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    Thermo Fisher real time pcr system
    Validation of vorinostat-induced transcriptional responses by <t>qRT-PCR.</t> Transcriptional responses of BJ and BJ LTSTERas fibroblasts following DMSO (black) and 25 μ M vorinostat treatment (red) were validated by qRT-PCR using the same template RNA as that used in the microarray study. Log2 transformed expression intensities of HG U133 plus 2.0 probe sets corresponding to ( a ) BAK1 , BAD, BIK and BCL2A1 and ( b ) BCL2L11 and BMF are shown. Gene names and probe set IDs are indicated in the title of each panel. Normalized probe set intensity values ( Y -axis) are presented, which allows direct comparison of basal expression (time 0 h), and log2 transcriptional responses over time between cell types (BJ and BJ LTSTERas). Data represent the summarized probe set intensities from three independent microarray time course experiments. qRT-PCR verification of selected genes is shown. Transcript expression was calculated relative to the control gene RPL32 (that does not respond to vorinostat or DMSO treatment), and fold-change mRNA expression over time was calculated relative to the 0 h time point. Data are presented as the mean±S.E.M. of three independent experiments. For both Affymetrix and qRT-PCR results, data sets for BJ cells are in the left panels while data sets for BJ LTSTERas are in the right panels. ( c ) Whole cell protein extracts from BJ and BJ LTSTERas fibroblasts treated with DMSO or 25 μ M vorinostat for 0–48 h were separated by <t>SDS-PAGE</t> and analyzed for the expression of BMF, BIK and BIM by western blotting. The expression of α -Tubulin was analyzed to confirm equal protein loading in each lane. The asterisk (*) denotes a non-specific band in the BIK western blot, which acts as a positive control for activity of the BIK antibody. The arrow denotes the location of BIK. Images are representative of two independent experiments
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    Images

    1) Product Images from "HDAC inhibitors induce tumor-cell-selective pro-apoptotic transcriptional responses"

    Article Title: HDAC inhibitors induce tumor-cell-selective pro-apoptotic transcriptional responses

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.9

    Validation of vorinostat-induced transcriptional responses by qRT-PCR. Transcriptional responses of BJ and BJ LTSTERas fibroblasts following DMSO (black) and 25 μ M vorinostat treatment (red) were validated by qRT-PCR using the same template RNA as that used in the microarray study. Log2 transformed expression intensities of HG U133 plus 2.0 probe sets corresponding to ( a ) BAK1 , BAD, BIK and BCL2A1 and ( b ) BCL2L11 and BMF are shown. Gene names and probe set IDs are indicated in the title of each panel. Normalized probe set intensity values ( Y -axis) are presented, which allows direct comparison of basal expression (time 0 h), and log2 transcriptional responses over time between cell types (BJ and BJ LTSTERas). Data represent the summarized probe set intensities from three independent microarray time course experiments. qRT-PCR verification of selected genes is shown. Transcript expression was calculated relative to the control gene RPL32 (that does not respond to vorinostat or DMSO treatment), and fold-change mRNA expression over time was calculated relative to the 0 h time point. Data are presented as the mean±S.E.M. of three independent experiments. For both Affymetrix and qRT-PCR results, data sets for BJ cells are in the left panels while data sets for BJ LTSTERas are in the right panels. ( c ) Whole cell protein extracts from BJ and BJ LTSTERas fibroblasts treated with DMSO or 25 μ M vorinostat for 0–48 h were separated by SDS-PAGE and analyzed for the expression of BMF, BIK and BIM by western blotting. The expression of α -Tubulin was analyzed to confirm equal protein loading in each lane. The asterisk (*) denotes a non-specific band in the BIK western blot, which acts as a positive control for activity of the BIK antibody. The arrow denotes the location of BIK. Images are representative of two independent experiments
    Figure Legend Snippet: Validation of vorinostat-induced transcriptional responses by qRT-PCR. Transcriptional responses of BJ and BJ LTSTERas fibroblasts following DMSO (black) and 25 μ M vorinostat treatment (red) were validated by qRT-PCR using the same template RNA as that used in the microarray study. Log2 transformed expression intensities of HG U133 plus 2.0 probe sets corresponding to ( a ) BAK1 , BAD, BIK and BCL2A1 and ( b ) BCL2L11 and BMF are shown. Gene names and probe set IDs are indicated in the title of each panel. Normalized probe set intensity values ( Y -axis) are presented, which allows direct comparison of basal expression (time 0 h), and log2 transcriptional responses over time between cell types (BJ and BJ LTSTERas). Data represent the summarized probe set intensities from three independent microarray time course experiments. qRT-PCR verification of selected genes is shown. Transcript expression was calculated relative to the control gene RPL32 (that does not respond to vorinostat or DMSO treatment), and fold-change mRNA expression over time was calculated relative to the 0 h time point. Data are presented as the mean±S.E.M. of three independent experiments. For both Affymetrix and qRT-PCR results, data sets for BJ cells are in the left panels while data sets for BJ LTSTERas are in the right panels. ( c ) Whole cell protein extracts from BJ and BJ LTSTERas fibroblasts treated with DMSO or 25 μ M vorinostat for 0–48 h were separated by SDS-PAGE and analyzed for the expression of BMF, BIK and BIM by western blotting. The expression of α -Tubulin was analyzed to confirm equal protein loading in each lane. The asterisk (*) denotes a non-specific band in the BIK western blot, which acts as a positive control for activity of the BIK antibody. The arrow denotes the location of BIK. Images are representative of two independent experiments

    Techniques Used: Quantitative RT-PCR, Microarray, Transformation Assay, Expressing, SDS Page, Western Blot, Positive Control, Activity Assay

    2) Product Images from "Prox1 represses IL-2 gene expression by interacting with NFAT2"

    Article Title: Prox1 represses IL-2 gene expression by interacting with NFAT2

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17278

    Prox1 is associated with IL-2 promoter Chromatin was extracted from ( A ) Jurkat cells and ( B ) naïve CD4 + T cells unstimulated or stimulated with anti-CD3/CD28 Dynabeads for 24 h, and precipitated with anti-Prox1 antibody or isotype IgG. The DNA sequence containing minimal IL-2 promoter (–256 to -46, product size: 211 bp) was analyzed by PCR. Data represent one of three separate experiments.
    Figure Legend Snippet: Prox1 is associated with IL-2 promoter Chromatin was extracted from ( A ) Jurkat cells and ( B ) naïve CD4 + T cells unstimulated or stimulated with anti-CD3/CD28 Dynabeads for 24 h, and precipitated with anti-Prox1 antibody or isotype IgG. The DNA sequence containing minimal IL-2 promoter (–256 to -46, product size: 211 bp) was analyzed by PCR. Data represent one of three separate experiments.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    Overexpression of Prox1 inhibits IL-2 expression ( A and B ) Lentiviral vector expressing Prox1 gene and GFP, or GFP alone, were generated. Jurkat cells were infected with Prox1 lentivirus or the control lentivirus. The GFP + Jurkat cells were sorted 48 h post-infection, and stimulated with anti-CD3/CD28 Dynabeads for indicated times. (A) Prox1 and (B) IL-2 mRNA levels were assessed by real-time PCR, while ( C ) IL-2 protein levels at 24 h were measured by ELISA. ( D ) Jurkat cells were transiently transfected with increasing amount of Prox1 plasmids (0.125, 0.25 and 0.5 μg/ml) or control vector (pcDNA3), and stimulated with anti-CD3/CD28 Dynabeads for 24 h. The IL-2 protein levels were measured by ELISA. The data from represent the mean ± SEM of three experiments, ** P
    Figure Legend Snippet: Overexpression of Prox1 inhibits IL-2 expression ( A and B ) Lentiviral vector expressing Prox1 gene and GFP, or GFP alone, were generated. Jurkat cells were infected with Prox1 lentivirus or the control lentivirus. The GFP + Jurkat cells were sorted 48 h post-infection, and stimulated with anti-CD3/CD28 Dynabeads for indicated times. (A) Prox1 and (B) IL-2 mRNA levels were assessed by real-time PCR, while ( C ) IL-2 protein levels at 24 h were measured by ELISA. ( D ) Jurkat cells were transiently transfected with increasing amount of Prox1 plasmids (0.125, 0.25 and 0.5 μg/ml) or control vector (pcDNA3), and stimulated with anti-CD3/CD28 Dynabeads for 24 h. The IL-2 protein levels were measured by ELISA. The data from represent the mean ± SEM of three experiments, ** P

