japonica Cultivars Reveals Major Differences in the Root Early Response to Osmotic Stress"

    Article Title: Comparative Leaf and Root Transcriptomic Analysis of two Rice Japonica Cultivars Reveals Major Differences in the Root Early Response to Osmotic Stress

    Journal: Rice

    doi: 10.1186/s12284-016-0098-1

    Validation of the expression of selected genes from RNA-Seq using qRT-PCR. Fold changes in gene expression were transformed to a log 2 scale. The qRT-PCR data log 2 -values (X-axis ) were plotted against the RNA-Seq log 2 values ( Y-axis ). The function of the regression line and the R 2 value are shown
    Figure Legend Snippet: Validation of the expression of selected genes from RNA-Seq using qRT-PCR. Fold changes in gene expression were transformed to a log 2 scale. The qRT-PCR data log 2 -values (X-axis ) were plotted against the RNA-Seq log 2 values ( Y-axis ). The function of the regression line and the R 2 value are shown

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

    16) Product Images from "The cotton WRKY transcription factor (GhWRKY33) reduces transgenic Arabidopsis resistance to drought stress"

    Article Title: The cotton WRKY transcription factor (GhWRKY33) reduces transgenic Arabidopsis resistance to drought stress

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37035-2

    Quantitative RT-PCR analysis of expression of the drought stress-related and ABA-responsive genes in GhWRKY33 transgenic Arabidopsis. Total RNA was isolated from 10-day-old seedlings grown without (CK) or with 250 mM mannitol treatment for 72 h or with 100 μM ABA treatment for 6 h. Transcript levels of RD29A, DREB2A, ERD15, SOS2, RAB18 and ABI1 in the transgenic lines and wild type were determined by quantitative RT-PCR using AtACTIN2 as a quantification control. Mean values and standard errors (bars) were shown from three independent experiments. One and two asterisks represent there was a significant difference (P
    Figure Legend Snippet: Quantitative RT-PCR analysis of expression of the drought stress-related and ABA-responsive genes in GhWRKY33 transgenic Arabidopsis. Total RNA was isolated from 10-day-old seedlings grown without (CK) or with 250 mM mannitol treatment for 72 h or with 100 μM ABA treatment for 6 h. Transcript levels of RD29A, DREB2A, ERD15, SOS2, RAB18 and ABI1 in the transgenic lines and wild type were determined by quantitative RT-PCR using AtACTIN2 as a quantification control. Mean values and standard errors (bars) were shown from three independent experiments. One and two asterisks represent there was a significant difference (P

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

    17) Product Images from "Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-to-mesenchymal transition"

    Article Title: Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-to-mesenchymal transition

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.006510

    Activation of canonical Wnt signaling causes mesenchymal transition in cultured endothelial cells. The effects of canonical Wnt signaling activation were investigated in adult BAECs and the mouse brain bEnd.3 endothelial cell line by treatment with the canonical Wnt signaling activator BIO. (A) BIO induces a mesenchymal phenotype in BAECs within 24 hours of treatment. (B) Real-time quantitative RT-PCR analysis using RNA samples from BIO-treated BAECs. BIO induces the expression of canonical Wnt signaling gene targets ( Axin2 , TCF7 ) and EndMT-associated genes ( Slug , SMA , Col1A1 ), whereas it leads to the downregulation of the endothelial-specific gene CD31 . Induction of canonical-Wnt-pathway target genes ( Axin2 , TCF7 ) and downregulation of CD31 occurs within 24 hours of BIO treatment, whereas induction of SMA and Col1A1 is not observed until after 3 days of exposure to BIO. (C) IF analysis of bEnd.3 cells treated for 48 hours with BIO (+) or its inactive analog MeBIO (–) and stained with antibodies recognizing CD31 (green) and SMA (red). BIO treatment leads to downregulation of CD31 and upregulation of SMA, consistent with the molecular data obtained in BAECs. (D) IF analysis of bEnd.3 cells treated for 1, 4 or 24 hours with BIO (+) or MeBIO (–) and stained with antibodies recognizing β-catenin (red). BIO treatment causes nuclear accumulation of β-catenin, a hallmark of canonical Wnt signaling activation. DAPI stain (blue) marks cellular nuclei. All BIO treatment time points (1, 4 or 24 hours) showed nuclear β-catenin (arrowheads). The images are from the 4-hour exposure. (E) Luciferase analysis using protein extracts isolated from bEnd.3 cells transiently transfected with Super TOPFlash and pRL-TK Renilla Luciferase Reporter for normalization. bEnd.3 cells were treated with BIO, MeBIO or vehicle (DMSO) for 6 hours. BIO addition increases luciferase activity, indicating induction of the canonical Wnt pathway.
    Figure Legend Snippet: Activation of canonical Wnt signaling causes mesenchymal transition in cultured endothelial cells. The effects of canonical Wnt signaling activation were investigated in adult BAECs and the mouse brain bEnd.3 endothelial cell line by treatment with the canonical Wnt signaling activator BIO. (A) BIO induces a mesenchymal phenotype in BAECs within 24 hours of treatment. (B) Real-time quantitative RT-PCR analysis using RNA samples from BIO-treated BAECs. BIO induces the expression of canonical Wnt signaling gene targets ( Axin2 , TCF7 ) and EndMT-associated genes ( Slug , SMA , Col1A1 ), whereas it leads to the downregulation of the endothelial-specific gene CD31 . Induction of canonical-Wnt-pathway target genes ( Axin2 , TCF7 ) and downregulation of CD31 occurs within 24 hours of BIO treatment, whereas induction of SMA and Col1A1 is not observed until after 3 days of exposure to BIO. (C) IF analysis of bEnd.3 cells treated for 48 hours with BIO (+) or its inactive analog MeBIO (–) and stained with antibodies recognizing CD31 (green) and SMA (red). BIO treatment leads to downregulation of CD31 and upregulation of SMA, consistent with the molecular data obtained in BAECs. (D) IF analysis of bEnd.3 cells treated for 1, 4 or 24 hours with BIO (+) or MeBIO (–) and stained with antibodies recognizing β-catenin (red). BIO treatment causes nuclear accumulation of β-catenin, a hallmark of canonical Wnt signaling activation. DAPI stain (blue) marks cellular nuclei. All BIO treatment time points (1, 4 or 24 hours) showed nuclear β-catenin (arrowheads). The images are from the 4-hour exposure. (E) Luciferase analysis using protein extracts isolated from bEnd.3 cells transiently transfected with Super TOPFlash and pRL-TK Renilla Luciferase Reporter for normalization. bEnd.3 cells were treated with BIO, MeBIO or vehicle (DMSO) for 6 hours. BIO addition increases luciferase activity, indicating induction of the canonical Wnt pathway.

    Techniques Used: Activation Assay, Cell Culture, Quantitative RT-PCR, Expressing, Staining, Luciferase, Isolation, Transfection, Activity Assay

    Activation of the canonical Wnt signaling pathway after experimental MI. MI was induced in TOPGAL mice by permanent LAD ligation. Sham-operated animals underwent the same surgical procedure without blood vessel ligation. (A) Schematic outline of the experimental time line and corresponding stages of the ischemic injury response. (B) Hearts were isolated at 24 hours, 4 days, 7 days and 3 weeks post-MI, and stained with X-gal to assess canonical Wnt activity. Upper panels: front views with visible sutures; middle panels: rear views; lower panels: higher-magnification images of interest areas and infarct sites. In sham-operated control hearts, β-galactosidase activity staining is indistinguishable from normal (i.e. non-operated) hearts at all time points after surgery, with canonical Wnt activity around the great vessels. The displayed sham examples are from 7 days after surgery. Similarly, there are no visible changes in β-galactosidase staining 24 hours after LAD occlusion, compared with non-operated heart. By contrast, starting at day-4 post-MI, numerous X-gal-positive cells (arrows) appear throughout the heart around blood vessels. By 7-days post-MI, X-gal-positive cells with canonical Wnt activity are present around the infarct and peri-infarct areas (arrows). β-galactosidase (canonical Wnt pathway) activity is undetectable in the injury area 3 weeks after experimental MI. (C) Real-time quantitative RT-PCR analysis using RNA isolated from mouse hearts at the indicated time points after LAD occlusion. RNA samples from sham-operated animals isolated at equivalent time points after surgery served as controls. Sham samples had comparable expression levels at all stages after injury. In MI samples, peak expression levels of putative target genes of canonical Wnt signaling coincided with the time of highest β-galactosidase activity, at day-7 post-MI, except Myc , which was induced earlier (at 24 hours). *P
    Figure Legend Snippet: Activation of the canonical Wnt signaling pathway after experimental MI. MI was induced in TOPGAL mice by permanent LAD ligation. Sham-operated animals underwent the same surgical procedure without blood vessel ligation. (A) Schematic outline of the experimental time line and corresponding stages of the ischemic injury response. (B) Hearts were isolated at 24 hours, 4 days, 7 days and 3 weeks post-MI, and stained with X-gal to assess canonical Wnt activity. Upper panels: front views with visible sutures; middle panels: rear views; lower panels: higher-magnification images of interest areas and infarct sites. In sham-operated control hearts, β-galactosidase activity staining is indistinguishable from normal (i.e. non-operated) hearts at all time points after surgery, with canonical Wnt activity around the great vessels. The displayed sham examples are from 7 days after surgery. Similarly, there are no visible changes in β-galactosidase staining 24 hours after LAD occlusion, compared with non-operated heart. By contrast, starting at day-4 post-MI, numerous X-gal-positive cells (arrows) appear throughout the heart around blood vessels. By 7-days post-MI, X-gal-positive cells with canonical Wnt activity are present around the infarct and peri-infarct areas (arrows). β-galactosidase (canonical Wnt pathway) activity is undetectable in the injury area 3 weeks after experimental MI. (C) Real-time quantitative RT-PCR analysis using RNA isolated from mouse hearts at the indicated time points after LAD occlusion. RNA samples from sham-operated animals isolated at equivalent time points after surgery served as controls. Sham samples had comparable expression levels at all stages after injury. In MI samples, peak expression levels of putative target genes of canonical Wnt signaling coincided with the time of highest β-galactosidase activity, at day-7 post-MI, except Myc , which was induced earlier (at 24 hours). *P

    Techniques Used: Activation Assay, Mouse Assay, Ligation, Isolation, Staining, Activity Assay, Quantitative RT-PCR, Expressing

    18) Product Images from "The attenuated hepatocellular carcinoma-specific Listeria vaccine Lmdd-MPFG prevents tumor occurrence through immune regulation of dendritic cells"

    Article Title: The attenuated hepatocellular carcinoma-specific Listeria vaccine Lmdd-MPFG prevents tumor occurrence through immune regulation of dendritic cells

    Journal: Oncotarget

    doi:

    TLRs and NLRs in LM-promoted dendritic maturation BMDCs were collected for 24 h treatment (control, LPS, LM, and LM-HK). Messenger RNA levels of NLRs (A) and TLRs (B) were detected in each group by quantitative real-time PCR. Protein extracts were prepared and relative NLR and TLR protein levels were detected by western blot assays (C). Densitometry values relative to internal controls are displayed in the histograms (D). Activation of several signaling pathways was also analyzed by western blot (E). Summary statistics are depicted in the histogram (F). All data are presented as the mean ± SEM (*p
    Figure Legend Snippet: TLRs and NLRs in LM-promoted dendritic maturation BMDCs were collected for 24 h treatment (control, LPS, LM, and LM-HK). Messenger RNA levels of NLRs (A) and TLRs (B) were detected in each group by quantitative real-time PCR. Protein extracts were prepared and relative NLR and TLR protein levels were detected by western blot assays (C). Densitometry values relative to internal controls are displayed in the histograms (D). Activation of several signaling pathways was also analyzed by western blot (E). Summary statistics are depicted in the histogram (F). All data are presented as the mean ± SEM (*p

    Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Activation Assay

    19) Product Images from "MiR-199a/b-5p inhibits hepatocellular carcinoma progression by post-transcriptionally suppressing ROCK1"

    Article Title: MiR-199a/b-5p inhibits hepatocellular carcinoma progression by post-transcriptionally suppressing ROCK1

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18052

    ROCK1 regulates HCC migration, and invasion (A) ROCK1 mRNA expression levels in 50 normal liver and 374 HCC tumor samples from the TCGA RNA-Seq database. (B) qRT-PCR analysis of ROCK1 mRNA levels in 35 HCC and matched normal liver samples. (C) qRT-PCR analysis of ROCK1 mRNA levels in immortalized normal hepatic cell line, QSG-7701 and HCC cell lines, SMMC-7721 and HepG2. (D) Western blot analysis of ROCK1 protein in immortalized normal hepatic cell line, QSG-7701 and HCC cell lines, SMMC-7721 and HepG2. (E) Transwell assays showing cell migration and invasion potential of control and ROCK1 siRNA transfected SMMC-7721 and HepG2 cell lines. (F) qRT-PCR analysis of miR-199a/b-5p expression in ROCK1 knockdown SMMC-7721 cells transfected with miR-NC or miR-199a/b-5p. (G) In vivo xenograft tumor nude mice model determining the effects of ROCK1 knockdown on HCC metastasis. Control and ROCK1 siRNA transfected SMMC-7721 cells were injected into the caudal vein of mice and examined after 14 days at the In Vivo Imaging System (IVIS). Kaplan-Meier survival curves of mice implanted with control and ROCK1 siRNA transfected SMMC-7721 cells are shown. The results are expressed as mean ± SD (*p
    Figure Legend Snippet: ROCK1 regulates HCC migration, and invasion (A) ROCK1 mRNA expression levels in 50 normal liver and 374 HCC tumor samples from the TCGA RNA-Seq database. (B) qRT-PCR analysis of ROCK1 mRNA levels in 35 HCC and matched normal liver samples. (C) qRT-PCR analysis of ROCK1 mRNA levels in immortalized normal hepatic cell line, QSG-7701 and HCC cell lines, SMMC-7721 and HepG2. (D) Western blot analysis of ROCK1 protein in immortalized normal hepatic cell line, QSG-7701 and HCC cell lines, SMMC-7721 and HepG2. (E) Transwell assays showing cell migration and invasion potential of control and ROCK1 siRNA transfected SMMC-7721 and HepG2 cell lines. (F) qRT-PCR analysis of miR-199a/b-5p expression in ROCK1 knockdown SMMC-7721 cells transfected with miR-NC or miR-199a/b-5p. (G) In vivo xenograft tumor nude mice model determining the effects of ROCK1 knockdown on HCC metastasis. Control and ROCK1 siRNA transfected SMMC-7721 cells were injected into the caudal vein of mice and examined after 14 days at the In Vivo Imaging System (IVIS). Kaplan-Meier survival curves of mice implanted with control and ROCK1 siRNA transfected SMMC-7721 cells are shown. The results are expressed as mean ± SD (*p

    Techniques Used: Migration, Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Transfection, In Vivo, Mouse Assay, Injection, In Vivo Imaging

    20) Product Images from "MiR-31 promotes mammary stem cell expansion and breast tumorigenesis by suppressing Wnt signaling antagonists"

    Article Title: MiR-31 promotes mammary stem cell expansion and breast tumorigenesis by suppressing Wnt signaling antagonists