    Techniques Used: Over Expression, Expressing, Plasmid Preparation, Generated, Infection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection

    Expression of Prox1 mRNA and protein in T cells ( A, B and C ) PBMCs, naïve CD4 + T cells and Jurkat cells were isolated, and stimulated with PHA and anti-CD3/CD28 Dynabeads for 24 h respectively. (A) Prox1 and (C) IL-2 mRNA levels were measured by RT-PCR, while (B) Prox1 protein levels were assessed by Western blot. For (A), (B) and (C), the data represent one of three independent experiments. ( D and E ) Naïve CD4 + T cells were stimulated with anti-CD3/CD28 Dynabeads for indicated times. (D) Prox1 and (E) IL-2 mRNA levels were measured by real-time PCR. Gene expression is normalized against the amount of GAPDH mRNA. The data from represent the mean ± SEM of three experiments.
    Figure Legend Snippet: Expression of Prox1 mRNA and protein in T cells ( A, B and C ) PBMCs, naïve CD4 + T cells and Jurkat cells were isolated, and stimulated with PHA and anti-CD3/CD28 Dynabeads for 24 h respectively. (A) Prox1 and (C) IL-2 mRNA levels were measured by RT-PCR, while (B) Prox1 protein levels were assessed by Western blot. For (A), (B) and (C), the data represent one of three independent experiments. ( D and E ) Naïve CD4 + T cells were stimulated with anti-CD3/CD28 Dynabeads for indicated times. (D) Prox1 and (E) IL-2 mRNA levels were measured by real-time PCR. Gene expression is normalized against the amount of GAPDH mRNA. The data from represent the mean ± SEM of three experiments.

    Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction

    3) Product Images from "Signal transducer and activator of transcription 3 (STAT3) promoter methylation and expression in pituitary adenoma"

    Article Title: Signal transducer and activator of transcription 3 (STAT3) promoter methylation and expression in pituitary adenoma

    Journal: BMC Medical Genetics

    doi: 10.1186/s12881-017-0434-3

    Analysis of STAT3 gene MS-PCR products on 2% agarose gel. M indicates methylated alleles, U unmethylated alleles. M cont. – positive methylation control (Standard Bisulfite Converted Universal Methylated Human DNA), U cont. – negative methylation control (normal human peripheral lymphocytes), H 2 O – water control, I-VI designate PA samples
    Figure Legend Snippet: Analysis of STAT3 gene MS-PCR products on 2% agarose gel. M indicates methylated alleles, U unmethylated alleles. M cont. – positive methylation control (Standard Bisulfite Converted Universal Methylated Human DNA), U cont. – negative methylation control (normal human peripheral lymphocytes), H 2 O – water control, I-VI designate PA samples

    Techniques Used: Mass Spectrometry, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Methylation

    4) Product Images from "High incidence of leukemia in large animals after stem cell gene therapy with a HOXB4-expressing retroviral vector"

    Article Title: High incidence of leukemia in large animals after stem cell gene therapy with a HOXB4-expressing retroviral vector

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI34371

    Retroviral integration induced mutagenesis is associated with leukemogenesis. ( A ) Southern blot analyses of marrow samples from G374, G450, and K00339 demonstrate monoclonality. Marrow DNA was digested with BglII, which cuts the transgene once, releasing a unique band for each integrant. Digestion with SacI, which cuts the transgene twice, showed a 3.9-kb band for all the integrants. ( B – D ) Integration sites were determined by LAM-PCR, and the schematic representations of integration sites for G374 ( B ), G450 ( C ), and K00339 ( D ) are shown. Exons are represented by black boxes. MSCV indicates the integration site, and the arrow indicates the orientation. ATG denotes the translation start site. Note that exons and introns are not to scale. ( E – G ) Dysregulated expression of genes in close vicinity of the integration sites for G374 ( E ), G450 ( F ), and K00339 ( G ). SYBR Green real-time RT-PCR was performed to determine the expression levels of dog MYB ( E ), dog ZFP36L2 and thyroid adenoma associated ( THADA ) ( F ), and macaque PRDM16 , SSBP2 , and SOCS1 ( G ). Canine PRDM16 ( E ) and macaque MAGOH2 ( G ) expression was undetectable in samples from normal control animals. ( G ) Expression of NSMCE1 was undetectable in K00339 and control animals. Marrow mRNA samples from normal animals were used as controls. M, DNA ladder.
    Figure Legend Snippet: Retroviral integration induced mutagenesis is associated with leukemogenesis. ( A ) Southern blot analyses of marrow samples from G374, G450, and K00339 demonstrate monoclonality. Marrow DNA was digested with BglII, which cuts the transgene once, releasing a unique band for each integrant. Digestion with SacI, which cuts the transgene twice, showed a 3.9-kb band for all the integrants. ( B – D ) Integration sites were determined by LAM-PCR, and the schematic representations of integration sites for G374 ( B ), G450 ( C ), and K00339 ( D ) are shown. Exons are represented by black boxes. MSCV indicates the integration site, and the arrow indicates the orientation. ATG denotes the translation start site. Note that exons and introns are not to scale. ( E – G ) Dysregulated expression of genes in close vicinity of the integration sites for G374 ( E ), G450 ( F ), and K00339 ( G ). SYBR Green real-time RT-PCR was performed to determine the expression levels of dog MYB ( E ), dog ZFP36L2 and thyroid adenoma associated ( THADA ) ( F ), and macaque PRDM16 , SSBP2 , and SOCS1 ( G ). Canine PRDM16 ( E ) and macaque MAGOH2 ( G ) expression was undetectable in samples from normal control animals. ( G ) Expression of NSMCE1 was undetectable in K00339 and control animals. Marrow mRNA samples from normal animals were used as controls. M, DNA ladder.

    Techniques Used: Mutagenesis, Southern Blot, Laser Capture Microdissection, Polymerase Chain Reaction, Expressing, SYBR Green Assay, Quantitative RT-PCR

    5) Product Images from "Insight into the role of PIKK family members and NF-кB in DNAdamage-induced senescence and senescence-associated secretory phenotype of colon cancer cells"

    Article Title: Insight into the role of PIKK family members and NF-кB in DNAdamage-induced senescence and senescence-associated secretory phenotype of colon cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0069-5

    The impact of simultaneous ATM, ATR, and DNA-PKc downregulation on senescence-associated secretory phenotype RT-PCR analysis of expression of selected senescence-associated secretory phenotype genes ( IL-8 , VEGF , RANTES ) in cells transfected either with negative siRNA or with combination of siRNAs targeting ATM , ATR , and PRKDC , treated with doxorubicin for 5 days. Results were normalized to the level of GAPDH mRNA. Normalized relative expression levels are shown (mean value of three independent experiments). Bars represent standard deviation
    Figure Legend Snippet: The impact of simultaneous ATM, ATR, and DNA-PKc downregulation on senescence-associated secretory phenotype RT-PCR analysis of expression of selected senescence-associated secretory phenotype genes ( IL-8 , VEGF , RANTES ) in cells transfected either with negative siRNA or with combination of siRNAs targeting ATM , ATR , and PRKDC , treated with doxorubicin for 5 days. Results were normalized to the level of GAPDH mRNA. Normalized relative expression levels are shown (mean value of three independent experiments). Bars represent standard deviation

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Standard Deviation

    The influence of downregulation of gene RELA (encoding NF-κB p65 subunit) on senescence of HCT116 p53+/+ and p53−/− cells HCT116 p53+/+ and p53−/− cells were transfected with negative siRNA or RELA siRNA (30 nM). Two days after transfection, the cells were treated with doxorubicin (100 nM) for 5 days. a Whole cell extracts were prepared at indicated time points after treatment with doxorubicin. The level of p65 and p21 proteins was estimated by Western blotting; GAPDH was used as a loading control. b ELISA analysis of of IL-8 secreted by HCT116 p53+/+ (left graph) and p53−/− cells (right graph) transfected with negative siRNA or with siRNA targeting RELA on days 1 and 5 of doxorubicin treatment; mean value of three independent experiments. Error bars represent standard deviation. c RT-PCR analysis of expression of IL-8 in cells transfected either with negative siRNA or with RELA siRNA, treated with doxorubicin for 5 days. Results were normalized to the level of GAPDH mRNA. Mean values of three independent experiments. Error bars represent standard deviation. d RT-PCR analysis of expression of selected NF-κB-regulated genes ( RANTES and GROα ) in cells transfected either with negative siRNA or with RELA siRNA, treated with doxorubicin for 5 days. Results were normalized to the level of GAPDH mRNA. Mean values of three independent experiments. Error bars represent standard deviation. e SA-β-Gal activity measured by flow cytometry. Representative histograms illustrating C 12 FDG fluorescence in HCT116 p53+/+ (left) and p53−/− (right) cells
    Figure Legend Snippet: The influence of downregulation of gene RELA (encoding NF-κB p65 subunit) on senescence of HCT116 p53+/+ and p53−/− cells HCT116 p53+/+ and p53−/− cells were transfected with negative siRNA or RELA siRNA (30 nM). Two days after transfection, the cells were treated with doxorubicin (100 nM) for 5 days. a Whole cell extracts were prepared at indicated time points after treatment with doxorubicin. The level of p65 and p21 proteins was estimated by Western blotting; GAPDH was used as a loading control. b ELISA analysis of of IL-8 secreted by HCT116 p53+/+ (left graph) and p53−/− cells (right graph) transfected with negative siRNA or with siRNA targeting RELA on days 1 and 5 of doxorubicin treatment; mean value of three independent experiments. Error bars represent standard deviation. c RT-PCR analysis of expression of IL-8 in cells transfected either with negative siRNA or with RELA siRNA, treated with doxorubicin for 5 days. Results were normalized to the level of GAPDH mRNA. Mean values of three independent experiments. Error bars represent standard deviation. d RT-PCR analysis of expression of selected NF-κB-regulated genes ( RANTES and GROα ) in cells transfected either with negative siRNA or with RELA siRNA, treated with doxorubicin for 5 days. Results were normalized to the level of GAPDH mRNA. Mean values of three independent experiments. Error bars represent standard deviation. e SA-β-Gal activity measured by flow cytometry. Representative histograms illustrating C 12 FDG fluorescence in HCT116 p53+/+ (left) and p53−/− (right) cells

    Techniques Used: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Expressing, Activity Assay, Flow Cytometry, Cytometry, Fluorescence

    6) Product Images from "Dicer Ablation Impairs Prostate Stem Cell Activity and Causes Prostate Atrophy"

    Article Title: Dicer Ablation Impairs Prostate Stem Cell Activity and Causes Prostate Atrophy

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1002/stem.455

    Dicer ablation results in a reduction of miRNA expression in the prostate. (A): PCR analysis confirms the homologous recombination at the DNA level. (B): Expression of eight miRNAs in WT, Dicer Het, and cKO prostate by Taqman miRNA assays. SnoRNA234 was
    Figure Legend Snippet: Dicer ablation results in a reduction of miRNA expression in the prostate. (A): PCR analysis confirms the homologous recombination at the DNA level. (B): Expression of eight miRNAs in WT, Dicer Het, and cKO prostate by Taqman miRNA assays. SnoRNA234 was

    Techniques Used: Expressing, Polymerase Chain Reaction, Homologous Recombination

    7) Product Images from "KPNA2 promotes metabolic reprogramming in glioblastomas by regulation of c-myc"

    Article Title: KPNA2 promotes metabolic reprogramming in glioblastomas by regulation of c-myc

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0861-9

    KPNA2 promoted the glycolytic metabolism in the glioblastoma cells. a Levels of KPNA2 were analyzed by qRT-PCR and immunoblotting in the U87 glioblastoma cells transfected with the lentiviruses expressing small hairpin RNAs of KPNA2 (shKPNA2), wild-type KPNA2 (KPNA2), or a vector with scrambled nonspecific shRNAs(SC). NC is for the original U87 cells that without any handles, GAPDH served as a loading control. b mRNA levels and c enzymatic activities of HK2, PKM2 and PFK1 were determined in the U87 cells transfected with KPNA2-shRNA(siKPNA2), scrambled shRNA(SC) or wild-type KPNA2(KPNA2). Each bar represented the mean ± s.d. from three independent experiments. * P
    Figure Legend Snippet: KPNA2 promoted the glycolytic metabolism in the glioblastoma cells. a Levels of KPNA2 were analyzed by qRT-PCR and immunoblotting in the U87 glioblastoma cells transfected with the lentiviruses expressing small hairpin RNAs of KPNA2 (shKPNA2), wild-type KPNA2 (KPNA2), or a vector with scrambled nonspecific shRNAs(SC). NC is for the original U87 cells that without any handles, GAPDH served as a loading control. b mRNA levels and c enzymatic activities of HK2, PKM2 and PFK1 were determined in the U87 cells transfected with KPNA2-shRNA(siKPNA2), scrambled shRNA(SC) or wild-type KPNA2(KPNA2). Each bar represented the mean ± s.d. from three independent experiments. * P

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

    8) Product Images from "A dosage-dependent pleiotropic role of Dicer in prostate cancer growth and metastasis"

    Article Title: A dosage-dependent pleiotropic role of Dicer in prostate cancer growth and metastasis

    Journal: Oncogene

    doi: 10.1038/onc.2013.281

    Disrupting Dicer activity inhibits disease progression in the Pten null mouse model for prostate cancer (A) Quantitative PCR analyses of ratio of exon24/exon21 in genomic DNAs from 15-week-old Pten −/− , Pten −/− Dicer −/+ , and Pten −/− Dicer −/− mice. (B) qRT-PCR analysis of 6 representative microRNAs in 15-week-old Pten −/− , Pten −/− Dicer −/+ , and Pten −/− Dicer −/− mice. *: p
    Figure Legend Snippet: Disrupting Dicer activity inhibits disease progression in the Pten null mouse model for prostate cancer (A) Quantitative PCR analyses of ratio of exon24/exon21 in genomic DNAs from 15-week-old Pten −/− , Pten −/− Dicer −/+ , and Pten −/− Dicer −/− mice. (B) qRT-PCR analysis of 6 representative microRNAs in 15-week-old Pten −/− , Pten −/− Dicer −/+ , and Pten −/− Dicer −/− mice. *: p

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

    9) Product Images from "Expression of serotonin 2A, 2C, 6 and 7 receptor and IL-6 mRNA in experimental toxoplasmic encephalitis in mice"

    Article Title: Expression of serotonin 2A, 2C, 6 and 7 receptor and IL-6 mRNA in experimental toxoplasmic encephalitis in mice

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2019.e02890

    Relative mRNA expression level of IL-6, serotonin 2A receptor (5-HTR2A), serotonin 2C receptor (5-HTR2C), serotonin 6 receptor (5-HTR6) and serotonin 7 receptor (5-HTR7) in the brains of healthy or T. gondii -infected mice. Gene expression was detected by quantitative real-time PCR. β-Actin was used as a reference gene. The gene-specific primers are listed in the Material and Methods. The relative expression levels were calculated by the 2 (-ΔΔCT) method. Statistical analysis was performed using a one-way analysis of variance and Tukey's multiple comparison test. The values represent means ± S.D. ***p
    Figure Legend Snippet: Relative mRNA expression level of IL-6, serotonin 2A receptor (5-HTR2A), serotonin 2C receptor (5-HTR2C), serotonin 6 receptor (5-HTR6) and serotonin 7 receptor (5-HTR7) in the brains of healthy or T. gondii -infected mice. Gene expression was detected by quantitative real-time PCR. β-Actin was used as a reference gene. The gene-specific primers are listed in the Material and Methods. The relative expression levels were calculated by the 2 (-ΔΔCT) method. Statistical analysis was performed using a one-way analysis of variance and Tukey's multiple comparison test. The values represent means ± S.D. ***p

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

    10) Product Images from "Reduced expression of enolase-1 correlates with high intracellular glucose levels and increased senescence in cisplatin-resistant ovarian cancer cells"

    Article Title: Reduced expression of enolase-1 correlates with high intracellular glucose levels and increased senescence in cisplatin-resistant ovarian cancer cells