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01059-5

    Identification of miR-31 direct targets. a , b qRT-PCR analysis for Axin1 , Dkk1 , Gsk3β , Smad3 and Smad4 in Control ( n = 3) and miR-31 KO ( n = 3) a , as well as Control ( n = 3) and DTG ( n = 3) b mammary epithelium. c Western blotting for Dkk1, Axin1, Gsk3β and Smad4 in Control ( n = 3) and miR-31 KO ( n = 3), as well as Control ( n = 3) and DTG ( n = 3) mammary epithelium. β-Tubulin was used as a loading control. d Ratio of luciferase activity of miR-31 mimics vs. scrambled RNA in wild type (WT) and mutant (Mut) 3’-UTR constructs based on 3 independent experiments. e RNA crosslinking, immunoprecipitation, and qRT-PCR (CLIP-PCR) assay for Dkk1 , Axin1 , Gsk3β , Smad3 and Smad4 upon Ago2 antibody immunoprecipitates in response to miR-31 inhibitor and scramble RNA (NC). IgG was used as a negative control. f qRT-PCR analysis for Smad3 , Smad4 , Axin1 , Dkk1 and Gsk3β in PyVT ( n = 3) and PyVT/KO ( n = 3) tumors. g Western blotting for Dkk1, Axin1, Gsk3β, Smad4 and p-Smad2/3 in PyVT ( n = 3) and PyVT/KO ( n = 3) tumors at 12 weeks of age. β-Tubulin was used as a loading control. h Immunofluoresence for Dkk1 in PyVT ( n = 3) and PyVT/KO ( n = 3) tumors at 12 weeks of age. Scale bar, 50 μm. i Flow cytometry profiles of CD24-FITC and CD29-PE in cell suspensions of mammary epithelium from K5-rtTA ( n = 3), DTG ( n = 3), Dkk1 ( n = 3) and DTG/ Dkk1 ( n = 3) mice. Arrows indicated CD24 + CD29 high cell population. n = 3 biological replicates. Data represented as mean ± S.D. n = 3. Two tailed unpaired t -test for a , b , d , f (* P
    Figure Legend Snippet: Identification of miR-31 direct targets. a , b qRT-PCR analysis for Axin1 , Dkk1 , Gsk3β , Smad3 and Smad4 in Control ( n = 3) and miR-31 KO ( n = 3) a , as well as Control ( n = 3) and DTG ( n = 3) b mammary epithelium. c Western blotting for Dkk1, Axin1, Gsk3β and Smad4 in Control ( n = 3) and miR-31 KO ( n = 3), as well as Control ( n = 3) and DTG ( n = 3) mammary epithelium. β-Tubulin was used as a loading control. d Ratio of luciferase activity of miR-31 mimics vs. scrambled RNA in wild type (WT) and mutant (Mut) 3’-UTR constructs based on 3 independent experiments. e RNA crosslinking, immunoprecipitation, and qRT-PCR (CLIP-PCR) assay for Dkk1 , Axin1 , Gsk3β , Smad3 and Smad4 upon Ago2 antibody immunoprecipitates in response to miR-31 inhibitor and scramble RNA (NC). IgG was used as a negative control. f qRT-PCR analysis for Smad3 , Smad4 , Axin1 , Dkk1 and Gsk3β in PyVT ( n = 3) and PyVT/KO ( n = 3) tumors. g Western blotting for Dkk1, Axin1, Gsk3β, Smad4 and p-Smad2/3 in PyVT ( n = 3) and PyVT/KO ( n = 3) tumors at 12 weeks of age. β-Tubulin was used as a loading control. h Immunofluoresence for Dkk1 in PyVT ( n = 3) and PyVT/KO ( n = 3) tumors at 12 weeks of age. Scale bar, 50 μm. i Flow cytometry profiles of CD24-FITC and CD29-PE in cell suspensions of mammary epithelium from K5-rtTA ( n = 3), DTG ( n = 3), Dkk1 ( n = 3) and DTG/ Dkk1 ( n = 3) mice. Arrows indicated CD24 + CD29 high cell population. n = 3 biological replicates. Data represented as mean ± S.D. n = 3. Two tailed unpaired t -test for a , b , d , f (* P

    Techniques Used: Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Mutagenesis, Construct, Cross-linking Immunoprecipitation, Polymerase Chain Reaction, Negative Control, Flow Cytometry, Cytometry, Mouse Assay, Two Tailed Test

    Loss of miR-31 represses metastasis to the lung. a Quantification of metastasis-free survival rates of PyVT and PyVT/KO mice at 12 and 16 weeks of age. 12 wks: Py VT ( n = 16), KO/PyVT ( n = 9); 16 wks: PyVT ( n = 20), KO/PyVT ( n = 10). b Representative histology of lungs from PyVT and PyVT/KO mice at 12 and 16 weeks of age. Arrows point to metastatic foci in the lungs. Quantification of metastatic tumors in the lungs. 12 wks: PyVT ( n = 16), KO/PyVT ( n = 9); 16 wks: PyVT ( n = 20), KO/PyVT ( n = 10). Scale bar, 100 μm. c Immunofluorescence for Vimentin in PyVT and PyVT/KO tumors at 12 weeks of age. Scale bar, 50 μm. d Immunohistochemistry for Slug in PyVT and PyVT/KO tumors at 12 weeks of age. Scale bar, 25 μm. e qRT-PCR analysis for Twist1 , Bmi1 , Zeb1 and Fzd3 in PyVT and PyVT/KO tumors at 12 weeks of age. n = 3 biological replicates. f Immunofluorescence for P-cadherin in tumors (top panels) and normal mammary ducts (bottom panels) from PyVT and PyVT/KO mice at 12 weeks of age. Scale bar, 25 μm. g Immunofluorescence for β1-integrin (ITGB1) and IHC for α2-integrin (ITGA2) in PyVT and PyVT/KO tumors at 18 weeks of age. Scale bar, 25 μm. h Western blotting for ITGA2 and ITGB1 in PyVT and PyVT/KO tumors at 18 weeks of age. GAPDH was used as a loading control. i Morphology of PyVT and PyVT/KO tumor cells into three-dimensional matrigel culture at indicated time points. Arrows point to protrusions. j Immunofluorescence for Vimentin in BT549 breast cancer cells treated with Scramble RNA and miR-31 mimics. Scale bar, 50 μm. n = 3 technical replicates. Data represented as mean ± S.D. Sample size: PyVT ( n = 3) and PyVT/KO ( n = 3) for c – i . Two tailed unpaired t -test for e (* P
    Figure Legend Snippet: Loss of miR-31 represses metastasis to the lung. a Quantification of metastasis-free survival rates of PyVT and PyVT/KO mice at 12 and 16 weeks of age. 12 wks: Py VT ( n = 16), KO/PyVT ( n = 9); 16 wks: PyVT ( n = 20), KO/PyVT ( n = 10). b Representative histology of lungs from PyVT and PyVT/KO mice at 12 and 16 weeks of age. Arrows point to metastatic foci in the lungs. Quantification of metastatic tumors in the lungs. 12 wks: PyVT ( n = 16), KO/PyVT ( n = 9); 16 wks: PyVT ( n = 20), KO/PyVT ( n = 10). Scale bar, 100 μm. c Immunofluorescence for Vimentin in PyVT and PyVT/KO tumors at 12 weeks of age. Scale bar, 50 μm. d Immunohistochemistry for Slug in PyVT and PyVT/KO tumors at 12 weeks of age. Scale bar, 25 μm. e qRT-PCR analysis for Twist1 , Bmi1 , Zeb1 and Fzd3 in PyVT and PyVT/KO tumors at 12 weeks of age. n = 3 biological replicates. f Immunofluorescence for P-cadherin in tumors (top panels) and normal mammary ducts (bottom panels) from PyVT and PyVT/KO mice at 12 weeks of age. Scale bar, 25 μm. g Immunofluorescence for β1-integrin (ITGB1) and IHC for α2-integrin (ITGA2) in PyVT and PyVT/KO tumors at 18 weeks of age. Scale bar, 25 μm. h Western blotting for ITGA2 and ITGB1 in PyVT and PyVT/KO tumors at 18 weeks of age. GAPDH was used as a loading control. i Morphology of PyVT and PyVT/KO tumor cells into three-dimensional matrigel culture at indicated time points. Arrows point to protrusions. j Immunofluorescence for Vimentin in BT549 breast cancer cells treated with Scramble RNA and miR-31 mimics. Scale bar, 50 μm. n = 3 technical replicates. Data represented as mean ± S.D. Sample size: PyVT ( n = 3) and PyVT/KO ( n = 3) for c – i . Two tailed unpaired t -test for e (* P

    Techniques Used: Mouse Assay, Immunofluorescence, Immunohistochemistry, Quantitative RT-PCR, Western Blot, Two Tailed Test

    MiR-31 activates Wnt and represses TGFβ signaling pathways. a Wnt signals were evaluated by Axin2-LacZ reporter activity in mammary ducts from Control ( n = 3) and DTG ( n = 3) mice, as well as Control ( n = 3) and miR-31 KO ( n = 3) mice. Blue, LacZ signals. Scale bar, 100 μm. b Immunohistochemistry for β-Catenin in Control ( n = 3) and DTG ( n = 3) mammary glands at 12 weeks of age and 14.5 d.p.c. Arrows, nuclear localized β-Catenin. Scale bar, 25 μm. c Western blotting for β-Catenin in Control ( n = 3) and miR-31 KO ( n = 3) mice. GAPDH was used as a loading control. d Immunofluorescence for β-Catenin in HC11 mouse mammary epithelial cells in the presence of Dox ( miR-31 overexpression). Control, without Dox. Red, β-Catenin; Blue, DAPI. n = 3 technical replicates. e Western blotting for Wnt target LBH in miR-31 overexpressing (Dox) or miR-31 inhibited ( anti-miR-31 ) HC11 mammary epithelial cells. The Control HC11 cells were treated with Scramble RNA. n = 3 technical replicates. f TOP/FOPflash luciferase assays demonstrate that Dox induced miR-31 overexpression activates Wnt reporter gene expression and that miR-31 inhibitor ( anti-miR-31 ) markedly represses Wnt reporter gene expression. Scramble RNA was used as negative control (NC). n = 3 technical replicates. g Western blotting for Smad3, p-Smad2/3 and p21 in mammary epithelial cells of Control ( n = 3) and miR-31 KO ( n = 3) mice at 10 weeks of age. h qRT-PCR analysis for TGFβ components and downstream target genes, Cdkn2b (encoding p15), Cdkn1a (encoding p21), Cdkn1c (encoding p57) and Tgfbr1 in mammary epithelial cells of Control ( n = 3) and miR-31 KO ( n = 3) mice at 10 weeks of age. i HC11 mammary epithelial cells were transfected with CAGA luciferase reporter vector, combined with scrambled RNA (negative control, NC), miR-31 mimics or miR-31 inhibitor ( anti-miR-31 ) for 24 h and then harvested for luciferase activity determination. n = 3 biological replicates. Data represented as mean ± S.D. n = 3. Two tailed unpaired t -test for f , h , i (* P
    Figure Legend Snippet: MiR-31 activates Wnt and represses TGFβ signaling pathways. a Wnt signals were evaluated by Axin2-LacZ reporter activity in mammary ducts from Control ( n = 3) and DTG ( n = 3) mice, as well as Control ( n = 3) and miR-31 KO ( n = 3) mice. Blue, LacZ signals. Scale bar, 100 μm. b Immunohistochemistry for β-Catenin in Control ( n = 3) and DTG ( n = 3) mammary glands at 12 weeks of age and 14.5 d.p.c. Arrows, nuclear localized β-Catenin. Scale bar, 25 μm. c Western blotting for β-Catenin in Control ( n = 3) and miR-31 KO ( n = 3) mice. GAPDH was used as a loading control. d Immunofluorescence for β-Catenin in HC11 mouse mammary epithelial cells in the presence of Dox ( miR-31 overexpression). Control, without Dox. Red, β-Catenin; Blue, DAPI. n = 3 technical replicates. e Western blotting for Wnt target LBH in miR-31 overexpressing (Dox) or miR-31 inhibited ( anti-miR-31 ) HC11 mammary epithelial cells. The Control HC11 cells were treated with Scramble RNA. n = 3 technical replicates. f TOP/FOPflash luciferase assays demonstrate that Dox induced miR-31 overexpression activates Wnt reporter gene expression and that miR-31 inhibitor ( anti-miR-31 ) markedly represses Wnt reporter gene expression. Scramble RNA was used as negative control (NC). n = 3 technical replicates. g Western blotting for Smad3, p-Smad2/3 and p21 in mammary epithelial cells of Control ( n = 3) and miR-31 KO ( n = 3) mice at 10 weeks of age. h qRT-PCR analysis for TGFβ components and downstream target genes, Cdkn2b (encoding p15), Cdkn1a (encoding p21), Cdkn1c (encoding p57) and Tgfbr1 in mammary epithelial cells of Control ( n = 3) and miR-31 KO ( n = 3) mice at 10 weeks of age. i HC11 mammary epithelial cells were transfected with CAGA luciferase reporter vector, combined with scrambled RNA (negative control, NC), miR-31 mimics or miR-31 inhibitor ( anti-miR-31 ) for 24 h and then harvested for luciferase activity determination. n = 3 biological replicates. Data represented as mean ± S.D. n = 3. Two tailed unpaired t -test for f , h , i (* P

    Techniques Used: Activity Assay, Mouse Assay, Immunohistochemistry, Western Blot, Immunofluorescence, Over Expression, Luciferase, Expressing, Negative Control, Quantitative RT-PCR, Transfection, Plasmid Preparation, Two Tailed Test

    Expression pattern of miR-31 in mammary gland and tumors. a qRT-PCR for miR-31 , miR-22 and miR205 in Lin - CD24 + CD29 high , Lin - CD24 + CD29 low , Lin - CD24 - CD29 + and Lin - CD24 - CD29 - populations at 12 weeks of age. n = 3 biological replicates. b In situ hybridization for miR-31 in 12-week-old WT mammary gland ducts and tertiary branches. DTG mammary ducts, a positive control. The DTG mice have been administered with Dox at 1 week of age. miR-31 KO mammary ducts, a negative control. Scale bar, 25 μm. c qRT-PCR for miR-31 in WT mammary epithelial cells at 6, 10 weeks, P14.5 (14.5 days post pregnancy), P18.5, L1 (1 day post lactation) and Inv (10 days post involution). n = 3 at each time points. d The schematic diagram showing two potential p65 (NF-κB) binding sites (-1746 bp and -1375 bp) in the miR-31 promoter. TSS, transcription start site. e , f qRT-PCR for miR-31 e and western blotting for RANKL, p-p65, p65 f in HC11 mouse mammary epithelial cells treated with RANKL siRNA (SiRANKL), and scramble RNA (NC). g Luciferase activity in lysates of HC11 mouse mammary epithelial cells transfected with luciferase reporter plasmids of pGL3-basic plasmid, miR-31 promoter or miR-31 mutant promoter with mutation at the -1375 (p65-mut-1) or -1746 (p65-mut-2) binding site, treated with scramble RNA (negative control, NC) and RANKL siRNA. h ChIP assays carried out on HC11 mammary epithelial cells using antibodies against p65 under indicated conditions. i – k In situ hybridization and qRT-PCR analysis for miR-31 i and western blotting for p-Ikkα, p-p65 and p65 j , and immunohistochemistry for RANKL k in WT mouse mammary gland and PyVT tumors. Scale bar, 25 μm. l western blotting for p-Akt, p-Ikkα, p-p65 and p65 in MCF7 breast cancer cells treated with Pten inhibitor bpV(pic) at indicated concentrations for 12 h. m qRT-PCR for miR-31 in MCF7 breast cancer cells treated with PTEN inhibitor bpV(pic) at indicated concentrations for 12 h. Data represented as mean ± S.D. Sample size: WT ( n = 3) and PyVT ( n = 3) for i – k . Two tailed unpaired t -test for e , g , i , m (* P
    Figure Legend Snippet: Expression pattern of miR-31 in mammary gland and tumors. a qRT-PCR for miR-31 , miR-22 and miR205 in Lin - CD24 + CD29 high , Lin - CD24 + CD29 low , Lin - CD24 - CD29 + and Lin - CD24 - CD29 - populations at 12 weeks of age. n = 3 biological replicates. b In situ hybridization for miR-31 in 12-week-old WT mammary gland ducts and tertiary branches. DTG mammary ducts, a positive control. The DTG mice have been administered with Dox at 1 week of age. miR-31 KO mammary ducts, a negative control. Scale bar, 25 μm. c qRT-PCR for miR-31 in WT mammary epithelial cells at 6, 10 weeks, P14.5 (14.5 days post pregnancy), P18.5, L1 (1 day post lactation) and Inv (10 days post involution). n = 3 at each time points. d The schematic diagram showing two potential p65 (NF-κB) binding sites (-1746 bp and -1375 bp) in the miR-31 promoter. TSS, transcription start site. e , f qRT-PCR for miR-31 e and western blotting for RANKL, p-p65, p65 f in HC11 mouse mammary epithelial cells treated with RANKL siRNA (SiRANKL), and scramble RNA (NC). g Luciferase activity in lysates of HC11 mouse mammary epithelial cells transfected with luciferase reporter plasmids of pGL3-basic plasmid, miR-31 promoter or miR-31 mutant promoter with mutation at the -1375 (p65-mut-1) or -1746 (p65-mut-2) binding site, treated with scramble RNA (negative control, NC) and RANKL siRNA. h ChIP assays carried out on HC11 mammary epithelial cells using antibodies against p65 under indicated conditions. i – k In situ hybridization and qRT-PCR analysis for miR-31 i and western blotting for p-Ikkα, p-p65 and p65 j , and immunohistochemistry for RANKL k in WT mouse mammary gland and PyVT tumors. Scale bar, 25 μm. l western blotting for p-Akt, p-Ikkα, p-p65 and p65 in MCF7 breast cancer cells treated with Pten inhibitor bpV(pic) at indicated concentrations for 12 h. m qRT-PCR for miR-31 in MCF7 breast cancer cells treated with PTEN inhibitor bpV(pic) at indicated concentrations for 12 h. Data represented as mean ± S.D. Sample size: WT ( n = 3) and PyVT ( n = 3) for i – k . Two tailed unpaired t -test for e , g , i , m (* P

    Techniques Used: Expressing, Quantitative RT-PCR, In Situ Hybridization, Positive Control, Mouse Assay, Negative Control, Binding Assay, Western Blot, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Chromatin Immunoprecipitation, Immunohistochemistry, Two Tailed Test

    21) Product Images from "Cryptochrome Genes Are Highly Expressed in the Ovary of the African Clawed Frog, Xenopus tropicalis"

    Article Title: Cryptochrome Genes Are Highly Expressed in the Ovary of the African Clawed Frog, Xenopus tropicalis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0009273

    Cry mRNA levels and their daily variations in X. tropicalis tissues estimated by quantitative RT-PCR. Each tissue (n = 4) was collected at ZT6 and ZT18. Each Cry mRNA level was calculated as a value relative to that of the Xtβ2M or XtHprt1 gene. Error bars represent ±SEM. (A) Daily changes in the Cry mRNA levels in eleven tissues. Messenger RNA levels are shown as a ratio to XtHprt1 mRNA levels, which showed relatively small changes between ZT6 and ZT18 in many tissues (except for the ovary). The Gusb gene was used as another internal control gene. * p
    Figure Legend Snippet: Cry mRNA levels and their daily variations in X. tropicalis tissues estimated by quantitative RT-PCR. Each tissue (n = 4) was collected at ZT6 and ZT18. Each Cry mRNA level was calculated as a value relative to that of the Xtβ2M or XtHprt1 gene. Error bars represent ±SEM. (A) Daily changes in the Cry mRNA levels in eleven tissues. Messenger RNA levels are shown as a ratio to XtHprt1 mRNA levels, which showed relatively small changes between ZT6 and ZT18 in many tissues (except for the ovary). The Gusb gene was used as another internal control gene. * p

    Techniques Used: Quantitative RT-PCR

    22) Product Images from "Induction of E6AP by microRNA-302c dysregulation inhibits TGF-β-dependent fibrogenesis in hepatic stellate cells"

    Article Title: Induction of E6AP by microRNA-302c dysregulation inhibits TGF-β-dependent fibrogenesis in hepatic stellate cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-57322-w

    Decreased miR-302c level upon TGF-β treatment. ( A ) The locations of the predicted miRNA binding sites within the 3′-UTR of E6AP mRNA. ( B ) Real-time RT-PCR assays for the miR-302a/b/c/d-3p in quiescent or activated primary HSCs. Data were normalized against the levels of U6 small RNA. The data represents the mean ± SE ( n = 3, significant as compared with primary quiescent HSCs, * p
    Figure Legend Snippet: Decreased miR-302c level upon TGF-β treatment. ( A ) The locations of the predicted miRNA binding sites within the 3′-UTR of E6AP mRNA. ( B ) Real-time RT-PCR assays for the miR-302a/b/c/d-3p in quiescent or activated primary HSCs. Data were normalized against the levels of U6 small RNA. The data represents the mean ± SE ( n = 3, significant as compared with primary quiescent HSCs, * p

    Techniques Used: Binding Assay, Quantitative RT-PCR

    23) Product Images from "EFEMP1 is repressed by estrogen and inhibits the epithelial-mesenchymal transition via Wnt/β-catenin signaling in endometrial carcinoma"

    Article Title: EFEMP1 is repressed by estrogen and inhibits the epithelial-mesenchymal transition via Wnt/β-catenin signaling in endometrial carcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8263

    EFEMP1 suppresses EMT in EC cells A. Immunoblotting analysis of the expression of the four EMT-related markers (E-cadherin, Vimentin, Snail and β-catenin) in HEC-1B and RL95-2 cells transfected with plasmids expressing exEFEMP1 and shEFEMP1, respectively. β-actin was used as an internal control. B. The changes in cellular morphology induced by TGF-β that resulted in EMT were evaluated in different groups using phase contrast microscopy. C. Four cell types, HEC-1B-NC, HEC-1B-exEFEMP1, RL95-2-NC and RL95-2-shEFEMP1, were incubated for 48 h in the absence or presence of TGF-β (2 ng/ml) to induce EMT. Then, total RNA was isolated and subjected to quantitative real-time PCR to monitor the expression of EMT-related marker transcripts, which were normalized to β-actin levels. Data shown are the mean (±SD; n=3), *p
    Figure Legend Snippet: EFEMP1 suppresses EMT in EC cells A. Immunoblotting analysis of the expression of the four EMT-related markers (E-cadherin, Vimentin, Snail and β-catenin) in HEC-1B and RL95-2 cells transfected with plasmids expressing exEFEMP1 and shEFEMP1, respectively. β-actin was used as an internal control. B. The changes in cellular morphology induced by TGF-β that resulted in EMT were evaluated in different groups using phase contrast microscopy. C. Four cell types, HEC-1B-NC, HEC-1B-exEFEMP1, RL95-2-NC and RL95-2-shEFEMP1, were incubated for 48 h in the absence or presence of TGF-β (2 ng/ml) to induce EMT. Then, total RNA was isolated and subjected to quantitative real-time PCR to monitor the expression of EMT-related marker transcripts, which were normalized to β-actin levels. Data shown are the mean (±SD; n=3), *p

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

    24) Product Images from "Chromatin-associated RNAi components contribute to transcriptional regulation in Drosophila"

    Article Title: Chromatin-associated RNAi components contribute to transcriptional regulation in Drosophila

    Journal: Nature

    doi: 10.1038/nature10492

    Pol II promoter-proximal pausing on hsp70 is reduced in Dcr2 RNAi cells a-c ) Quantitative RT-PCR analysis of heat shock gene transcripts. RNA from S2 cells treated with EGFP dsRNA (control) or Dcr2 dsRNA ( a ), or AGO2 dsRNA ( b ) were analyzed with primers specific for the indicated heat shock genes. c ) Induction of Dcr2-flag transgene is able to revert the phenotype induced by Dcr2-depletion. S2 cells stably transformed with a Dcr2-flag transgene were treated with EGFP dsRNA (control) or Dcr2 dsRNA. The Dcr2-flag expression is induced only in the Dcr2 RNAi sample by the addition of copper. The samples were analyzed before and after 72h induction of the transgene. The transcript levels are shown with respect to the EGFP control (experiment/control ratio). n=3, bars represent the mean±standard deviation. d ) hsp70 DNA-FISH on polytene chromosomes from wild type (WT), homozygous Dcr2 L811fsX or homozygous Ago2 414 mutant larvae; lower pictures show DNA staining; upper pictures show the merge of DNA (blue) and FISH (green) signals. e ) Schematic representation of the hsp70 transcription unit with the position of the PCR amplicons used in this study. The numbers indicate the middle of each amplicon with respect to the transcription start site. f-h ) ChIP analysis of the hsp70 heat shock gene. Chromatin from S2 cells or S2 cells after exposure to heat shock (HS) was immunoprecipitated with anti-Pol II 4H8 (recognises the Ser5-phosphorylated CTD domain), anti-Dcr2 or anti-AGO2 antibodies. n=3, bars represent the mean± standard deviation. i-j) ChIP analysis of the hsp70 heat shock gene. Chromatin from S2 cells treated with EGFP dsRNA (control) or Dcr2 dsRNA was immunoprecipitated with anti-Pol II 4H8 or anti-NELF-E antibodies. The resulting DNA has been analyzed by quantitative PCR. Protein binding is expressed as a percentage of input subtracted by the background signal. n=3, bars represent the mean± standard deviation. Differences in Pol II ChIP values in 2f and 2i are due to different batches of antibody used in these assays. k) Permanganate mapping of open transcription bubbles on hsp70 . Permanganate reacts with single-stranded thymine residues, like those in a open transcription bubble, revealing a transcriptionally engaged RNA polymerase. The autoradiograph includes a G/A ladder, used to determine the position of the bands, and the permanganate reactivity of thymine residues observed in S2 cells treated with EGFP dsRNA (control) or Dcr2 dsRNA. The hyper-reactive thymines +22 and +30 are labeled. Two independent biological samples have been analyzed. Shown is a representative picture l) Quantification of the autoradiograph.
    Figure Legend Snippet: Pol II promoter-proximal pausing on hsp70 is reduced in Dcr2 RNAi cells a-c ) Quantitative RT-PCR analysis of heat shock gene transcripts. RNA from S2 cells treated with EGFP dsRNA (control) or Dcr2 dsRNA ( a ), or AGO2 dsRNA ( b ) were analyzed with primers specific for the indicated heat shock genes. c ) Induction of Dcr2-flag transgene is able to revert the phenotype induced by Dcr2-depletion. S2 cells stably transformed with a Dcr2-flag transgene were treated with EGFP dsRNA (control) or Dcr2 dsRNA. The Dcr2-flag expression is induced only in the Dcr2 RNAi sample by the addition of copper. The samples were analyzed before and after 72h induction of the transgene. The transcript levels are shown with respect to the EGFP control (experiment/control ratio). n=3, bars represent the mean±standard deviation. d ) hsp70 DNA-FISH on polytene chromosomes from wild type (WT), homozygous Dcr2 L811fsX or homozygous Ago2 414 mutant larvae; lower pictures show DNA staining; upper pictures show the merge of DNA (blue) and FISH (green) signals. e ) Schematic representation of the hsp70 transcription unit with the position of the PCR amplicons used in this study. The numbers indicate the middle of each amplicon with respect to the transcription start site. f-h ) ChIP analysis of the hsp70 heat shock gene. Chromatin from S2 cells or S2 cells after exposure to heat shock (HS) was immunoprecipitated with anti-Pol II 4H8 (recognises the Ser5-phosphorylated CTD domain), anti-Dcr2 or anti-AGO2 antibodies. n=3, bars represent the mean± standard deviation. i-j) ChIP analysis of the hsp70 heat shock gene. Chromatin from S2 cells treated with EGFP dsRNA (control) or Dcr2 dsRNA was immunoprecipitated with anti-Pol II 4H8 or anti-NELF-E antibodies. The resulting DNA has been analyzed by quantitative PCR. Protein binding is expressed as a percentage of input subtracted by the background signal. n=3, bars represent the mean± standard deviation. Differences in Pol II ChIP values in 2f and 2i are due to different batches of antibody used in these assays. k) Permanganate mapping of open transcription bubbles on hsp70 . Permanganate reacts with single-stranded thymine residues, like those in a open transcription bubble, revealing a transcriptionally engaged RNA polymerase. The autoradiograph includes a G/A ladder, used to determine the position of the bands, and the permanganate reactivity of thymine residues observed in S2 cells treated with EGFP dsRNA (control) or Dcr2 dsRNA. The hyper-reactive thymines +22 and +30 are labeled. Two independent biological samples have been analyzed. Shown is a representative picture l) Quantification of the autoradiograph.

    Techniques Used: Quantitative RT-PCR, Stable Transfection, Transformation Assay, Expressing, Standard Deviation, Fluorescence In Situ Hybridization, Mutagenesis, Staining, Polymerase Chain Reaction, Amplification, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Protein Binding, Autoradiography, Labeling

    25) Product Images from "The Hepatitis Delta Virus accumulation requires paraspeckle components and affects NEAT1 level and PSP1 localization"

    Article Title: The Hepatitis Delta Virus accumulation requires paraspeckle components and affects NEAT1 level and PSP1 localization

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24500-1

    HDV replication decreases PSP1 co-localization with NEAT1 . ( a ) Co-localization of PSP1 and NEAT1 foci in 293-HDV cells treated or not with tetracycline, as demonstrated by immunostaining using an antibody against PSP1 (green) and in situ hybridization for NEAT1 (red). DAPI (blue) was used to visualize the nucleus. Arrows indicate foci where PSP1 co-localized with NEAT1 . ( b ) Orthogonal view of the 293-HDV cell not treated with tetracycline cell displayed in ( a ), as demonstrated by immunostaining using an antibody against PSP1 (green) and in situ hybridization for NEAT1 (red). DAPI (blue) was used to visualize the nucleus. ( c ) Percentage of NEAT1 foci co-localizing with PSP1 foci in 293-HDV cells treated or not with tetracycline. Average and standard deviation was calculated on 25 randomly selected cells per condition from two independent experiments. ( d ) Decrease of the interaction between NEAT1_2 and PSP1 upon HDV replication in HEK-293 cells. 293-HDV cells were treated (+) or not (−) with tetracycline and the complexes cross-linked with formaldehyde. The lysates were used for RIP using α-PSF and α-PSP1 antibodies, and the α-β actin antibody was used as a negative control. The isolated RNA was reverse-transcribed with random primers and amplified by PCR with primers targeting the NEAT1_2 gene. The resulting PCR product was resolved on an agarose gel. The 100 bp DNA Ladder (NEB) was used as marker.
    Figure Legend Snippet: HDV replication decreases PSP1 co-localization with NEAT1 . ( a ) Co-localization of PSP1 and NEAT1 foci in 293-HDV cells treated or not with tetracycline, as demonstrated by immunostaining using an antibody against PSP1 (green) and in situ hybridization for NEAT1 (red). DAPI (blue) was used to visualize the nucleus. Arrows indicate foci where PSP1 co-localized with NEAT1 . ( b ) Orthogonal view of the 293-HDV cell not treated with tetracycline cell displayed in ( a ), as demonstrated by immunostaining using an antibody against PSP1 (green) and in situ hybridization for NEAT1 (red). DAPI (blue) was used to visualize the nucleus. ( c ) Percentage of NEAT1 foci co-localizing with PSP1 foci in 293-HDV cells treated or not with tetracycline. Average and standard deviation was calculated on 25 randomly selected cells per condition from two independent experiments. ( d ) Decrease of the interaction between NEAT1_2 and PSP1 upon HDV replication in HEK-293 cells. 293-HDV cells were treated (+) or not (−) with tetracycline and the complexes cross-linked with formaldehyde. The lysates were used for RIP using α-PSF and α-PSP1 antibodies, and the α-β actin antibody was used as a negative control. The isolated RNA was reverse-transcribed with random primers and amplified by PCR with primers targeting the NEAT1_2 gene. The resulting PCR product was resolved on an agarose gel. The 100 bp DNA Ladder (NEB) was used as marker.