    Journal: American Journal of Translational Research

    doi:

    ENO1 protein and mRNA levels in a panel of ovarian cancer cells. (A) Western blot analysis was performed using protein extracts (30-50 µg) of cisplatin-sensitive (A2780, OV-90, and OVCAR3) and cisplatin-resistant (A280CP20, A2780CIS, OV-90CIS, and OVCAR3CIS) ovarian cancer cells. (B) Densitometry analysis of band intensities, shown in (A). Fold changes in protein levels were calculated relative to the cisplatin sensitive pair. Averages ± SEM are shown for three independent experiments (C) ENO1 mRNA expression levels were assessed by qPCR. β-actin was used as a PCR internal control. Fold changes in mRNA levels were calculated relative to the cisplatin sensitive pair. Averages ± SEM are shown for three independent experiments. *P
    Figure Legend Snippet: ENO1 protein and mRNA levels in a panel of ovarian cancer cells. (A) Western blot analysis was performed using protein extracts (30-50 µg) of cisplatin-sensitive (A2780, OV-90, and OVCAR3) and cisplatin-resistant (A280CP20, A2780CIS, OV-90CIS, and OVCAR3CIS) ovarian cancer cells. (B) Densitometry analysis of band intensities, shown in (A). Fold changes in protein levels were calculated relative to the cisplatin sensitive pair. Averages ± SEM are shown for three independent experiments (C) ENO1 mRNA expression levels were assessed by qPCR. β-actin was used as a PCR internal control. Fold changes in mRNA levels were calculated relative to the cisplatin sensitive pair. Averages ± SEM are shown for three independent experiments. *P

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

    11) Product Images from "Involvement of Interleukin-6 and Androgen Receptor Signaling in Pancreatic Cancer"

    Article Title: Involvement of Interleukin-6 and Androgen Receptor Signaling in Pancreatic Cancer

    Journal: Genes & Cancer

    doi: 10.1177/1947601910383417

    Migration of pancreatic cancer cell lines was investigated using a wound-healing scratch assay ( A-E ). Knockdown of AR suppressed migration of pancreatic cancer cells in the presence of IL-6 and DHT ( F-H ). KP-2 ( A ), SUIT-2 ( B ), Panc-1 ( C ), AsPC-1 ( D ), and MIAPaCa-2 ( E ) cells were subjected to wound-healing scratch assay. Cells were incubated with or without IL-6 (100 ng/mL) and/or DHT (10 nM) for approximately 24 hours. The migration thus observed was presented as a ratio of migration, considering migration in untreated control as 1. Scion images were used for analysis. The error bars represent mean ± SD of 3 independent experiments. Results of semiquantitative RT-PCR analysis for AR and GAPDH mRNAs are shown in the lower part below each graph ( A-E ). KP-2 cells were transfected with 10 nM AR siRNA (si-AR) or 10 nM control siRNA (si-C) (Thermo Fisher Scientific Dharmacon), subjected to wound-healing scratch assay, and incubated with IL-6 (100 ng/mL) and DHT (10 nM) for 24 hours. ( F ) Light microscopy pictures show KP-2 cells transfected with 10 nM si-C or 10 nM si-AR and incubated with 100 ng/mL IL-6 and 10 nM DHT at 0 hours (left panel) and 24 hours later (right panels). ( G ) The migration observed was presented as a ratio of migration, considering migration in transfection with si-C as 1. The error bars represent mean ± SD of 3 independent experiments. ( H ) Knockdown of AR expression in KP-2 cells. Whole cell lysates from KP-2 cells after si-C or si-AR treatment were immunoblotted with antibodies against AR or GAPDH.
    Figure Legend Snippet: Migration of pancreatic cancer cell lines was investigated using a wound-healing scratch assay ( A-E ). Knockdown of AR suppressed migration of pancreatic cancer cells in the presence of IL-6 and DHT ( F-H ). KP-2 ( A ), SUIT-2 ( B ), Panc-1 ( C ), AsPC-1 ( D ), and MIAPaCa-2 ( E ) cells were subjected to wound-healing scratch assay. Cells were incubated with or without IL-6 (100 ng/mL) and/or DHT (10 nM) for approximately 24 hours. The migration thus observed was presented as a ratio of migration, considering migration in untreated control as 1. Scion images were used for analysis. The error bars represent mean ± SD of 3 independent experiments. Results of semiquantitative RT-PCR analysis for AR and GAPDH mRNAs are shown in the lower part below each graph ( A-E ). KP-2 cells were transfected with 10 nM AR siRNA (si-AR) or 10 nM control siRNA (si-C) (Thermo Fisher Scientific Dharmacon), subjected to wound-healing scratch assay, and incubated with IL-6 (100 ng/mL) and DHT (10 nM) for 24 hours. ( F ) Light microscopy pictures show KP-2 cells transfected with 10 nM si-C or 10 nM si-AR and incubated with 100 ng/mL IL-6 and 10 nM DHT at 0 hours (left panel) and 24 hours later (right panels). ( G ) The migration observed was presented as a ratio of migration, considering migration in transfection with si-C as 1. The error bars represent mean ± SD of 3 independent experiments. ( H ) Knockdown of AR expression in KP-2 cells. Whole cell lysates from KP-2 cells after si-C or si-AR treatment were immunoblotted with antibodies against AR or GAPDH.

    Techniques Used: Migration, Wound Healing Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Transfection, Light Microscopy, Expressing

    AR and IL-6R expression in pancreatic cancer cell lines. ( A ) Relative expression of AR mRNA. RNAs from MIAPaCa-2, Panc-1, AsPC-1, SUIT-2, KP-2 cells, and LNCaP (AR-positive) cells were subjected to real-time RT-PCR for AR and GAPDH gene expression. AR/GAPDH mRNA in MIAPaCa-2 was set as 1-fold. The results were based on the relative expression level of the housekeeping gene GAPDH as an internal control. ( B ) IL-6Rα, GP130, AR, and GAPDH protein expression. Total cell lysates (5 µg) of MIAPaCa-2, Panc-1, AsPC-1, SUIT-2, KP-2, and LNCaP were subjected to 10% SDS-PAGE for immunoblotting with specific antibodies against IL-6Rα (Santa Cruz Biotechnology), GP130 (Cell Signaling Technology), AR (Santa Cruz Biotechnology), and GAPDH (Santa Cruz Biotechnology).
    Figure Legend Snippet: AR and IL-6R expression in pancreatic cancer cell lines. ( A ) Relative expression of AR mRNA. RNAs from MIAPaCa-2, Panc-1, AsPC-1, SUIT-2, KP-2 cells, and LNCaP (AR-positive) cells were subjected to real-time RT-PCR for AR and GAPDH gene expression. AR/GAPDH mRNA in MIAPaCa-2 was set as 1-fold. The results were based on the relative expression level of the housekeeping gene GAPDH as an internal control. ( B ) IL-6Rα, GP130, AR, and GAPDH protein expression. Total cell lysates (5 µg) of MIAPaCa-2, Panc-1, AsPC-1, SUIT-2, KP-2, and LNCaP were subjected to 10% SDS-PAGE for immunoblotting with specific antibodies against IL-6Rα (Santa Cruz Biotechnology), GP130 (Cell Signaling Technology), AR (Santa Cruz Biotechnology), and GAPDH (Santa Cruz Biotechnology).