    Techniques Used: Immunostaining, In Situ Hybridization, Standard Deviation, Negative Control, Isolation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker

    Association of HDV RNA with PSF, p54nrb and PSP1 in HEK-293 cells. HDV replication in 293-HDV cells was induced (+) or not (−) by tetracycline, then the cells were treated with formaldehyde to cross-link the RNA-protein complexes and lyzed. The lysates were used for RIP using α-PSF, α-p54nrb and α-PSP1 antibodies. The α-β-actin antibody was used for RIP as a negative control. Following co-immunoprecipitation, the cross-links were reversed by heating the samples and RT-PCR was carried out to amplify a 300 bp fragment of the HDV RNA genome located on the terminal domain containing the ribozyme sequences. The resulting PCR product was resolved on an agarose gel. The 100 bp DNA Ladder (NEB) was used as marker.
    Figure Legend Snippet: Association of HDV RNA with PSF, p54nrb and PSP1 in HEK-293 cells. HDV replication in 293-HDV cells was induced (+) or not (−) by tetracycline, then the cells were treated with formaldehyde to cross-link the RNA-protein complexes and lyzed. The lysates were used for RIP using α-PSF, α-p54nrb and α-PSP1 antibodies. The α-β-actin antibody was used for RIP as a negative control. Following co-immunoprecipitation, the cross-links were reversed by heating the samples and RT-PCR was carried out to amplify a 300 bp fragment of the HDV RNA genome located on the terminal domain containing the ribozyme sequences. The resulting PCR product was resolved on an agarose gel. The 100 bp DNA Ladder (NEB) was used as marker.

    Techniques Used: Negative Control, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker

    26) Product Images from "Analysis of the Salmonella regulatory network suggests involvement of SsrB and H-NS in σE-regulated SPI-2 gene expression"

    Article Title: Analysis of the Salmonella regulatory network suggests involvement of SsrB and H-NS in σE-regulated SPI-2 gene expression

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.00027

    Validation of microarray results by qRT-PCR . Groups of genes both up- and down-regulated by σ E were selected from three growth conditions (LB log, LPM 4 h, and LPM 20 h) based on microarray results, and validated by qRT-PCR using primers designed inside those genes. The results are plotted on a log 2 -scale comparing WT strain to rpoE -deletion strains. Values are normalized with gyrB mRNA levels and represent the average of RNA prepared from independent biological triplicates.
    Figure Legend Snippet: Validation of microarray results by qRT-PCR . Groups of genes both up- and down-regulated by σ E were selected from three growth conditions (LB log, LPM 4 h, and LPM 20 h) based on microarray results, and validated by qRT-PCR using primers designed inside those genes. The results are plotted on a log 2 -scale comparing WT strain to rpoE -deletion strains. Values are normalized with gyrB mRNA levels and represent the average of RNA prepared from independent biological triplicates.

    Techniques Used: Microarray, Quantitative RT-PCR

    27) Product Images from "PIAS1 protects against myocardial ischemia-reperfusion injury by stimulating PPARγ SUMOylation"

    Article Title: PIAS1 protects against myocardial ischemia-reperfusion injury by stimulating PPARγ SUMOylation

    Journal: BMC Cell Biology

    doi: 10.1186/s12860-018-0176-x

    PPARy SUMOylation antagonizes injury after H/R by suppressing NF-κB activation. a H9C2 cells were cultured under normoxic or H/R conditions, and cell lysates were then immunoprecipitated with anti-PPARγ antibody, followed by blotting with anti-SUMO1 or anti-PPARγ antibody. Whole-cell lysates were blotted with anti-SUMO1 antibody. b 293 T cells were cotransfected with HA-SUMO1 and Flag-PPARγ wild-type or SUMO site mutants (K77R and K365R) plasmids as indicated. The cell lysates were immunoprecipitated with anti-Flag antibody, followed by blotting with anti-HA or anti-Flag antibody. Whole-cell lysates were blotted with anti-HA antibody. c PPARγ-knockdown H9C2 cells were constructed by sh-RNA lentivirus. Sh-PPARγ cells were transfected with vector, PPARγ-WT or K77R plasmids as indicated; then, cells were cultured under normoxic or H/R conditions. Cells lysates were blotted with anti-PPARγ or anti-GAPDH antibody. d TUNEL staining of apoptotic H9C2 cells transfected with vector, PPARγ-WT and K77R plasmids as indicated under normoxic or H/R conditions. Scale bars, 100um. e Quantification of apoptotic rates based on TUNEL staining ( n = 5). f RT-PCR analysis of IL-1β, IL-6 and TNFα in sh-PPARγ cells transfected with vector, PPARγ-WT and K77R plasmids as indicated after being subjected to normoxic or H/R conditions ( n = 3). g Western blot analysis of phosphorylated IκBα protein in sh-PPARγ cells transfected with vector, PPARγ-WT and K77R plasmids as indicated after being subjected to normoxic or H/R conditions. Quantification of the densitometry of the western blot band is shown above ( n = 3). h Representative immunofluorescence images of p65 proteins (green) in sh-PPARγ cells transfected with vector, PPARγ-WT and K77R plasmids as indicated under normoxic or H/R conditions. Scale bars, 100um. i Quantification of nuclear p65-positive cells based on TUNEL staining ( n = 5). Experiments in a , b , c , f and g were performed three times, and experiments in d , e , h and i were performed five times. Data presented are means ± SD, ** P
    Figure Legend Snippet: PPARy SUMOylation antagonizes injury after H/R by suppressing NF-κB activation. a H9C2 cells were cultured under normoxic or H/R conditions, and cell lysates were then immunoprecipitated with anti-PPARγ antibody, followed by blotting with anti-SUMO1 or anti-PPARγ antibody. Whole-cell lysates were blotted with anti-SUMO1 antibody. b 293 T cells were cotransfected with HA-SUMO1 and Flag-PPARγ wild-type or SUMO site mutants (K77R and K365R) plasmids as indicated. The cell lysates were immunoprecipitated with anti-Flag antibody, followed by blotting with anti-HA or anti-Flag antibody. Whole-cell lysates were blotted with anti-HA antibody. c PPARγ-knockdown H9C2 cells were constructed by sh-RNA lentivirus. Sh-PPARγ cells were transfected with vector, PPARγ-WT or K77R plasmids as indicated; then, cells were cultured under normoxic or H/R conditions. Cells lysates were blotted with anti-PPARγ or anti-GAPDH antibody. d TUNEL staining of apoptotic H9C2 cells transfected with vector, PPARγ-WT and K77R plasmids as indicated under normoxic or H/R conditions. Scale bars, 100um. e Quantification of apoptotic rates based on TUNEL staining ( n = 5). f RT-PCR analysis of IL-1β, IL-6 and TNFα in sh-PPARγ cells transfected with vector, PPARγ-WT and K77R plasmids as indicated after being subjected to normoxic or H/R conditions ( n = 3). g Western blot analysis of phosphorylated IκBα protein in sh-PPARγ cells transfected with vector, PPARγ-WT and K77R plasmids as indicated after being subjected to normoxic or H/R conditions. Quantification of the densitometry of the western blot band is shown above ( n = 3). h Representative immunofluorescence images of p65 proteins (green) in sh-PPARγ cells transfected with vector, PPARγ-WT and K77R plasmids as indicated under normoxic or H/R conditions. Scale bars, 100um. i Quantification of nuclear p65-positive cells based on TUNEL staining ( n = 5). Experiments in a , b , c , f and g were performed three times, and experiments in d , e , h and i were performed five times. Data presented are means ± SD, ** P

    Techniques Used: Activation Assay, Cell Culture, Immunoprecipitation, Construct, Transfection, Plasmid Preparation, TUNEL Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence

    28) Product Images from "Stable Form of JAB1 Enhances Proliferation and Maintenance of Hematopoietic Progenitors *"

    Article Title: Stable Form of JAB1 Enhances Proliferation and Maintenance of Hematopoietic Progenitors *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M804539200

    Down-regulation of p16 INK4a transcription in bone marrow of Jab1 transgenic mice. A and B , total RNA was isolated from bone marrow cells of WT, JAB1-Tg, and JAB1 heterozygous ( Jab1 +/– ) mice and subjected to a quantitative RT-PCR analysis ( A ). The reactions were performed within a linear range, and the results were confirmed with the real time PCR technique. Quantified results are the means ± S.D. of three independent experiments ( B ). The relative amount normalized to the expression level of β-actin in wild-type cells was set to 1.0. C , total RNA was isolated from KSL cells, which were sorted from bone marrow of WT and JAB1-Tg mice by flow cytometer and subjected to a quantitative RT-PCR analysis. Total JAB1 mRNA was measured as a control.
    Figure Legend Snippet: Down-regulation of p16 INK4a transcription in bone marrow of Jab1 transgenic mice. A and B , total RNA was isolated from bone marrow cells of WT, JAB1-Tg, and JAB1 heterozygous ( Jab1 +/– ) mice and subjected to a quantitative RT-PCR analysis ( A ). The reactions were performed within a linear range, and the results were confirmed with the real time PCR technique. Quantified results are the means ± S.D. of three independent experiments ( B ). The relative amount normalized to the expression level of β-actin in wild-type cells was set to 1.0. C , total RNA was isolated from KSL cells, which were sorted from bone marrow of WT and JAB1-Tg mice by flow cytometer and subjected to a quantitative RT-PCR analysis. Total JAB1 mRNA was measured as a control.

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

    29) Product Images from "The adhesion molecule L1 regulates transendothelial migration and trafficking of dendritic cells"

    Article Title: The adhesion molecule L1 regulates transendothelial migration and trafficking of dendritic cells

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20081211

    TNF-α induces L1 expression in endothelium. (A) HUVEC or 1G11 cells were starved of serum and endothelial growth factors and then treated with 20 ng/ml TNF-α for 3 h, followed by FACS analysis for L1 expression. (B) HUVEC were treated with 20 ng/ml TNF-α for the indicated time lengths, followed by FACS analysis for L1 expression. The data refer to the percentage of L1-positive cells in a representative experiment. Each experiment was repeated three times with similar results. (C) HUVECs were treated with 20 ng/ml TNF-α for the indicated time lengths before isolation of RNA and quantitative RT-PCR analysis for L1 expression. Data represent the means ± SEM of three experiments performed. *, P
    Figure Legend Snippet: TNF-α induces L1 expression in endothelium. (A) HUVEC or 1G11 cells were starved of serum and endothelial growth factors and then treated with 20 ng/ml TNF-α for 3 h, followed by FACS analysis for L1 expression. (B) HUVEC were treated with 20 ng/ml TNF-α for the indicated time lengths, followed by FACS analysis for L1 expression. The data refer to the percentage of L1-positive cells in a representative experiment. Each experiment was repeated three times with similar results. (C) HUVECs were treated with 20 ng/ml TNF-α for the indicated time lengths before isolation of RNA and quantitative RT-PCR analysis for L1 expression. Data represent the means ± SEM of three experiments performed. *, P

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

    30) Product Images from "Dihydroartemisinin Exerts Its Anticancer Activity through Depleting Cellular Iron via Transferrin Receptor-1"

    Article Title: Dihydroartemisinin Exerts Its Anticancer Activity through Depleting Cellular Iron via Transferrin Receptor-1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042703

    DHA caused iron depletion and weakened iron uptake pathway. (A) HepG2 cells treated with different concentrations of DHA for 24 hours or with 25 µM DHA for different times were harvested. Total proteins were extracted and western blotting was performed. (B) HepG2 cells were pretreated with DFO (250 µM) or FAC (100 µg/ml) or left untreated for 30 min and then DHA (25 µM) were added to further treatment. After 24 hours, cell lysates were prepared and immunoblotted. (C) HepG2 cells were treated as (A) and harvested to extract total RNA. The mRNA levels of endogenous DMT1, Steap3 and Fpn1 were determined by quantitative RT-PCR. *, P
    Figure Legend Snippet: DHA caused iron depletion and weakened iron uptake pathway. (A) HepG2 cells treated with different concentrations of DHA for 24 hours or with 25 µM DHA for different times were harvested. Total proteins were extracted and western blotting was performed. (B) HepG2 cells were pretreated with DFO (250 µM) or FAC (100 µg/ml) or left untreated for 30 min and then DHA (25 µM) were added to further treatment. After 24 hours, cell lysates were prepared and immunoblotted. (C) HepG2 cells were treated as (A) and harvested to extract total RNA. The mRNA levels of endogenous DMT1, Steap3 and Fpn1 were determined by quantitative RT-PCR. *, P

    Techniques Used: Western Blot, Quantitative RT-PCR

    The effects of DHA were partially dependent on TfR1 internalization. (A) HepG2 cells were preincubated with 25 µg/ml nystatin or not for 30 min and treated with 25 µM DHA for another 4 hours. Cells were lysed to western blot assay. (B) HepG2 cells were preincubated with 25 µg/ml nystatin or not for 30 min and treated with 25 µM DHA for another 24 hours. Total RNA was harvested and quantitative RT-PCR was performed. *, P
    Figure Legend Snippet: The effects of DHA were partially dependent on TfR1 internalization. (A) HepG2 cells were preincubated with 25 µg/ml nystatin or not for 30 min and treated with 25 µM DHA for another 4 hours. Cells were lysed to western blot assay. (B) HepG2 cells were preincubated with 25 µg/ml nystatin or not for 30 min and treated with 25 µM DHA for another 24 hours. Total RNA was harvested and quantitative RT-PCR was performed. *, P

    Techniques Used: Western Blot, Quantitative RT-PCR

    31) Product Images from "Che-1 sustains hypoxic response of colorectal cancer cells by affecting Hif-1α stabilization"

    Article Title: Che-1 sustains hypoxic response of colorectal cancer cells by affecting Hif-1α stabilization

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0497-1

    Che-1 modulates HIF-1α protein expression. a WB analysis with the indicated Abs of TCEs from HCT116 and HT29 cells transiently transfected with stealth siRNA negative control (siControl) or siRNA Che-1 (siChe-1) and exposed to hypoxia where indicated. b qRT–PCR performed in HCT116 cells transiently transfected and treated as in a . Values were normalized to RPL19 mRNA expression. Error bars represent the standard error of three different experiments. * P ≤ 0,02, ** P ≤ 0.01. c WB analysis with the indicated Abs of TCEs from HCT116 cells transiently transfected as in a . d SIAH-2 expression levels are reported as FPKM reconstructed from Cufflinks RNA-Seq analysis from HCT116 transiently transfected with siControl or siChe-1 and exposed to hypoxia. e qRT–PCR performed as in b . Values were normalized to RPL19 mRNA expression. Error bars represent the standard error of three different experiments. * P ≤ 0,02, n.s., not significant
    Figure Legend Snippet: Che-1 modulates HIF-1α protein expression. a WB analysis with the indicated Abs of TCEs from HCT116 and HT29 cells transiently transfected with stealth siRNA negative control (siControl) or siRNA Che-1 (siChe-1) and exposed to hypoxia where indicated. b qRT–PCR performed in HCT116 cells transiently transfected and treated as in a . Values were normalized to RPL19 mRNA expression. Error bars represent the standard error of three different experiments. * P ≤ 0,02, ** P ≤ 0.01. c WB analysis with the indicated Abs of TCEs from HCT116 cells transiently transfected as in a . d SIAH-2 expression levels are reported as FPKM reconstructed from Cufflinks RNA-Seq analysis from HCT116 transiently transfected with siControl or siChe-1 and exposed to hypoxia. e qRT–PCR performed as in b . Values were normalized to RPL19 mRNA expression. Error bars represent the standard error of three different experiments. * P ≤ 0,02, n.s., not significant

    Techniques Used: Expressing, Western Blot, Transfection, Negative Control, Quantitative RT-PCR, RNA Sequencing Assay

    32) Product Images from "Ectopic Overexpression of SsCBF1, a CRT/DRE-Binding Factor from the Nightshade Plant Solanum lycopersicoides, Confers Freezing and Salt Tolerance in Transgenic Arabidopsis"

    Article Title: Ectopic Overexpression of SsCBF1, a CRT/DRE-Binding Factor from the Nightshade Plant Solanum lycopersicoides, Confers Freezing and Salt Tolerance in Transgenic Arabidopsis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061810

    Freezing tolerance and delayed flowering phenotype of 35S:SsCBF1 transgenic Arabidopsis . ( A ) Schematic representation of the construct used for Arabidopsis transformation of the SsCBF1 gene. ( B ) Relative expression of SsCBF1 in Col-0 and two T 3 generation transgenic lines (#11 and #18). Total RNA was extracted from 10-day-old seedlings, then analyzed by semi-quantitative RT-PCR. ACTIN7 gene was used as an internal control. ( C ) Comparison of freezing tolerance between Col-0 and two transgenic lines. See Methods for details. The right diagram indicates different genotypes used in the assay. ( D ) Late flowering phenotype of transgenic Arabidopsis overexpressing SsCBF1 . Col-0 and two SsCBF1 OE lines were grown under the same conditions as described in Methods. Four-week-old plants were photographed.
    Figure Legend Snippet: Freezing tolerance and delayed flowering phenotype of 35S:SsCBF1 transgenic Arabidopsis . ( A ) Schematic representation of the construct used for Arabidopsis transformation of the SsCBF1 gene. ( B ) Relative expression of SsCBF1 in Col-0 and two T 3 generation transgenic lines (#11 and #18). Total RNA was extracted from 10-day-old seedlings, then analyzed by semi-quantitative RT-PCR. ACTIN7 gene was used as an internal control. ( C ) Comparison of freezing tolerance between Col-0 and two transgenic lines. See Methods for details. The right diagram indicates different genotypes used in the assay. ( D ) Late flowering phenotype of transgenic Arabidopsis overexpressing SsCBF1 . Col-0 and two SsCBF1 OE lines were grown under the same conditions as described in Methods. Four-week-old plants were photographed.

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

    SsCBF1 transcript levels in response to various abiotic stresses. ( A ) Low-temperature induced expression pattern of SsCBF1 in S. lycopersicoides . S. lycopersicoides seedlings grown under standard conditions were transferred to a climate chamber set at 4°C for a 24 time course under constant light. The aerial parts were harvested for RNA extraction and qRT-PCR analysis. Zero time samples were taken prior to treatment. Transcript levels of SsCBF1 were normalized to the ACTIN2 expression. ( B ) Drought induced expression pattern of SsCBF1 in S. lycopersicoides . Detached young leaves of S. lycopersicoides were placed on a dry filter paper for drought treatment. Samples were collected according to the time course. The SsCBF1 mRNA levels were analyzed as in (A). ( C ) High-salinity induced expression pattern of SsCBF1 in S. lycopersicoides . Detached young leaves of S. lycopersicoides were placed on the filter paper soaked with NaCl solution (250 mM, 0.02% Tween-20) for a high-salinity treatment. Samples were collected at the indicated time points. The SsCBF1 mRNA levels were analyzed as in (A). Data shown are average and SD of triplicate reactions. Shown are representative data from one biological replicate and three biological replicates were conducted with similar results.
    Figure Legend Snippet: SsCBF1 transcript levels in response to various abiotic stresses. ( A ) Low-temperature induced expression pattern of SsCBF1 in S. lycopersicoides . S. lycopersicoides seedlings grown under standard conditions were transferred to a climate chamber set at 4°C for a 24 time course under constant light. The aerial parts were harvested for RNA extraction and qRT-PCR analysis. Zero time samples were taken prior to treatment. Transcript levels of SsCBF1 were normalized to the ACTIN2 expression. ( B ) Drought induced expression pattern of SsCBF1 in S. lycopersicoides . Detached young leaves of S. lycopersicoides were placed on a dry filter paper for drought treatment. Samples were collected according to the time course. The SsCBF1 mRNA levels were analyzed as in (A). ( C ) High-salinity induced expression pattern of SsCBF1 in S. lycopersicoides . Detached young leaves of S. lycopersicoides were placed on the filter paper soaked with NaCl solution (250 mM, 0.02% Tween-20) for a high-salinity treatment. Samples were collected at the indicated time points. The SsCBF1 mRNA levels were analyzed as in (A). Data shown are average and SD of triplicate reactions. Shown are representative data from one biological replicate and three biological replicates were conducted with similar results.

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

    33) Product Images from "Protocadherin-18 Is a Novel Differentiation Marker and an Inhibitory Signaling Receptor for CD8+ Effector Memory T Cells"

    Article Title: Protocadherin-18 Is a Novel Differentiation Marker and an Inhibitory Signaling Receptor for CD8+ Effector Memory T Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036101

    Protocadherin qRT-PCR and gene array analyses in activated Cm cells. (5a) pcdh18 qRT-PCR analysis of FACS-purified Cm and Em cells from young, aged, or ‘memory’ mice (from Fig. 4d ) was as indicated (bottom panels). Gene array of FACS-purified Cm cells from a pool of 10 aged mice was performed by the NYU Center for Health Informatics and Bioinformatics. Purified Cm were activated in vitro and total cells in each culture were isolated for RNA extraction. cDNAs were hybridized to GeneChip arrays using an Affymatrix platform and the data were processed as described in Materials and Methods . (5b) Bone marrow CD8 + T cells were purified by positive selection magnetic immunobeading from young or aged mice, activated in vitro with ConA, and qRT-PCR performed as before.
    Figure Legend Snippet: Protocadherin qRT-PCR and gene array analyses in activated Cm cells. (5a) pcdh18 qRT-PCR analysis of FACS-purified Cm and Em cells from young, aged, or ‘memory’ mice (from Fig. 4d ) was as indicated (bottom panels). Gene array of FACS-purified Cm cells from a pool of 10 aged mice was performed by the NYU Center for Health Informatics and Bioinformatics. Purified Cm were activated in vitro and total cells in each culture were isolated for RNA extraction. cDNAs were hybridized to GeneChip arrays using an Affymatrix platform and the data were processed as described in Materials and Methods . (5b) Bone marrow CD8 + T cells were purified by positive selection magnetic immunobeading from young or aged mice, activated in vitro with ConA, and qRT-PCR performed as before.

    Techniques Used: Quantitative RT-PCR, FACS, Purification, Mouse Assay, In Vitro, Isolation, RNA Extraction, Selection

    RT-PCR analysis of protocadherin 18 in tissues and immune cells. (2a) Amino acid sequence comparison of the p56 lck Y505 motif in protocadherins. (2b) RT-PCR analysis of mouse tissues. The indicated tissues and organs were isolated from a control mouse, RNA was extracted and used to prepare cDNA, and PCR performed using control (pcdh8 and β-actin) or pch18 primers as described in ‘ Materials and Methods ’. (2c) RT-PCR analysis of spleen cells. Spleens were isolated from a 7 week old mouse and the indicated cells were purified by FACS (top panel). TIL were isolated from an MCA38 tumor. CTLL-2 is a CD8 + lytic cell line. RNA was isolated and used to program RT-PCR as described in ‘ Materials and Methods ’. Similarly spleen CD8 + T cells were purified and activated in vitro with anti-CD3 for the indicated times before analysis (bottom panel). (2d) TIL qRT-PCR analyses. Single cell suspensions of MCA38 tumors were prepared and CD4 + or CD8 + TIL were isolated by magnetic immunobeading. (Liver tissue was isolated and used for RNA isolation in certain control analyses. TIL were labeled with anti-CD4 or CD8 Ab and further purified by FACS (example of flow cytometry analysis shown in left panels) before RNA isolation and qRT-PCR analysis. TIL used to prepare RNA immediately after isolation are indicated in as ‘nonlytic’ or ‘TIL 0 hr’. As indicated some TIL samples were cultured in vitro for 8 or 24 h before RNA isolation during which time TIL recover both proximal TCR-mediated signaling and lytic function [5] , [7] . PCR analyses of various target RNAs are shown and include several control reactions that demonstrate specificity of the expression patterns observed (e.g. pcdh18 and pcdh12 in CD8 + TIL, Dab1 and Dab2a in CD8 + TIL, granzyme B in CD8 + TIL and liver, as well as TNF, IFN, IL-2, PD-1, and PD-1L). Data show SD from three independent experiments. (2e) qRT-PCR analysis of purified spleen cells. Spleen immune cells were isolated by FACS from young (4 week) or old ( > 48 week) control mice and RNA was isolated and used to program pcdh18 qRT-PCR as described in ‘ Materials and Methods ’. The data shown are representative of multiple repetitions.
    Figure Legend Snippet: RT-PCR analysis of protocadherin 18 in tissues and immune cells. (2a) Amino acid sequence comparison of the p56 lck Y505 motif in protocadherins. (2b) RT-PCR analysis of mouse tissues. The indicated tissues and organs were isolated from a control mouse, RNA was extracted and used to prepare cDNA, and PCR performed using control (pcdh8 and β-actin) or pch18 primers as described in ‘ Materials and Methods ’. (2c) RT-PCR analysis of spleen cells. Spleens were isolated from a 7 week old mouse and the indicated cells were purified by FACS (top panel). TIL were isolated from an MCA38 tumor. CTLL-2 is a CD8 + lytic cell line. RNA was isolated and used to program RT-PCR as described in ‘ Materials and Methods ’. Similarly spleen CD8 + T cells were purified and activated in vitro with anti-CD3 for the indicated times before analysis (bottom panel). (2d) TIL qRT-PCR analyses. Single cell suspensions of MCA38 tumors were prepared and CD4 + or CD8 + TIL were isolated by magnetic immunobeading. (Liver tissue was isolated and used for RNA isolation in certain control analyses. TIL were labeled with anti-CD4 or CD8 Ab and further purified by FACS (example of flow cytometry analysis shown in left panels) before RNA isolation and qRT-PCR analysis. TIL used to prepare RNA immediately after isolation are indicated in as ‘nonlytic’ or ‘TIL 0 hr’. As indicated some TIL samples were cultured in vitro for 8 or 24 h before RNA isolation during which time TIL recover both proximal TCR-mediated signaling and lytic function [5] , [7] . PCR analyses of various target RNAs are shown and include several control reactions that demonstrate specificity of the expression patterns observed (e.g. pcdh18 and pcdh12 in CD8 + TIL, Dab1 and Dab2a in CD8 + TIL, granzyme B in CD8 + TIL and liver, as well as TNF, IFN, IL-2, PD-1, and PD-1L). Data show SD from three independent experiments. (2e) qRT-PCR analysis of purified spleen cells. Spleen immune cells were isolated by FACS from young (4 week) or old ( > 48 week) control mice and RNA was isolated and used to program pcdh18 qRT-PCR as described in ‘ Materials and Methods ’. The data shown are representative of multiple repetitions.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Isolation, Polymerase Chain Reaction, Purification, FACS, In Vitro, Quantitative RT-PCR, Labeling, Flow Cytometry, Cytometry, Cell Culture, Expressing, Mouse Assay

    Biochemical and functional analyses of lytic T cells transfected with pcdh18. (6a) Flow cytometry analysis of primary lytic effector cells generated from spleens of 7 week old mice (prepared as described in ‘ Materials and Methods ’). PI lo cells are > 80% CD8 + , PI hi cells are ∼40% CD8 + (top panel). > 70% of CD8 + CD44 hi cells are CD62L hi CD127 hi (bottom panel). (6b) (top) Reciprocal immunoblot of p56 lck isolated from nonlytic TIL. Analysis was performed as described in Figure 1a (after conjugation with cognate MCA38 tumor cells for 0, 5, or 15 min as indicted and immune precipitated with anti-p56 lck Ab 2102- left panels- or Ab 3A5- right panels) and blots were probed with anti-pY or anti-Pcdh18 as indicated. (bottom) Expression of pcdh18 protein in transfected effector cells by flow cytometry was as described in ‘ Materials and Methods ’. Cells were stained with control or anti-pcdh18 Ab as indicated (top). (6c) Phase contrast microscopy of transfected cells. Effector cells were transfected as indicated and cultured in vitro in the presence or absence of IL-2 for ∼24 h before microscopy. Arrows indicate cell clusters. (6d) RNA was extracted from transfected effector cells (‘control’ or ‘pcdh18’), nonlytic TIL (‘TIL 0 h’), or lytic TIL that were activated with anti-CD3 for 4 hours (‘TIL 24 h’) and used for cytokine qRT-PCR, or cells were assessed for viability (PI and Annexin V staining), as described in ‘ Materials and Methods ’. In the viability assay two different plasmid constructs were used in separate experiments (bottom left panel) or the Y842F mutant (bottom right panel). (6e) Transfected effector cells were assayed for lytic function by re-directed cytolysis assay as described in ‘ Materials and Methods ’. (6f) Transfected cells were assayed for binding of anti-Zap70 pY493 after permeabilization following activation with anti-TCR for 2 min as described in ‘ Material and Methods ’.
    Figure Legend Snippet: Biochemical and functional analyses of lytic T cells transfected with pcdh18. (6a) Flow cytometry analysis of primary lytic effector cells generated from spleens of 7 week old mice (prepared as described in ‘ Materials and Methods ’). PI lo cells are > 80% CD8 + , PI hi cells are ∼40% CD8 + (top panel). > 70% of CD8 + CD44 hi cells are CD62L hi CD127 hi (bottom panel). (6b) (top) Reciprocal immunoblot of p56 lck isolated from nonlytic TIL. Analysis was performed as described in Figure 1a (after conjugation with cognate MCA38 tumor cells for 0, 5, or 15 min as indicted and immune precipitated with anti-p56 lck Ab 2102- left panels- or Ab 3A5- right panels) and blots were probed with anti-pY or anti-Pcdh18 as indicated. (bottom) Expression of pcdh18 protein in transfected effector cells by flow cytometry was as described in ‘ Materials and Methods ’. Cells were stained with control or anti-pcdh18 Ab as indicated (top). (6c) Phase contrast microscopy of transfected cells. Effector cells were transfected as indicated and cultured in vitro in the presence or absence of IL-2 for ∼24 h before microscopy. Arrows indicate cell clusters. (6d) RNA was extracted from transfected effector cells (‘control’ or ‘pcdh18’), nonlytic TIL (‘TIL 0 h’), or lytic TIL that were activated with anti-CD3 for 4 hours (‘TIL 24 h’) and used for cytokine qRT-PCR, or cells were assessed for viability (PI and Annexin V staining), as described in ‘ Materials and Methods ’. In the viability assay two different plasmid constructs were used in separate experiments (bottom left panel) or the Y842F mutant (bottom right panel). (6e) Transfected effector cells were assayed for lytic function by re-directed cytolysis assay as described in ‘ Materials and Methods ’. (6f) Transfected cells were assayed for binding of anti-Zap70 pY493 after permeabilization following activation with anti-TCR for 2 min as described in ‘ Material and Methods ’.