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

    12) Product Images from "MiR-196a exerts its oncogenic effect in glioblastoma multiforme by inhibition of IκBα both in vitro and in vivo"

    Article Title: MiR-196a exerts its oncogenic effect in glioblastoma multiforme by inhibition of IκBα both in vitro and in vivo

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/not307

    Effect of miR-196a on cell proliferation and apoptosis in vitro. (A) miR-196a expression was quantified by qRT–PCR in U87MG and T98G cells 48 h after transfection of miR-196a mimics, AMO-miR-196a, or negative control oligo. (B) Cell viability
    Figure Legend Snippet: Effect of miR-196a on cell proliferation and apoptosis in vitro. (A) miR-196a expression was quantified by qRT–PCR in U87MG and T98G cells 48 h after transfection of miR-196a mimics, AMO-miR-196a, or negative control oligo. (B) Cell viability

    Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Transfection, Negative Control

    13) Product Images from "Transcriptional Regulation of N-acetylaspartate Metabolism in the 5xFAD Model of Alzheimer's Disease: Evidence for Neuron-Glia Communication During Energetic Crisis"

    Article Title: Transcriptional Regulation of N-acetylaspartate Metabolism in the 5xFAD Model of Alzheimer's Disease: Evidence for Neuron-Glia Communication During Energetic Crisis

    Journal: Molecular and cellular neurosciences

    doi: 10.1016/j.mcn.2015.03.009

    ( A ) In-house QRT-PCR confirmation of the most significantly downregulated Complex I gene, Ndusf2 , confirms 4-month array data (5xFAD values expressed as fold-2 month wild type values, +/- SEM, n=5. ** p≤0.01). Reduced Ndusf2 expression correlates
    Figure Legend Snippet: ( A ) In-house QRT-PCR confirmation of the most significantly downregulated Complex I gene, Ndusf2 , confirms 4-month array data (5xFAD values expressed as fold-2 month wild type values, +/- SEM, n=5. ** p≤0.01). Reduced Ndusf2 expression correlates

    Techniques Used: Quantitative RT-PCR, Expressing

    ( A ) Increased aspa expression in 5xFAD brains at 2 months of age relative to wild type as assessed by QRT-PCR. Aspa expression presented as fold-wild type 2 month levels for each group, mean +/- SEM, n=5. Immunhistochemical evidence of increases in ASPA
    Figure Legend Snippet: ( A ) Increased aspa expression in 5xFAD brains at 2 months of age relative to wild type as assessed by QRT-PCR. Aspa expression presented as fold-wild type 2 month levels for each group, mean +/- SEM, n=5. Immunhistochemical evidence of increases in ASPA

    Techniques Used: Expressing, Quantitative RT-PCR

    Whole brain homogenates analyzed by HPLC show a significant reduction in NAA from 2-4 months of age in 5xFAD brains ( 1A ; mean +/- SEM shown for each genotype at each age, n=7). QRT-PCR analysis of Nat8L expression 2-4 months of age showing a significant
    Figure Legend Snippet: Whole brain homogenates analyzed by HPLC show a significant reduction in NAA from 2-4 months of age in 5xFAD brains ( 1A ; mean +/- SEM shown for each genotype at each age, n=7). QRT-PCR analysis of Nat8L expression 2-4 months of age showing a significant

    Techniques Used: High Performance Liquid Chromatography, Quantitative RT-PCR, Expressing

    14) Product Images from "Rapid enhancement of α-tocopherol content in Nicotiana benthamiana by transient expression of Arabidopsis thaliana Tocopherol cyclase and Homogentisate phytyl transferase genes"

    Article Title: Rapid enhancement of α-tocopherol content in Nicotiana benthamiana by transient expression of Arabidopsis thaliana Tocopherol cyclase and Homogentisate phytyl transferase genes

    Journal: 3 Biotech

    doi: 10.1007/s13205-018-1496-4

    Transient expression of AtHPT, AtTC and AtTC + AtHPT post agroinfiltration by reverse transcription PCR. Top panel: Target genes Lane 1 Wild type control Lane 2 HPT Lane 3 TC Lane 3 HPT + TC with TC primers Lane 3 HPT + TC with HPT primers Lane 6 HPT + TC with both HPT TC primers. Bottom panel: Reference gene F-box
    Figure Legend Snippet: Transient expression of AtHPT, AtTC and AtTC + AtHPT post agroinfiltration by reverse transcription PCR. Top panel: Target genes Lane 1 Wild type control Lane 2 HPT Lane 3 TC Lane 3 HPT + TC with TC primers Lane 3 HPT + TC with HPT primers Lane 6 HPT + TC with both HPT TC primers. Bottom panel: Reference gene F-box

    Techniques Used: Expressing, Polymerase Chain Reaction

    15) Product Images from "Clinical profile of hearing loss in children with congenital cytomegalovirus (CMV) infection: CMV DNA diagnosis using preserved umbilical cord"

    Article Title: Clinical profile of hearing loss in children with congenital cytomegalovirus (CMV) infection: CMV DNA diagnosis using preserved umbilical cord

    Journal: Acta Oto-Laryngologica

    doi: 10.3109/00016489.2011.583268

    An original amplification plot of real-time PCR in case no. 12 with positive CMV DNA. CMV DNA in positive control and case 12 (A) and genomic DNA (GJB2 gene) in positive control, case no. 12 and negative control (B) are amplified. These results show that our real-time PCR method is precise. Blank, sample without any added DNA.
    Figure Legend Snippet: An original amplification plot of real-time PCR in case no. 12 with positive CMV DNA. CMV DNA in positive control and case 12 (A) and genomic DNA (GJB2 gene) in positive control, case no. 12 and negative control (B) are amplified. These results show that our real-time PCR method is precise. Blank, sample without any added DNA.

    Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Positive Control, Negative Control

    16) Product Images from "HIF-2α mediates hypoxia-induced LIF expression in human colorectal cancer cells"

    Article Title: HIF-2α mediates hypoxia-induced LIF expression in human colorectal cancer cells

    Journal: Oncotarget

    doi:

    Hypoxia transactivates hypoxia-responsive elements (HREs) in the LIF promoter through HIF-2α (A) The human LIF gene contains 4 putative HREs in its promoter region. (B) Hypoxia activates the luciferase activity of reporter vectors containing HRE-C or HRE-D sites in the LIF promoter. RKO and HCT116 cells were transfected with the luciferase reporter vectors, and then subjected to hypoxia treatment for 36 h before measuring luciferase activities. Luciferase reporter vectors containing the HRE site in the VEGF promoter was included as a positive control. (C) and (D) HIF-2α but not HIF-1α binds to HRE-C and HRE-D sites in the LIF promoter under the hypoxic condition in RKO cells as determined by ChIP assays. Cells were cultured under the hypoxic or normoxic conditions for 36 h before assays. The HRE site in the VEGF promoter serves as a positive control. The amount of DNA fragments pulled-down was determined by real-time PCR (C) or conventional PCR (D) . Data are presented as mean ± SD ( n = 3). *: p
    Figure Legend Snippet: Hypoxia transactivates hypoxia-responsive elements (HREs) in the LIF promoter through HIF-2α (A) The human LIF gene contains 4 putative HREs in its promoter region. (B) Hypoxia activates the luciferase activity of reporter vectors containing HRE-C or HRE-D sites in the LIF promoter. RKO and HCT116 cells were transfected with the luciferase reporter vectors, and then subjected to hypoxia treatment for 36 h before measuring luciferase activities. Luciferase reporter vectors containing the HRE site in the VEGF promoter was included as a positive control. (C) and (D) HIF-2α but not HIF-1α binds to HRE-C and HRE-D sites in the LIF promoter under the hypoxic condition in RKO cells as determined by ChIP assays. Cells were cultured under the hypoxic or normoxic conditions for 36 h before assays. The HRE site in the VEGF promoter serves as a positive control. The amount of DNA fragments pulled-down was determined by real-time PCR (C) or conventional PCR (D) . Data are presented as mean ± SD ( n = 3). *: p

    Techniques Used: Luciferase, Activity Assay, Transfection, Positive Control, Chromatin Immunoprecipitation, Cell Culture, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Hypoxia induces LIF expression levels in human colorectal cancer cell lines Human colorectal cancer cell lines RKO and HCT116 cells were cultured under the hypoxic condition for the indicated time periods. (A) The mRNA expression levels of LIF in these cells were determined by Taqman real-time PCR and normalized with actin (left panels). The mRNA expression levels of VEGF in these cells were determined as a positive control (right panels). (B) The LIF protein levels were determined by Elisa assays (left panels) and Western-blot assays (right panels), respectively. Data are presented as mean ± SD ( n = 3). *: p
    Figure Legend Snippet: Hypoxia induces LIF expression levels in human colorectal cancer cell lines Human colorectal cancer cell lines RKO and HCT116 cells were cultured under the hypoxic condition for the indicated time periods. (A) The mRNA expression levels of LIF in these cells were determined by Taqman real-time PCR and normalized with actin (left panels). The mRNA expression levels of VEGF in these cells were determined as a positive control (right panels). (B) The LIF protein levels were determined by Elisa assays (left panels) and Western-blot assays (right panels), respectively. Data are presented as mean ± SD ( n = 3). *: p

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Positive Control, Enzyme-linked Immunosorbent Assay, Western Blot

    HIF-2α transcriptionally regulates LIF expression (A) Ectopic HIF-2α expression increases LIF and VEGF mRNA expression levels in RKO and HCT116 cells. The mRNA expression levels of LIF and VEGF were determined by Taqman real-time PCR and normalized with actin. (B) Ectopic HIF-1α expression increases VEGF mRNA expression levels but has no obvious effect on LIF mRNA expression levels in RKO and HCT116 cells. (C) Ectopic HIF-2α expression increases LIF protein levels in RKO and HCT116 cells as determined by Western-blot assays. (D) and (E) HIF-2α binds to HRE-C and HRE-D sites in the LIF promoter in RKO cells transfected with HIF-2α expression plasmids as determined by ChIP assays. The amount of DNA fragments pulled-down was determined by real-time PCR (D) or conventional PCR (E) . The HRE site in the VEGF promoter serves as a positive control. (F) HIF-2α activates luciferase activity of reporter vectors containing HRE-C or HRE-D sites in the LIF promoter in RKO cells transfected with HIF-2α expression plasmids. Luciferase reporter vectors containing the HRE site in the VEGF promoter was included as a positive control. Data are presented as mean ± SD ( n = 3). *: p
    Figure Legend Snippet: HIF-2α transcriptionally regulates LIF expression (A) Ectopic HIF-2α expression increases LIF and VEGF mRNA expression levels in RKO and HCT116 cells. The mRNA expression levels of LIF and VEGF were determined by Taqman real-time PCR and normalized with actin. (B) Ectopic HIF-1α expression increases VEGF mRNA expression levels but has no obvious effect on LIF mRNA expression levels in RKO and HCT116 cells. (C) Ectopic HIF-2α expression increases LIF protein levels in RKO and HCT116 cells as determined by Western-blot assays. (D) and (E) HIF-2α binds to HRE-C and HRE-D sites in the LIF promoter in RKO cells transfected with HIF-2α expression plasmids as determined by ChIP assays. The amount of DNA fragments pulled-down was determined by real-time PCR (D) or conventional PCR (E) . The HRE site in the VEGF promoter serves as a positive control. (F) HIF-2α activates luciferase activity of reporter vectors containing HRE-C or HRE-D sites in the LIF promoter in RKO cells transfected with HIF-2α expression plasmids. Luciferase reporter vectors containing the HRE site in the VEGF promoter was included as a positive control. Data are presented as mean ± SD ( n = 3). *: p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Positive Control, Luciferase, Activity Assay

    HIF-2α mediates the induction of LIF expression by hypoxia Knockdown of endogenous HIF-2α but not HIF-1α largely abolishes the induction of LIF expression by hypoxia in RKO cells. Cells with knockdown of endogenous HIF-1α, HIF-2α by siRNA oligos or transfected with control siRNA were treated with hypoxia for 36 h. Two different siRNA oligos against HIF-1α and HIF-2α, respectively, were used, and similar results were obtained. (A) The mRNA expression levels of LIF (left panel) and VEGF (right panel) were determined by Taqman real-time PCR and normalized with actin. (B) The LIF protein levels were determined by Western-blot assays. (C) The knockdown of HIF-1α (left panel) and HIF-2α (right panel) in cells was confirmed at the mRNA level by Taqman real-time PCR and normalized with actin. Data are presented as mean ± SD ( n = 3). *: p
    Figure Legend Snippet: HIF-2α mediates the induction of LIF expression by hypoxia Knockdown of endogenous HIF-2α but not HIF-1α largely abolishes the induction of LIF expression by hypoxia in RKO cells. Cells with knockdown of endogenous HIF-1α, HIF-2α by siRNA oligos or transfected with control siRNA were treated with hypoxia for 36 h. Two different siRNA oligos against HIF-1α and HIF-2α, respectively, were used, and similar results were obtained. (A) The mRNA expression levels of LIF (left panel) and VEGF (right panel) were determined by Taqman real-time PCR and normalized with actin. (B) The LIF protein levels were determined by Western-blot assays. (C) The knockdown of HIF-1α (left panel) and HIF-2α (right panel) in cells was confirmed at the mRNA level by Taqman real-time PCR and normalized with actin. Data are presented as mean ± SD ( n = 3). *: p

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot

    17) Product Images from "Next-generation sequencing identified SPATC1L as a possible candidate gene for both early-onset and age-related hearing loss"

    Article Title: Next-generation sequencing identified SPATC1L as a possible candidate gene for both early-onset and age-related hearing loss

    Journal: European Journal of Human Genetics

    doi: 10.1038/s41431-018-0229-9

    SPATC1L mRNA and protein levels in Hek293 transfected cells and Spatc1l expression in mouse whole cochlea and other tissues at different time points. a qRT-PCR analysis of relative mRNA expression of SPATC1L wild type and mutants after 48 h of transfection in Hek293 cells. Results are expressed as a fold-change of expression levels, and are normalized to the relative amount of the internal standard Neo. Error bars indicate 95% confidence intervals. b Western blot analysis of SPATC1L wild type and mutant proteins. Hsp90 was applied to determine equal loading. c The graph shows expression of Spatc1l in mouse whole cochlea at P3, P8, P12 and 2 months. Results are reported as fold change in gene expression over β actin, used as an internal control. The gene shows an age-related expression. d The graph shows Spatc1l expression at 2 months of age in different mouse tissues, including liver, cochlea, spleen, lung, kidney, brain, testis and heart. Results are reported as fold change in gene expression over β actin, used as an internal control
    Figure Legend Snippet: SPATC1L mRNA and protein levels in Hek293 transfected cells and Spatc1l expression in mouse whole cochlea and other tissues at different time points. a qRT-PCR analysis of relative mRNA expression of SPATC1L wild type and mutants after 48 h of transfection in Hek293 cells. Results are expressed as a fold-change of expression levels, and are normalized to the relative amount of the internal standard Neo. Error bars indicate 95% confidence intervals. b Western blot analysis of SPATC1L wild type and mutant proteins. Hsp90 was applied to determine equal loading. c The graph shows expression of Spatc1l in mouse whole cochlea at P3, P8, P12 and 2 months. Results are reported as fold change in gene expression over β actin, used as an internal control. The gene shows an age-related expression. d The graph shows Spatc1l expression at 2 months of age in different mouse tissues, including liver, cochlea, spleen, lung, kidney, brain, testis and heart. Results are reported as fold change in gene expression over β actin, used as an internal control

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

    18) Product Images from "MiR-31 regulates the cisplatin resistance by targeting Src in gallbladder cancer"

    Article Title: MiR-31 regulates the cisplatin resistance by targeting Src in gallbladder cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.13067

    Effect of miR-31 on cisplatin sensitivity and invasion capacity of DDP-resistant cells A. The affirmation of the level of miR-31 mRNA expressed in GBC-SD/DDP and NOZ/DDP cells transfected with miR-31 mimic by qRT-PCR. B. The cell viability plotted against the concentration of DDP in GBC-SD/DPP and NOZ/DPP cells transfected with miR-31 or vector. C. Colony formation assay of DDP-resistant cells transfected with miR-31 or vector after exposure to DDP. D. The DDP-induced apoptosis rate of GBC-SD/DPP and NOZ/DPP cells transfected with miR-31 or vector analyzed by flow cytometry. E. The invasive ability of DDP-resistant cells transfected with miR-31 or vector after treatment with DDP in the transwell invasion assay. Data were presented as mean ± SD. * P
    Figure Legend Snippet: Effect of miR-31 on cisplatin sensitivity and invasion capacity of DDP-resistant cells A. The affirmation of the level of miR-31 mRNA expressed in GBC-SD/DDP and NOZ/DDP cells transfected with miR-31 mimic by qRT-PCR. B. The cell viability plotted against the concentration of DDP in GBC-SD/DPP and NOZ/DPP cells transfected with miR-31 or vector. C. Colony formation assay of DDP-resistant cells transfected with miR-31 or vector after exposure to DDP. D. The DDP-induced apoptosis rate of GBC-SD/DPP and NOZ/DPP cells transfected with miR-31 or vector analyzed by flow cytometry. E. The invasive ability of DDP-resistant cells transfected with miR-31 or vector after treatment with DDP in the transwell invasion assay. Data were presented as mean ± SD. * P