    Techniques Used: Functional Assay, Transfection, Flow Cytometry, Cytometry, Generated, Mouse Assay, Isolation, Conjugation Assay, Expressing, Staining, Microscopy, Cell Culture, In Vitro, Quantitative RT-PCR, Viability Assay, Plasmid Preparation, Construct, Mutagenesis, Binding Assay, Activation Assay

    RNA analyses of spleen cells after induction of memory in vivo . (a) qRT-PCR analysis of spleen CD8 + T cells isolated from BL/6 mice previously infected with either 5,000 recombinant Listeria monocytogenes , buffer controls, or EL4 cells at a subtumorigenic dose (‘regressor tumor’). 28 days after exposure spleen CD8 + T cells were purified, activated in vitro with Con A for the indicated times, RNA prepared and qRT-PCR performed as described in ‘ Materials and Methods ’. (This analysis has been performed using mice previously infected for times up to 1 year with similar results). Insert shows gel analysis of pcdh18 RT-PCR expression from L. monocytogenes -immune mice. (3b) qRT-PCR analysis of purified CD8 + T cells from mice injected with L. monocytogenes or allogeneic H-2D spleen cells. (3c) qRT-PCR analysis of purified CD8 + T cells from mice originally infected with L. monocytogenes which were challenged by in vivo infection by L. monocytogenes . Spleens were isolated at the indicated times following challenge. Age-matched naive mice received only primary exposure given at the time of secondary challenge. Nonlytic TIL are shown for comparison. (3d) qRT-PCR analysis of purified control CD8 + spleen T cells and activated in vitro with anti-CD3e for the indicated times before RNA isolation and analysis by qRT-PCR. The data shown are representative of multiple repetitions.
    Figure Legend Snippet: RNA analyses of spleen cells after induction of memory in vivo . (a) qRT-PCR analysis of spleen CD8 + T cells isolated from BL/6 mice previously infected with either 5,000 recombinant Listeria monocytogenes , buffer controls, or EL4 cells at a subtumorigenic dose (‘regressor tumor’). 28 days after exposure spleen CD8 + T cells were purified, activated in vitro with Con A for the indicated times, RNA prepared and qRT-PCR performed as described in ‘ Materials and Methods ’. (This analysis has been performed using mice previously infected for times up to 1 year with similar results). Insert shows gel analysis of pcdh18 RT-PCR expression from L. monocytogenes -immune mice. (3b) qRT-PCR analysis of purified CD8 + T cells from mice injected with L. monocytogenes or allogeneic H-2D spleen cells. (3c) qRT-PCR analysis of purified CD8 + T cells from mice originally infected with L. monocytogenes which were challenged by in vivo infection by L. monocytogenes . Spleens were isolated at the indicated times following challenge. Age-matched naive mice received only primary exposure given at the time of secondary challenge. Nonlytic TIL are shown for comparison. (3d) qRT-PCR analysis of purified control CD8 + spleen T cells and activated in vitro with anti-CD3e for the indicated times before RNA isolation and analysis by qRT-PCR. The data shown are representative of multiple repetitions.

    Techniques Used: In Vivo, Quantitative RT-PCR, Isolation, Mouse Assay, Infection, Recombinant, Purification, In Vitro, Reverse Transcription Polymerase Chain Reaction, Expressing, Injection

    34) Product Images from "SlDEAD31, a Putative DEAD-Box RNA Helicase Gene, Regulates Salt and Drought Tolerance and Stress-Related Genes in Tomato"

    Article Title: SlDEAD31, a Putative DEAD-Box RNA Helicase Gene, Regulates Salt and Drought Tolerance and Stress-Related Genes in Tomato

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0133849

    Expression patterns of SlDEAD30 and SlDEAD31 genes under various stress treatments including NaCl (200 mM, A, D), heat (40°C) and cold (4°C, B, E), dehydration, and wounding (C, F). Gene expression was detected by RT-PCR using total RNA from leaves or roots of 35-day-old tomato plants. Bars represent mean relative expression values ± SE ( n = 3). Values are presented relative to untreated plants (0 h). Asterisks indicate a significant difference (P
    Figure Legend Snippet: Expression patterns of SlDEAD30 and SlDEAD31 genes under various stress treatments including NaCl (200 mM, A, D), heat (40°C) and cold (4°C, B, E), dehydration, and wounding (C, F). Gene expression was detected by RT-PCR using total RNA from leaves or roots of 35-day-old tomato plants. Bars represent mean relative expression values ± SE ( n = 3). Values are presented relative to untreated plants (0 h). Asterisks indicate a significant difference (P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    35) Product Images from "Endothelial cell tropism is a determinant of H5N1 pathogenesis in mammalian species"

    Article Title: Endothelial cell tropism is a determinant of H5N1 pathogenesis in mammalian species

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006270

    Restriction of cell tropism alters cytokine and chemokine responses in the lungs. C57BL/6J mice (n = 8) were intranasally infected with a 25 PFU dose of the H5N1 viruses and total RNA from the lungs was isolated on day 5 pi via Trizol extraction. Expression levels of inflammatory genes (IFN-β, MIP-1α, CCL2, IL-6, IL-1β and TNFα) were measured by quantitative PCR analyses. Data is represented as fold expression relative to mock infected mice (mean ± SEM). Asterisk denotes statistical significance determined by one-way ANOVA and the values are denoted as *p
    Figure Legend Snippet: Restriction of cell tropism alters cytokine and chemokine responses in the lungs. C57BL/6J mice (n = 8) were intranasally infected with a 25 PFU dose of the H5N1 viruses and total RNA from the lungs was isolated on day 5 pi via Trizol extraction. Expression levels of inflammatory genes (IFN-β, MIP-1α, CCL2, IL-6, IL-1β and TNFα) were measured by quantitative PCR analyses. Data is represented as fold expression relative to mock infected mice (mean ± SEM). Asterisk denotes statistical significance determined by one-way ANOVA and the values are denoted as *p

    Techniques Used: Mouse Assay, Infection, Isolation, Expressing, Real-time Polymerase Chain Reaction

    36) Product Images from "Forkhead box A3 attenuated the progression of fibrosis in a rat model of biliary atresia"

    Article Title: Forkhead box A3 attenuated the progression of fibrosis in a rat model of biliary atresia

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.99

    Validation of RNA-seq data. ( a ) mRNA levels of CTGF, DAPK1, Foxa3 and EGFL7 were evaluated by qRT-PCR. Data represent mean values±S.D. from three independent experiments. Protein levels of Foxa3 and CTGF in livers of biliary atresia patients and control were assessed by western blotting ( b ) and immunohistochemistry staining ( c ) (*** P
    Figure Legend Snippet: Validation of RNA-seq data. ( a ) mRNA levels of CTGF, DAPK1, Foxa3 and EGFL7 were evaluated by qRT-PCR. Data represent mean values±S.D. from three independent experiments. Protein levels of Foxa3 and CTGF in livers of biliary atresia patients and control were assessed by western blotting ( b ) and immunohistochemistry staining ( c ) (*** P

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Staining

    37) Product Images from "NCoR1 restrains thymic negative selection by repressing Bim expression to spare thymocytes undergoing positive selection"

    Article Title: NCoR1 restrains thymic negative selection by repressing Bim expression to spare thymocytes undergoing positive selection

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00931-8

    NCoR1 binds to the Bim promoter and represses Bim expression. a RNA-Seq analysis of differentially expressed genes in NCoR1-deficient (cKO) and wild-type (WT) thymocytes with or without activation in vitro. b , c Quantitative RT-PCR analysis of Bim mRNA expression in freshly sorted cKO ( n = 6) and WT ( n = 6) CD4 + CD8 + double-positive (DP) thymocytes ( b ), and in total cKO ( n = 4) and WT ( n = 4) thymocytes ( c ) after in vitro TCR stimulation for the indicated amounts of time. d Immunoblot analysis of NCoR1 and Bim expression levels in extracts of cKO and WT thymocytes with or without stimulation for the indicated amounts of time. e Quantification of NCoR1 and Bim expression levels in ( d ). f ChIP analysis of the Bim promoter by anti-NCoR1 in cKO and WT thymocytes with or without activation in vitro. IgG served as a negative control. g ChIP analysis of the Bim promoter in cKO and WT thymocytes using the indicted antibodies. The data are representative of two independent experiments ( b , c , f , g ). Statistical significance was analyzed using the two-tailed Student’s t test (* P
    Figure Legend Snippet: NCoR1 binds to the Bim promoter and represses Bim expression. a RNA-Seq analysis of differentially expressed genes in NCoR1-deficient (cKO) and wild-type (WT) thymocytes with or without activation in vitro. b , c Quantitative RT-PCR analysis of Bim mRNA expression in freshly sorted cKO ( n = 6) and WT ( n = 6) CD4 + CD8 + double-positive (DP) thymocytes ( b ), and in total cKO ( n = 4) and WT ( n = 4) thymocytes ( c ) after in vitro TCR stimulation for the indicated amounts of time. d Immunoblot analysis of NCoR1 and Bim expression levels in extracts of cKO and WT thymocytes with or without stimulation for the indicated amounts of time. e Quantification of NCoR1 and Bim expression levels in ( d ). f ChIP analysis of the Bim promoter by anti-NCoR1 in cKO and WT thymocytes with or without activation in vitro. IgG served as a negative control. g ChIP analysis of the Bim promoter in cKO and WT thymocytes using the indicted antibodies. The data are representative of two independent experiments ( b , c , f , g ). Statistical significance was analyzed using the two-tailed Student’s t test (* P

    Techniques Used: Expressing, RNA Sequencing Assay, Activation Assay, In Vitro, Quantitative RT-PCR, Chromatin Immunoprecipitation, Negative Control, Two Tailed Test

    38) Product Images from "SPIN1 promotes tumorigenesis by blocking the uL18 (universal large ribosomal subunit protein 18)-MDM2-p53 pathway in human cancer"

    Article Title: SPIN1 promotes tumorigenesis by blocking the uL18 (universal large ribosomal subunit protein 18)-MDM2-p53 pathway in human cancer

    Journal: eLife

    doi: 10.7554/eLife.31275

    SPIN1 knockdown reduces rRNA expression and SPIN1-Y170A mutant retains activity to repress p53. ( A ) Scramble siRNA or SPIN1 siRNA was introduced into U2OS cells. RNA levels were analyzed using Q-PCR (*p value
    Figure Legend Snippet: SPIN1 knockdown reduces rRNA expression and SPIN1-Y170A mutant retains activity to repress p53. ( A ) Scramble siRNA or SPIN1 siRNA was introduced into U2OS cells. RNA levels were analyzed using Q-PCR (*p value

    Techniques Used: Expressing, Mutagenesis, Activity Assay, Polymerase Chain Reaction

    39) Product Images from "Yy1 Gene Dosage Effect and Bi-Allelic Expression of Peg3"

    Article Title: Yy1 Gene Dosage Effect and Bi-Allelic Expression of Peg3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0119493

    Maternal expression of Peg3 in mouse brain. ( A ) RT-PCR testing the maternal expression of Peg3 . The total RNA was isolated from the different parts of a 4-month-old male mouse with the paternally transmitted DelKO allele (midbrain, cerebellum, olfactory, hypothalamus, and pituitary). These RNA were analyzed with the two sets of primers amplifying exon 1–4 and exon 3–6, confirming the maternal expression of Peg3 in the midbrain and hypothalamus. ( B ) qRT-PCR analyses were also performed to measure the relative expression levels of the paternal and maternal alleles of Peg3 between the RNA samples isolated from the different parts of the adult brain, including the midbrain (M), cerebellum (C), olfactory (O), hypothalamus (H), and pituitary (P). Parts of the adult mouse brain showing Peg3 maternal allele expression have been marked with an asterisk (*).
    Figure Legend Snippet: Maternal expression of Peg3 in mouse brain. ( A ) RT-PCR testing the maternal expression of Peg3 . The total RNA was isolated from the different parts of a 4-month-old male mouse with the paternally transmitted DelKO allele (midbrain, cerebellum, olfactory, hypothalamus, and pituitary). These RNA were analyzed with the two sets of primers amplifying exon 1–4 and exon 3–6, confirming the maternal expression of Peg3 in the midbrain and hypothalamus. ( B ) qRT-PCR analyses were also performed to measure the relative expression levels of the paternal and maternal alleles of Peg3 between the RNA samples isolated from the different parts of the adult brain, including the midbrain (M), cerebellum (C), olfactory (O), hypothalamus (H), and pituitary (P). Parts of the adult mouse brain showing Peg3 maternal allele expression have been marked with an asterisk (*).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Quantitative RT-PCR

    Effects of Yy1 gene dosage on the Peg3 imprinted domain. ( A ) Genomic structure of the Peg3 imprinted domain: maternally expressed Zim1 and paternally expressed Peg3 and Usp29 . ( B ) The current study used two sets of RT-PCR primers for Peg3 : the first set amplifying exons 1–4 and the second set amplifying exons 3–6. ( C ) RT-PCR analyses of the progeny derived from Breeding I (female Yy1 heterozygotes X male Peg3 DelKO heterozygotes). RT-PCR amplifying exons 1–4 and exons 3–6 were performed using the total RNA isolated from the neonatal brains with 4 genotypes (lanes 1–4). In the case of RT-PCR amplifying exons 1–4, the PCR products from the pups with four genotypes represent the expression from the paternal allele since Peg3 is paternally expressed. In the case of RT-PCR amplifying exons 3–6, the PCR products from the pups with two genotypes (lane 3, Yy1-/+; lane 4, WT) still represent the expression from the paternal allele of Peg3 , but the products from the pups with the two other genotypes (lane 1, Yy1-/+ Peg3+/-; lane 2, Peg3+/-) represent the expression from the maternal allele. The mRNA from the paternal allele of Peg3 , DelKO, cannot be detected by the RT-PCR amplifying exons 3–6 since the exon 6 is deleted in the DelKO allele. ( D ) Quantitative RT-PCR analyses using the total RNA isolated from the pups with four genotypes: Yy1-/+ Peg3+/- (1), Peg3+/- (2), Yy1-/+ (3), WT (4). The expression values of each gene were normalized first with an internal control ( 28S ) and later with the values from the WT pup (lane 4). The expression levels of Peg3 were analyzed using the primer set amplifying exons 1–4, thus representing the expression levels of the paternal allele. This series of qRT-PCR analyses were repeated three independent times from cDNA synthesis to qRT-PCR. Error bars indicate standard deviations for observed triplicates.
    Figure Legend Snippet: Effects of Yy1 gene dosage on the Peg3 imprinted domain. ( A ) Genomic structure of the Peg3 imprinted domain: maternally expressed Zim1 and paternally expressed Peg3 and Usp29 . ( B ) The current study used two sets of RT-PCR primers for Peg3 : the first set amplifying exons 1–4 and the second set amplifying exons 3–6. ( C ) RT-PCR analyses of the progeny derived from Breeding I (female Yy1 heterozygotes X male Peg3 DelKO heterozygotes). RT-PCR amplifying exons 1–4 and exons 3–6 were performed using the total RNA isolated from the neonatal brains with 4 genotypes (lanes 1–4). In the case of RT-PCR amplifying exons 1–4, the PCR products from the pups with four genotypes represent the expression from the paternal allele since Peg3 is paternally expressed. In the case of RT-PCR amplifying exons 3–6, the PCR products from the pups with two genotypes (lane 3, Yy1-/+; lane 4, WT) still represent the expression from the paternal allele of Peg3 , but the products from the pups with the two other genotypes (lane 1, Yy1-/+ Peg3+/-; lane 2, Peg3+/-) represent the expression from the maternal allele. The mRNA from the paternal allele of Peg3 , DelKO, cannot be detected by the RT-PCR amplifying exons 3–6 since the exon 6 is deleted in the DelKO allele. ( D ) Quantitative RT-PCR analyses using the total RNA isolated from the pups with four genotypes: Yy1-/+ Peg3+/- (1), Peg3+/- (2), Yy1-/+ (3), WT (4). The expression values of each gene were normalized first with an internal control ( 28S ) and later with the values from the WT pup (lane 4). The expression levels of Peg3 were analyzed using the primer set amplifying exons 1–4, thus representing the expression levels of the paternal allele. This series of qRT-PCR analyses were repeated three independent times from cDNA synthesis to qRT-PCR. Error bars indicate standard deviations for observed triplicates.

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

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    Article Snippet: RNA isolation, microarray and quantitative RT-PCR analysis Total RNA was extracted with TRIzol reagent (Invitrogen) and purified on RNeasy Mini Kit columns (Qiagen). .. Microarray analysis of RNA expression was performed with Illumina's Sentrix Human-6 v2 Expression BeadChip Kit containing 46,713 probes/array targeting genes and known alternative splice variants from the RefSeq database release 17 and UniGene build 188.

    Article Title: The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri
    Article Snippet: Quantitative RT-PCR analysis Total RNA was extracted and DNA removed by TURBO DNA-free Kit (Ambion), and cDNA was reverse-transcribed using SuperScript VILO Master Mix (Invitrogen). .. RNA expression was quantitatively measured using the Livak (2−ΔΔCT ) method.