    Techniques Used: Transfection, Quantitative RT-PCR, Concentration Assay, Plasmid Preparation, Colony Assay, Flow Cytometry, Cytometry, Transwell Invasion Assay

    Src is a direct target gene of miR-31 and inversely correlated with miR-31 in GBC patients A. Sequence of the miR-31-binding site within the human Src 3′-UTR and a schematic diagram of the reporter construct showing the entire Src 3′-UTR sequence and the mutated Src 3′-UTR sequence. Src-WT: wild-type; Src-MUT: mutant type B. Luciferase assay of NOZ/DDP cells co-transfected with vector or miR-31 and a luciferase reporter containing the full length of Src 3′-UTR (WT) or a mutant (MUT). Luciferase activities were measured 24 hours post-transfection. Src-WT: wild-type; Src-MUT: mutant type C. The expression level of Src measured by qRT-PCR and normalized to GAPDH. D. The immunoblotting data of Src and p-Src(Y416) expression in DDP-resistant GBC cells transfected with miR-31 or vector. E. The mRNA expression of Src in tumor tissues and the adjacent non-tumor tissues from 41 GBC patients by qRT-PCR. F. An inverse correlation between miR-31 and Src mRNA expression by Pearson correlation analysis (Pearson's correlation: r= −0.56, P
    Figure Legend Snippet: Src is a direct target gene of miR-31 and inversely correlated with miR-31 in GBC patients A. Sequence of the miR-31-binding site within the human Src 3′-UTR and a schematic diagram of the reporter construct showing the entire Src 3′-UTR sequence and the mutated Src 3′-UTR sequence. Src-WT: wild-type; Src-MUT: mutant type B. Luciferase assay of NOZ/DDP cells co-transfected with vector or miR-31 and a luciferase reporter containing the full length of Src 3′-UTR (WT) or a mutant (MUT). Luciferase activities were measured 24 hours post-transfection. Src-WT: wild-type; Src-MUT: mutant type C. The expression level of Src measured by qRT-PCR and normalized to GAPDH. D. The immunoblotting data of Src and p-Src(Y416) expression in DDP-resistant GBC cells transfected with miR-31 or vector. E. The mRNA expression of Src in tumor tissues and the adjacent non-tumor tissues from 41 GBC patients by qRT-PCR. F. An inverse correlation between miR-31 and Src mRNA expression by Pearson correlation analysis (Pearson's correlation: r= −0.56, P

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

    MiR-31 increases chemosensitivity of NOZ/DDP cells in vivo A. The xenograft tumors excised from the nude mice subcutaneously injected NOZ/DDP cells transfected with miR-31 or vector with or without treatment with DDP. B. The effect of miR-31 on the volume of xenograft tumors treated with DDP. C. The effect of miR-31 on the weight of xenograft tumors treated with DDP. D, E. The expression levels of miR-31and Src mRNA of xenograft tumors quantified by qRT-PCR. Data were presented as mean ± SD. * P
    Figure Legend Snippet: MiR-31 increases chemosensitivity of NOZ/DDP cells in vivo A. The xenograft tumors excised from the nude mice subcutaneously injected NOZ/DDP cells transfected with miR-31 or vector with or without treatment with DDP. B. The effect of miR-31 on the volume of xenograft tumors treated with DDP. C. The effect of miR-31 on the weight of xenograft tumors treated with DDP. D, E. The expression levels of miR-31and Src mRNA of xenograft tumors quantified by qRT-PCR. Data were presented as mean ± SD. * P

    Techniques Used: In Vivo, Mouse Assay, Injection, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR

    Down-regulation of miR-31 in DDP-resistant GBC cells A. Heatmap representation of the expression difference of miRNAs in the GBC-SD, NOZ and the DDP-resistant GBC cells. The horizontal axis signifies the expression of miRNAs, and columns represent the biological replicates. Red, high expression; green, low expression. B. The expression level of miR-31 detected in DDP-resistant cells (GBC-SD/DDP and NOZ/DDP cells) and in its parental cells by qRT-PCR. Data were presented as mean ± SD. ** P
    Figure Legend Snippet: Down-regulation of miR-31 in DDP-resistant GBC cells A. Heatmap representation of the expression difference of miRNAs in the GBC-SD, NOZ and the DDP-resistant GBC cells. The horizontal axis signifies the expression of miRNAs, and columns represent the biological replicates. Red, high expression; green, low expression. B. The expression level of miR-31 detected in DDP-resistant cells (GBC-SD/DDP and NOZ/DDP cells) and in its parental cells by qRT-PCR. Data were presented as mean ± SD. ** P

    Techniques Used: Expressing, Quantitative RT-PCR

    19) Product Images from "Cooperative TRAIL production mediates IFNα/Smac mimetic-induced cell death in TNFα-resistant solid cancer cells"

    Article Title: Cooperative TRAIL production mediates IFNα/Smac mimetic-induced cell death in TNFα-resistant solid cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.6915

    IFNα and BV6 cooperate to increase TNFα and TRAIL production A. Cells were treated for 12 hours with IFNα (A172: 5 ng/ml, HT-29: 10 ng/ml) and/or 1 μM BV6. TNFα mRNA levels were determined by qRT-PCR and are shown as fold increase with mean + SD of four independent experiments performed in duplicate; 28S rRNA was used as loading control, * P
    Figure Legend Snippet: IFNα and BV6 cooperate to increase TNFα and TRAIL production A. Cells were treated for 12 hours with IFNα (A172: 5 ng/ml, HT-29: 10 ng/ml) and/or 1 μM BV6. TNFα mRNA levels were determined by qRT-PCR and are shown as fold increase with mean + SD of four independent experiments performed in duplicate; 28S rRNA was used as loading control, * P

    Techniques Used: Quantitative RT-PCR

    IFNα/BV6-induced cell death depends on TRAIL signaling in A172 cells A. - C. A172 cells were transiently transfected with 5 nM siRNA targeting TRAIL (siTRAIL1, siTRAIL2) or control siRNA. TRAIL mRNA levels were analyzed by qRT-PCR and are shown as fold increase with mean + SD of three (A172) or five (HT-29) independent experiments performed in duplicate, 28S rRNA was used as loading control A. . Cell death was determined after treatment for 72 hours with 5 ng/ml IFNα and/or 1 μM BV6 by PI staining using flow cytometry, mean + SD of five independent experiments performed in duplicate are shown; *, P
    Figure Legend Snippet: IFNα/BV6-induced cell death depends on TRAIL signaling in A172 cells A. - C. A172 cells were transiently transfected with 5 nM siRNA targeting TRAIL (siTRAIL1, siTRAIL2) or control siRNA. TRAIL mRNA levels were analyzed by qRT-PCR and are shown as fold increase with mean + SD of three (A172) or five (HT-29) independent experiments performed in duplicate, 28S rRNA was used as loading control A. . Cell death was determined after treatment for 72 hours with 5 ng/ml IFNα and/or 1 μM BV6 by PI staining using flow cytometry, mean + SD of five independent experiments performed in duplicate are shown; *, P

    Techniques Used: Transfection, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry

    20) Product Images from "Antimelanogenic effect of c-phycocyanin through modulation of tyrosinase expression by upregulation of ERK and downregulation of p38 MAPK signaling pathways"

    Article Title: Antimelanogenic effect of c-phycocyanin through modulation of tyrosinase expression by upregulation of ERK and downregulation of p38 MAPK signaling pathways