    Fluorescence:

    Article Title: Overexpression of Cotton RAV1 Gene in Arabidopsis Confers Transgenic Plants High Salinity and Drought Sensitivity
    Article Snippet: Quantitative RT-PCR analysis Total RNA was extracted from tissues of cotton and Arabidopsis , respectively, by Trizol kit (Invitrogen) according to the manufacturer’s protocol. .. The Ct (cycle threshold), defined as the PCR cycle at which a statistically significant increase of reporter fluorescence is first detected, is used as a measure for the starting copy numbers of the target gene.

    Synthesized:

    Article Title: Overexpression of Cotton RAV1 Gene in Arabidopsis Confers Transgenic Plants High Salinity and Drought Sensitivity
    Article Snippet: Quantitative RT-PCR analysis Total RNA was extracted from tissues of cotton and Arabidopsis , respectively, by Trizol kit (Invitrogen) according to the manufacturer’s protocol. .. First-strand cDNA was synthesized from 1 μg DNase-treated total RNA sample using oligo (dT) and Takara MLV-Reverse transcriptase.

    Article Title: AMPK Activation Reduces Hepatic Lipid Content by Increasing Fat Oxidation In Vivo
    Article Snippet: .. Isolation of Total mRNA and Quantitative RT-PCR Analysis Total RNA from mouse liver tissue was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), and single-strand cDNA was synthesized from 5 µg of total RNA with random hexamer primers (Applied Biosystems, Foster City, CA, USA) and Superscript II (Life Technologies, Carlsbad, CA, USA). .. Real-time RT-PCR reactions were carried out in a final volume of 20 µL containing 125 ng of reverse-transcribed total RNA, 500 nM of primers, and 10 µL of 2× PCR mix containing Sybr Green (Roche, Meylan, France).

    Article Title: Tonoplast- and Plasma Membrane-Localized Aquaporin-Family Transporters in Blue Hydrangea Sepals of Aluminum Hyperaccumulating Plant
    Article Snippet: Quantitative RT-PCR Analysis Total RNA was extracted from each tissue in hydrangea using TRIzol Reagent with the PureLink RNA Mini kit (Invitrogen). .. First-strand cDNA was synthesized from 2 µg of total RNA using SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen).

    Isolation:

    Article Title: Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome
    Article Snippet: .. RNA isolation, microarray and quantitative RT-PCR analysis Total RNA was extracted with TRIzol reagent (Invitrogen) and purified on RNeasy Mini Kit columns (Qiagen). .. The RNA quality was assessed with the RNA 6000 Nano assay kit using the Agilent 2100 Bioanalyzer.

    Article Title: Application of hairless mouse strain to bioluminescence imaging of Arc expression in mouse brain
    Article Snippet: .. Quantitative RT-PCR analysis Total RNA was isolated from adult C57BL/6 and Arc -Luc Tg HL mouse hippocampi 30 min after KA injection [20 mg/kg BW, intraperitoneally (i.p.)] with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). .. Saline was injected as a control condition.

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB
    Article Snippet: .. Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific). .. Real‐time PCR was performed using SYBR Premix Ex Taq II (Takara, Dalian, China).

    Article Title: Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase
    Article Snippet: .. Quantitative RT-PCR analysis Total RNA was isolated from whole lung according to the TRIzol reagent protocol (Invitrogen, NY, USA) and purified by E.Z.N.A. total RNA kit I (OMEGA, GA, USA). .. Reverse transcription PCR was performed using the qScript cDNA SuperMix (Quanta biosciences, MD, USA).

    Article Title: AMPK Activation Reduces Hepatic Lipid Content by Increasing Fat Oxidation In Vivo
    Article Snippet: .. Isolation of Total mRNA and Quantitative RT-PCR Analysis Total RNA from mouse liver tissue was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), and single-strand cDNA was synthesized from 5 µg of total RNA with random hexamer primers (Applied Biosystems, Foster City, CA, USA) and Superscript II (Life Technologies, Carlsbad, CA, USA). .. Real-time RT-PCR reactions were carried out in a final volume of 20 µL containing 125 ng of reverse-transcribed total RNA, 500 nM of primers, and 10 µL of 2× PCR mix containing Sybr Green (Roche, Meylan, France).

    Quantitative RT-PCR:

    Article Title: Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome
    Article Snippet: .. RNA isolation, microarray and quantitative RT-PCR analysis Total RNA was extracted with TRIzol reagent (Invitrogen) and purified on RNeasy Mini Kit columns (Qiagen). .. The RNA quality was assessed with the RNA 6000 Nano assay kit using the Agilent 2100 Bioanalyzer.

    Article Title: Plin4-Dependent Lipid Droplets Hamper Neuronal Mitophagy in the MPTP/p-Induced Mouse Model of Parkinson’s Disease
    Article Snippet: .. RNA Reverse-Transcription and Quantitative RT-PCR Analysis Total RNA was extracted with Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, United States). .. Total RNA (1 μg) of each sample was reverse-transcribed into cDNA and amplified using a PrimeScriptTM RT Master Mix (Takara, RR036A, Takara Biotechnology, China) according to the manufacturer’s directions.

    Article Title: MicroRNA miR-124 Controls the Choice between Neuronal and Astrocyte Differentiation by Fine-tuning Ezh2 Expression *
    Article Snippet: .. Semiquantitative and Quantitative RT-PCR Analysis Total RNA was purified using TRIzol (Invitrogen) as recommended. .. Reverse transcription (RT) was performed using SuperScript III (Invitrogen) with random hexamer. cDNA was amplified with specific primers for Ezh2 , Suz12 , Eed , and Hprt .

    Article Title: Application of hairless mouse strain to bioluminescence imaging of Arc expression in mouse brain
    Article Snippet: .. Quantitative RT-PCR analysis Total RNA was isolated from adult C57BL/6 and Arc -Luc Tg HL mouse hippocampi 30 min after KA injection [20 mg/kg BW, intraperitoneally (i.p.)] with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). .. Saline was injected as a control condition.

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB
    Article Snippet: .. Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific). .. Real‐time PCR was performed using SYBR Premix Ex Taq II (Takara, Dalian, China).

    Article Title: Overexpression of Cotton RAV1 Gene in Arabidopsis Confers Transgenic Plants High Salinity and Drought Sensitivity
    Article Snippet: .. Quantitative RT-PCR analysis Total RNA was extracted from tissues of cotton and Arabidopsis , respectively, by Trizol kit (Invitrogen) according to the manufacturer’s protocol. .. First-strand cDNA was synthesized from 1 μg DNase-treated total RNA sample using oligo (dT) and Takara MLV-Reverse transcriptase.

    Article Title: MiR-184 regulates insulin secretion through repression of Slc25a22
    Article Snippet: .. Quantitative RT-PCR analysis Total RNA was prepared from cells using Trizol (Invitrogen). .. Gene expression levels were measured with LightCycler 480 (Roche) using the SYBR Premix Ex Taq (TAKARA) according to the manufacturer’s instructions.

    Article Title: Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase
    Article Snippet: .. Quantitative RT-PCR analysis Total RNA was isolated from whole lung according to the TRIzol reagent protocol (Invitrogen, NY, USA) and purified by E.Z.N.A. total RNA kit I (OMEGA, GA, USA). .. Reverse transcription PCR was performed using the qScript cDNA SuperMix (Quanta biosciences, MD, USA).

    Article Title: CRL3IBTK Regulates the Tumor Suppressor Pdcd4 through Ubiquitylation Coupled to Proteasomal Degradation *
    Article Snippet: .. Quantitative RT-PCR Analysis Total RNA was extracted from cells with TRIzol reagent (Invitrogen). .. RNA aliquots (200 ng) were reverse transcribed with Random Examers (Roche Applied Science) and Superscript III Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol.

    Article Title: The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri
    Article Snippet: .. Quantitative RT-PCR analysis Total RNA was extracted and DNA removed by TURBO DNA-free Kit (Ambion), and cDNA was reverse-transcribed using SuperScript VILO Master Mix (Invitrogen). .. Quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) and the values normalized to 16S rRNA.

    Article Title: AMPK Activation Reduces Hepatic Lipid Content by Increasing Fat Oxidation In Vivo
    Article Snippet: .. Isolation of Total mRNA and Quantitative RT-PCR Analysis Total RNA from mouse liver tissue was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), and single-strand cDNA was synthesized from 5 µg of total RNA with random hexamer primers (Applied Biosystems, Foster City, CA, USA) and Superscript II (Life Technologies, Carlsbad, CA, USA). .. Real-time RT-PCR reactions were carried out in a final volume of 20 µL containing 125 ng of reverse-transcribed total RNA, 500 nM of primers, and 10 µL of 2× PCR mix containing Sybr Green (Roche, Meylan, France).

    Article Title: Tonoplast- and Plasma Membrane-Localized Aquaporin-Family Transporters in Blue Hydrangea Sepals of Aluminum Hyperaccumulating Plant
    Article Snippet: .. Quantitative RT-PCR Analysis Total RNA was extracted from each tissue in hydrangea using TRIzol Reagent with the PureLink RNA Mini kit (Invitrogen). .. First-strand cDNA was synthesized from 2 µg of total RNA using SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen).

    Purification:

    Article Title: Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome
    Article Snippet: .. RNA isolation, microarray and quantitative RT-PCR analysis Total RNA was extracted with TRIzol reagent (Invitrogen) and purified on RNeasy Mini Kit columns (Qiagen). .. The RNA quality was assessed with the RNA 6000 Nano assay kit using the Agilent 2100 Bioanalyzer.

    Article Title: MicroRNA miR-124 Controls the Choice between Neuronal and Astrocyte Differentiation by Fine-tuning Ezh2 Expression *
    Article Snippet: .. Semiquantitative and Quantitative RT-PCR Analysis Total RNA was purified using TRIzol (Invitrogen) as recommended. .. Reverse transcription (RT) was performed using SuperScript III (Invitrogen) with random hexamer. cDNA was amplified with specific primers for Ezh2 , Suz12 , Eed , and Hprt .

    Article Title: Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase
    Article Snippet: .. Quantitative RT-PCR analysis Total RNA was isolated from whole lung according to the TRIzol reagent protocol (Invitrogen, NY, USA) and purified by E.Z.N.A. total RNA kit I (OMEGA, GA, USA). .. Reverse transcription PCR was performed using the qScript cDNA SuperMix (Quanta biosciences, MD, USA).

    Real-time Polymerase Chain Reaction:

    Article Title: Plin4-Dependent Lipid Droplets Hamper Neuronal Mitophagy in the MPTP/p-Induced Mouse Model of Parkinson’s Disease
    Article Snippet: RNA Reverse-Transcription and Quantitative RT-PCR Analysis Total RNA was extracted with Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, United States). .. RT-PCR was measured using a QuantiTect SYBR® Green PCR kit (Qiagen, Germany) with an ABI 7300 StepOneTM Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, United States).

    Article Title: Application of hairless mouse strain to bioluminescence imaging of Arc expression in mouse brain
    Article Snippet: Quantitative RT-PCR analysis Total RNA was isolated from adult C57BL/6 and Arc -Luc Tg HL mouse hippocampi 30 min after KA injection [20 mg/kg BW, intraperitoneally (i.p.)] with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). .. Quantitative PCR amplification was performed using the Mx3005P qPCR system (Agilent Technologies, Santa Clara, CA, USA) with Thunderbird SYBR qPCR Mix (Toyobo, Tokyo, Japan) for comparative quantification.

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB
    Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific). .. Real‐time PCR was performed using SYBR Premix Ex Taq II (Takara, Dalian, China).

    Article Title: MiR-184 regulates insulin secretion through repression of Slc25a22
    Article Snippet: Quantitative RT-PCR analysis Total RNA was prepared from cells using Trizol (Invitrogen). .. The real-time PCR was performed using the following cycle parameters: initial enzyme activation at 95°C for 5 s; followed by 45 cycles of 95°C for 5 s, 60°C for 20 s. Data was analyzed with the LightCycler 480 software (Roche), determining the threshold cycle (Ct) by the second derivative max method.

    Article Title: Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase
    Article Snippet: Quantitative RT-PCR analysis Total RNA was isolated from whole lung according to the TRIzol reagent protocol (Invitrogen, NY, USA) and purified by E.Z.N.A. total RNA kit I (OMEGA, GA, USA). .. Real-time PCR was carried out according to a standard protocol using the PerfeCTaFast Mix II (Quanta) with the TaqMan probe for arginase 1 and iNOS (ABI, NY, USA), and products measured on an ABI Viia 7 PCR system (ABI).

    Article Title: CRL3IBTK Regulates the Tumor Suppressor Pdcd4 through Ubiquitylation Coupled to Proteasomal Degradation *
    Article Snippet: Quantitative RT-PCR Analysis Total RNA was extracted from cells with TRIzol reagent (Invitrogen). .. Real-time PCR was performed using iQ Green Super Mix (Bio-Rad) and carried out with the iCycler iQReal-Time detection system (Bio-Rad) under the following conditions: 95 °C for 1 min and then 40 cycles at 94 °C for 10 s and 60 °C for 30 s. Primers were as follows: IBtkα FW, 5′- GTCAGCCCTCCTGTTGTGGAT-3′; IBtkα REV, 5′-TGCATTCACTGGTTTGGGGGC-3′; Pdcd4 FW, 5′-CCATGGTGCTTCAATAGCATGT-3′; Pdcd4 REV, 5′-CCCAGCATTTTCTTCATCACCG-3′; Bcl-XL FW, 5′-GTGAGTCGGATCGCAGCTT-3′; Bcl-XL REV, 5′-GCTGCTGCATTGTTCCCATAG-3′; GAPDH FW, 5′-CAGCCTCAAGATCATCAGCA-3′; GAPDH REV, 5′-TGTGGTCATCAGTCCTTCCA-3′.

    Article Title: Tonoplast- and Plasma Membrane-Localized Aquaporin-Family Transporters in Blue Hydrangea Sepals of Aluminum Hyperaccumulating Plant
    Article Snippet: Quantitative RT-PCR Analysis Total RNA was extracted from each tissue in hydrangea using TRIzol Reagent with the PureLink RNA Mini kit (Invitrogen). .. Real-time PCR was performed using a KAPA SYBR FAST qPCR kit (KAPABIOSYSTEMS) and ABI 7500 Fast Real-Time PCR system (Applied Biosystems) according to the manufacturer's instructions.

    Activation Assay:

    Article Title: MiR-184 regulates insulin secretion through repression of Slc25a22
    Article Snippet: Quantitative RT-PCR analysis Total RNA was prepared from cells using Trizol (Invitrogen). .. The real-time PCR was performed using the following cycle parameters: initial enzyme activation at 95°C for 5 s; followed by 45 cycles of 95°C for 5 s, 60°C for 20 s. Data was analyzed with the LightCycler 480 software (Roche), determining the threshold cycle (Ct) by the second derivative max method.

    Random Hexamer Labeling:

    Article Title: MicroRNA miR-124 Controls the Choice between Neuronal and Astrocyte Differentiation by Fine-tuning Ezh2 Expression *
    Article Snippet: Semiquantitative and Quantitative RT-PCR Analysis Total RNA was purified using TRIzol (Invitrogen) as recommended. .. Reverse transcription (RT) was performed using SuperScript III (Invitrogen) with random hexamer. cDNA was amplified with specific primers for Ezh2 , Suz12 , Eed , and Hprt .

    Article Title: AMPK Activation Reduces Hepatic Lipid Content by Increasing Fat Oxidation In Vivo
    Article Snippet: .. Isolation of Total mRNA and Quantitative RT-PCR Analysis Total RNA from mouse liver tissue was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), and single-strand cDNA was synthesized from 5 µg of total RNA with random hexamer primers (Applied Biosystems, Foster City, CA, USA) and Superscript II (Life Technologies, Carlsbad, CA, USA). .. Real-time RT-PCR reactions were carried out in a final volume of 20 µL containing 125 ng of reverse-transcribed total RNA, 500 nM of primers, and 10 µL of 2× PCR mix containing Sybr Green (Roche, Meylan, France).

    Amplification:

    Article Title: Plin4-Dependent Lipid Droplets Hamper Neuronal Mitophagy in the MPTP/p-Induced Mouse Model of Parkinson’s Disease
    Article Snippet: RNA Reverse-Transcription and Quantitative RT-PCR Analysis Total RNA was extracted with Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, United States). .. Total RNA (1 μg) of each sample was reverse-transcribed into cDNA and amplified using a PrimeScriptTM RT Master Mix (Takara, RR036A, Takara Biotechnology, China) according to the manufacturer’s directions.

    Article Title: MicroRNA miR-124 Controls the Choice between Neuronal and Astrocyte Differentiation by Fine-tuning Ezh2 Expression *
    Article Snippet: Semiquantitative and Quantitative RT-PCR Analysis Total RNA was purified using TRIzol (Invitrogen) as recommended. .. Reverse transcription (RT) was performed using SuperScript III (Invitrogen) with random hexamer. cDNA was amplified with specific primers for Ezh2 , Suz12 , Eed , and Hprt .

    Article Title: Application of hairless mouse strain to bioluminescence imaging of Arc expression in mouse brain
    Article Snippet: Quantitative RT-PCR analysis Total RNA was isolated from adult C57BL/6 and Arc -Luc Tg HL mouse hippocampi 30 min after KA injection [20 mg/kg BW, intraperitoneally (i.p.)] with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). .. Quantitative PCR amplification was performed using the Mx3005P qPCR system (Agilent Technologies, Santa Clara, CA, USA) with Thunderbird SYBR qPCR Mix (Toyobo, Tokyo, Japan) for comparative quantification.

    Labeling:

    Article Title: Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome
    Article Snippet: RNA isolation, microarray and quantitative RT-PCR analysis Total RNA was extracted with TRIzol reagent (Invitrogen) and purified on RNeasy Mini Kit columns (Qiagen). .. RNA was labeled, hybridized and scanned according to Illumina GLP standards by Aros AB laboratory.

    Expressing:

    Article Title: Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome
    Article Snippet: RNA isolation, microarray and quantitative RT-PCR analysis Total RNA was extracted with TRIzol reagent (Invitrogen) and purified on RNeasy Mini Kit columns (Qiagen). .. Microarray analysis of RNA expression was performed with Illumina's Sentrix Human-6 v2 Expression BeadChip Kit containing 46,713 probes/array targeting genes and known alternative splice variants from the RefSeq database release 17 and UniGene build 188.

    Article Title: Overexpression of Cotton RAV1 Gene in Arabidopsis Confers Transgenic Plants High Salinity and Drought Sensitivity
    Article Snippet: Quantitative RT-PCR analysis Total RNA was extracted from tissues of cotton and Arabidopsis , respectively, by Trizol kit (Invitrogen) according to the manufacturer’s protocol. .. Real-time quantitative RT-PCR analysis of cotton gene expression was performed using the fluorescent intercalating dye SYBR-Green in a detection system (Opticon2; MJ Research, New Haven, Connecticut, USA) as the method described earlier [ ].

    Article Title: MiR-184 regulates insulin secretion through repression of Slc25a22
    Article Snippet: Quantitative RT-PCR analysis Total RNA was prepared from cells using Trizol (Invitrogen). .. Gene expression levels were measured with LightCycler 480 (Roche) using the SYBR Premix Ex Taq (TAKARA) according to the manufacturer’s instructions.

    Polymerase Chain Reaction:

    Article Title: Plin4-Dependent Lipid Droplets Hamper Neuronal Mitophagy in the MPTP/p-Induced Mouse Model of Parkinson’s Disease
    Article Snippet: RNA Reverse-Transcription and Quantitative RT-PCR Analysis Total RNA was extracted with Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, United States). .. RT-PCR was measured using a QuantiTect SYBR® Green PCR kit (Qiagen, Germany) with an ABI 7300 StepOneTM Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, United States).

    Article Title: MicroRNA miR-124 Controls the Choice between Neuronal and Astrocyte Differentiation by Fine-tuning Ezh2 Expression *
    Article Snippet: Semiquantitative and Quantitative RT-PCR Analysis Total RNA was purified using TRIzol (Invitrogen) as recommended. .. Quantification of PCR product was done using image processing software, ImageJ (National Institutes of Health).

    Article Title: Overexpression of Cotton RAV1 Gene in Arabidopsis Confers Transgenic Plants High Salinity and Drought Sensitivity
    Article Snippet: Quantitative RT-PCR analysis Total RNA was extracted from tissues of cotton and Arabidopsis , respectively, by Trizol kit (Invitrogen) according to the manufacturer’s protocol. .. The Ct (cycle threshold), defined as the PCR cycle at which a statistically significant increase of reporter fluorescence is first detected, is used as a measure for the starting copy numbers of the target gene.

    Article Title: MiR-184 regulates insulin secretion through repression of Slc25a22
    Article Snippet: Quantitative RT-PCR analysis Total RNA was prepared from cells using Trizol (Invitrogen). .. The efficiency of PCR reaction shows 1.85–1.98 in the LightCycler 480 software.

    Article Title: Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase
    Article Snippet: Quantitative RT-PCR analysis Total RNA was isolated from whole lung according to the TRIzol reagent protocol (Invitrogen, NY, USA) and purified by E.Z.N.A. total RNA kit I (OMEGA, GA, USA). .. Reverse transcription PCR was performed using the qScript cDNA SuperMix (Quanta biosciences, MD, USA).

    Article Title: The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri
    Article Snippet: Quantitative RT-PCR analysis Total RNA was extracted and DNA removed by TURBO DNA-free Kit (Ambion), and cDNA was reverse-transcribed using SuperScript VILO Master Mix (Invitrogen). .. Quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) and the values normalized to 16S rRNA.

    Article Title: AMPK Activation Reduces Hepatic Lipid Content by Increasing Fat Oxidation In Vivo
    Article Snippet: Isolation of Total mRNA and Quantitative RT-PCR Analysis Total RNA from mouse liver tissue was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), and single-strand cDNA was synthesized from 5 µg of total RNA with random hexamer primers (Applied Biosystems, Foster City, CA, USA) and Superscript II (Life Technologies, Carlsbad, CA, USA). .. Real-time RT-PCR reactions were carried out in a final volume of 20 µL containing 125 ng of reverse-transcribed total RNA, 500 nM of primers, and 10 µL of 2× PCR mix containing Sybr Green (Roche, Meylan, France).

    Article Title: Tonoplast- and Plasma Membrane-Localized Aquaporin-Family Transporters in Blue Hydrangea Sepals of Aluminum Hyperaccumulating Plant
    Article Snippet: Quantitative RT-PCR Analysis Total RNA was extracted from each tissue in hydrangea using TRIzol Reagent with the PureLink RNA Mini kit (Invitrogen). .. The primer sets used for PCR were as follows: VALT, 5′-GGCCCTAGCAGAGTTCTTCTCT-3′ and 5′- AATGTAATGTTCCCACCAAGGA-3′ ; PALT1, 5′-ACCTGTAACTCCAGGGACTCCT-3′ and 5′-TATGAACTCAGCTCCCACCTTT-3′ ; 18SrRNA, 5′-GGAAGTTTGAGGCAATAACAGG-3′ and 5′-ATTGCAATGATCTATCCCCATC-3′ .

    Injection:

    Article Title: Application of hairless mouse strain to bioluminescence imaging of Arc expression in mouse brain
    Article Snippet: .. Quantitative RT-PCR analysis Total RNA was isolated from adult C57BL/6 and Arc -Luc Tg HL mouse hippocampi 30 min after KA injection [20 mg/kg BW, intraperitoneally (i.p.)] with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). .. Saline was injected as a control condition.

    Software:

    Article Title: MicroRNA miR-124 Controls the Choice between Neuronal and Astrocyte Differentiation by Fine-tuning Ezh2 Expression *
    Article Snippet: Semiquantitative and Quantitative RT-PCR Analysis Total RNA was purified using TRIzol (Invitrogen) as recommended. .. Quantification of PCR product was done using image processing software, ImageJ (National Institutes of Health).

    Article Title: MiR-184 regulates insulin secretion through repression of Slc25a22
    Article Snippet: Quantitative RT-PCR analysis Total RNA was prepared from cells using Trizol (Invitrogen). .. The real-time PCR was performed using the following cycle parameters: initial enzyme activation at 95°C for 5 s; followed by 45 cycles of 95°C for 5 s, 60°C for 20 s. Data was analyzed with the LightCycler 480 software (Roche), determining the threshold cycle (Ct) by the second derivative max method.

    Article Title: AMPK Activation Reduces Hepatic Lipid Content by Increasing Fat Oxidation In Vivo
    Article Snippet: Isolation of Total mRNA and Quantitative RT-PCR Analysis Total RNA from mouse liver tissue was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), and single-strand cDNA was synthesized from 5 µg of total RNA with random hexamer primers (Applied Biosystems, Foster City, CA, USA) and Superscript II (Life Technologies, Carlsbad, CA, USA). .. The relative amounts of the mRNAs studied were determined by means of the second-derivative maximum method, with LightCycler 480 analysis software and 18S RNA as the invariant control for all studies.

    Microarray:

    Article Title: Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome
    Article Snippet: .. RNA isolation, microarray and quantitative RT-PCR analysis Total RNA was extracted with TRIzol reagent (Invitrogen) and purified on RNeasy Mini Kit columns (Qiagen). .. The RNA quality was assessed with the RNA 6000 Nano assay kit using the Agilent 2100 Bioanalyzer.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Plin4-Dependent Lipid Droplets Hamper Neuronal Mitophagy in the MPTP/p-Induced Mouse Model of Parkinson’s Disease
    Article Snippet: RNA Reverse-Transcription and Quantitative RT-PCR Analysis Total RNA was extracted with Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, United States). .. RT-PCR was measured using a QuantiTect SYBR® Green PCR kit (Qiagen, Germany) with an ABI 7300 StepOneTM Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, United States).

    Article Title: Tonoplast- and Plasma Membrane-Localized Aquaporin-Family Transporters in Blue Hydrangea Sepals of Aluminum Hyperaccumulating Plant
    Article Snippet: Quantitative RT-PCR Analysis Total RNA was extracted from each tissue in hydrangea using TRIzol Reagent with the PureLink RNA Mini kit (Invitrogen). .. First-strand cDNA was synthesized from 2 µg of total RNA using SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen).

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    Thermo Fisher quantitative rt pcr analysis total rna
    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger <t>RNA</t> (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time <t>(qRT)‐PCR.</t> ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P
    Quantitative Rt Pcr Analysis Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

    Journal: Thoracic Cancer

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

    doi: 10.1111/1759-7714.12895

    Figure Lengend Snippet: Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

    Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

    Techniques: Expressing, Migration, In Vitro, Quantitative RT-PCR, Western Blot

    Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

    Journal: Thoracic Cancer

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

    doi: 10.1111/1759-7714.12895

    Figure Lengend Snippet: Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

    Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

    Techniques: Migration, In Vitro, Real-time Polymerase Chain Reaction, Expressing, shRNA, Western Blot

    Gene expression analysis by RNA sequencing of LT-HSCs and MEPs. (A) Unsupervised clustering of differentially expressed genes (P ≤ 0.05; log2-fold changes > 1.5). Each column represents data from one individual mouse. The color code for the genotypes of the individual mice is the same as in B. (B) PCA. The data for LT-HSCs and MEPs were derived from two independent experiments and combined ( n = 4 or 5 for SclCre , n = 7 or 4 for SclCre;Ezh2 +/Δ , n = 4 for SclCre;Ezh2 Δ/Δ , n = 6 or 4 for SclCre;V617F , n = 8 or 6 SclCre;V617F;Ezh2 +/Δ , and n = 6 for SclCre;V617F;Ezh2 Δ/Δ for LT-HSCs or MEP analysis). Each dot represents data from one individual mouse. (C) Competitive gene set enrichment analysis for gene expression signatures of interferon-γ, interferon-α, and fetal liver HSCs in LT-HSCs of SclCre;V617F;Ezh2 Δ/Δ compared with SclCre;V617F . (D) Plot showing the number of differentially expressed genes with cutoff of P ≤ 0.05. (E) Gene list of top 10 significant gene expression differences according to the absolute fold change. (F) Expression levels of Lin28b and Hmga2 . Each dot represents data from one individual mouse. (G) Relative expression of HMGA2 , IGF2BP3 , and Pcolce2 determined by qPCR in granulocyte RNA from patients with MPN that carry mutations in EZH2 or CALR . Each dot represents data from one individual patient. The mutations in patient granulocyte were determined by allele-specific PCR ( n = 4 for CALR mutation with WT EZH2 , n = 4 for CALR mutation with heterozygous mutation of EZH2 , n = 1 for CALR mutation with homozygous mutation of EZH2 , n = 8 for JAK2V617F mutation with WT EZH2 , n = 12 for JAK2V617F mutation with heterozygous mutation of EZH2 , and n = 4 for JAK2V617F mutation with h homozygous mutation of EZH2 ). (F and G) Horizontal lines indicate the mean of the values. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Loss of Ezh2 synergizes with JAK2-V617F in initiating myeloproliferative neoplasms and promoting myelofibrosis

    doi: 10.1084/jem.20151136

    Figure Lengend Snippet: Gene expression analysis by RNA sequencing of LT-HSCs and MEPs. (A) Unsupervised clustering of differentially expressed genes (P ≤ 0.05; log2-fold changes > 1.5). Each column represents data from one individual mouse. The color code for the genotypes of the individual mice is the same as in B. (B) PCA. The data for LT-HSCs and MEPs were derived from two independent experiments and combined ( n = 4 or 5 for SclCre , n = 7 or 4 for SclCre;Ezh2 +/Δ , n = 4 for SclCre;Ezh2 Δ/Δ , n = 6 or 4 for SclCre;V617F , n = 8 or 6 SclCre;V617F;Ezh2 +/Δ , and n = 6 for SclCre;V617F;Ezh2 Δ/Δ for LT-HSCs or MEP analysis). Each dot represents data from one individual mouse. (C) Competitive gene set enrichment analysis for gene expression signatures of interferon-γ, interferon-α, and fetal liver HSCs in LT-HSCs of SclCre;V617F;Ezh2 Δ/Δ compared with SclCre;V617F . (D) Plot showing the number of differentially expressed genes with cutoff of P ≤ 0.05. (E) Gene list of top 10 significant gene expression differences according to the absolute fold change. (F) Expression levels of Lin28b and Hmga2 . Each dot represents data from one individual mouse. (G) Relative expression of HMGA2 , IGF2BP3 , and Pcolce2 determined by qPCR in granulocyte RNA from patients with MPN that carry mutations in EZH2 or CALR . Each dot represents data from one individual patient. The mutations in patient granulocyte were determined by allele-specific PCR ( n = 4 for CALR mutation with WT EZH2 , n = 4 for CALR mutation with heterozygous mutation of EZH2 , n = 1 for CALR mutation with homozygous mutation of EZH2 , n = 8 for JAK2V617F mutation with WT EZH2 , n = 12 for JAK2V617F mutation with heterozygous mutation of EZH2 , and n = 4 for JAK2V617F mutation with h homozygous mutation of EZH2 ). (F and G) Horizontal lines indicate the mean of the values. *, P

    Article Snippet: Quantitative RT-PCR analysis Total RNA was extracted from 0.5 × 106 BM cells from SclCre mutant mice 12 wk after tamoxifen injection using TRIzol solution (Thermo Fisher Scientific) and reverse transcribed by High Capacity cDNA reverse transcription kit (Thermo Fisher Scientific) with an oligo dT primer.

    Techniques: Expressing, RNA Sequencing Assay, Mouse Assay, Derivative Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Mutagenesis