    Journal: Journal of Biomedical Science

    doi: 10.1186/1423-0127-18-74

    Cpc attenuated α-MSH-stimulated melanogenesis and elevated the abundance of intracellular cAMP . Cells were pretreated with 20 nM α-MSH for 30 mins, and then treated with Cpc (0.05, 0.1, 0.2 mg/mL) for 72 hrs. (A) Tyrosinase activity (black) and melanin content (grey) were measured. (B) The expression of tyrosinase was determined by immunoblotting analysis (black) and RT-PCR (grey), using β-actin and GAPDH as internal standards, respectively. (C) The cAMP concentration was measured by enzyme immunoassay at assigned time intervals (10, 30, 60 min) after Cpc treatment. Data were expressed at mean ± SD from three different experiments. The asterisk (*) indicates a significant difference from control group (*, P
    Figure Legend Snippet: Cpc attenuated α-MSH-stimulated melanogenesis and elevated the abundance of intracellular cAMP . Cells were pretreated with 20 nM α-MSH for 30 mins, and then treated with Cpc (0.05, 0.1, 0.2 mg/mL) for 72 hrs. (A) Tyrosinase activity (black) and melanin content (grey) were measured. (B) The expression of tyrosinase was determined by immunoblotting analysis (black) and RT-PCR (grey), using β-actin and GAPDH as internal standards, respectively. (C) The cAMP concentration was measured by enzyme immunoassay at assigned time intervals (10, 30, 60 min) after Cpc treatment. Data were expressed at mean ± SD from three different experiments. The asterisk (*) indicates a significant difference from control group (*, P

    Techniques Used: Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Effect of Cpc on cAMP/MAPK/ERK pathway and MITF expression at protein and mRNA levels . Immunoblot analysis was performed with cell extract proteins treated with (A) Cpc (0.1 mg/mL) at assigned time intervals for ERK1/2 (control (black); CPC-treated (grey)), and (B) different Cpc concentration (0.05, 0.1, 0.2 mg/mL) at 540 min for MEK. (C) Cell extract proteins at assigned time intervals treated with Cpc (0.1 mg/mL) were examined by Immunoblot analysis for MITF using β-actin as internal standards (control (black); CPC-treated (grey)). (D) Different levels of Cpc (0.05, 0.1, 0.2 mg/mL) treated MITF mRNA were analyzed by Q-PCR at 540 min. (E) Immunoblot analysis treated with Cpc (0.1 mg/mL), PD98059 (PD, 20 μM), and Cpc+PD at 72 hrs were performed for the evaluation of MITF and tyrosinase expression (MITF (black); tyrosinase (grey)). Data were expressed at mean ± SD from three different experiments. The asterisk (*) indicates a significant difference from control group (*, P
    Figure Legend Snippet: Effect of Cpc on cAMP/MAPK/ERK pathway and MITF expression at protein and mRNA levels . Immunoblot analysis was performed with cell extract proteins treated with (A) Cpc (0.1 mg/mL) at assigned time intervals for ERK1/2 (control (black); CPC-treated (grey)), and (B) different Cpc concentration (0.05, 0.1, 0.2 mg/mL) at 540 min for MEK. (C) Cell extract proteins at assigned time intervals treated with Cpc (0.1 mg/mL) were examined by Immunoblot analysis for MITF using β-actin as internal standards (control (black); CPC-treated (grey)). (D) Different levels of Cpc (0.05, 0.1, 0.2 mg/mL) treated MITF mRNA were analyzed by Q-PCR at 540 min. (E) Immunoblot analysis treated with Cpc (0.1 mg/mL), PD98059 (PD, 20 μM), and Cpc+PD at 72 hrs were performed for the evaluation of MITF and tyrosinase expression (MITF (black); tyrosinase (grey)). Data were expressed at mean ± SD from three different experiments. The asterisk (*) indicates a significant difference from control group (*, P

    Techniques Used: Expressing, Concentration Assay, Polymerase Chain Reaction

    Effect of Cpc on viability of B16F10 melanoma cell, tyrosinase activity and melanin contents . Cells were treated with Cpc (0.05, 0.1, 0.2 mg/mL) for 72 hrs. (A) Cell viability was determined by MTT assay as described in Materials and Methods. (B) Tyrosinase activity (black) and melanin content (grey) were measured. (C) The expression of tyrosinase was determined by immunoblotting analysis (black) and RT-PCR (grey), using β-actin and GAPDH as internal standards, respectively. Data were expressed at mean ± SD from three different experiments. The asterisk (*) indicates a significant difference from control group (*, P
    Figure Legend Snippet: Effect of Cpc on viability of B16F10 melanoma cell, tyrosinase activity and melanin contents . Cells were treated with Cpc (0.05, 0.1, 0.2 mg/mL) for 72 hrs. (A) Cell viability was determined by MTT assay as described in Materials and Methods. (B) Tyrosinase activity (black) and melanin content (grey) were measured. (C) The expression of tyrosinase was determined by immunoblotting analysis (black) and RT-PCR (grey), using β-actin and GAPDH as internal standards, respectively. Data were expressed at mean ± SD from three different experiments. The asterisk (*) indicates a significant difference from control group (*, P

    Techniques Used: Activity Assay, MTT Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

    21) Product Images from "Divergent Roles for Airway Epithelial MMP7 and Retinoic Acid in Experimental Asthma"

    Article Title: Divergent Roles for Airway Epithelial MMP7 and Retinoic Acid in Experimental Asthma

    Journal: Nature immunology

    doi: 10.1038/ni.1719

    Attenuated T H 2 cytokines and chemokines in Mmp7 −/− mice Mmp7 −/− mice and age and sex matched wild-type (n=5 per group) mice immunized as described in Fig. 2 and 24 h after the last immunization, concentrations of (a) IL-4, (b) IL-5 and (c) IL-13 were measured in BAL fluid using Luminex. (d) Level of CCL11 in BAL fluid was measured by ELISA. (e) Expression of Il-25 mRNA in the lung of WT and Mmp7 −/− CAA immunized mice was measured by real time RT-PCR. Data was normalized to actin. (Data represent mean values ± SD; representative of 3 independent experiments). * P
    Figure Legend Snippet: Attenuated T H 2 cytokines and chemokines in Mmp7 −/− mice Mmp7 −/− mice and age and sex matched wild-type (n=5 per group) mice immunized as described in Fig. 2 and 24 h after the last immunization, concentrations of (a) IL-4, (b) IL-5 and (c) IL-13 were measured in BAL fluid using Luminex. (d) Level of CCL11 in BAL fluid was measured by ELISA. (e) Expression of Il-25 mRNA in the lung of WT and Mmp7 −/− CAA immunized mice was measured by real time RT-PCR. Data was normalized to actin. (Data represent mean values ± SD; representative of 3 independent experiments). * P

    Techniques Used: Mouse Assay, Luminex, Enzyme-linked Immunosorbent Assay, Expressing, Cellular Antioxidant Activity Assay, Quantitative RT-PCR

    22) Product Images from "Progesterone receptor membrane associated component 1 enhances obesity progression in mice by facilitating lipid accumulation in adipocytes"

    Article Title: Progesterone receptor membrane associated component 1 enhances obesity progression in mice by facilitating lipid accumulation in adipocytes

    Journal: Communications Biology

    doi: 10.1038/s42003-020-01202-x

    Progesterone receptor membrane-associated component 1 (PGRMC1) contributes to the progression of adipocyte hypertrophy in mice. a Tissue weights of perirenal white adipose tissue (WAT), subcutaneous WAT, brown adipose tissue (BAT), and liver in C57BL/6J mice (WT or PGRMC1 adipose tissue-specific knockout (AKO)) fed normal diet (ND) or high-fat diet (HFD) for 16 weeks. ( n = 5–8). b Paraffin sections of mesenteric WAT and subcutaneous WAT from WT and PGRMC1-AKO mice fed ND or HFD stained with hematoxylin and eosin (scale bar: 500 μm). The graph depicts the average adipocyte area (at least 100 cells per mouse) ( n = 5). c The paraffin sections of BAT in WT and PGRMC1-AKO mice fed ND and HFD stained with hematoxylin and eosin (scale bar: 500 μm). d Analyses of mRNA expressions in mesenteric WAT of WT and PGRMC1-AKO mice fed ND or HFD by quantitative PCR (qPCR) ( n = 5–8). The graph shows relative fold change after normalizing with the mRNA level of GAPDH . e The analyses of protein expressions in mesenteric WAT of WT and PGRMC1-AKO mice fed ND or HFD by western blotting using the antibody against PGRMC1, CEBPβ, PPARγ, FABP4, or GAPDH. Data are represented as mean ± S.E. Statistical analysis was performed using ANOVA with Tukey’s T test. * P
    Figure Legend Snippet: Progesterone receptor membrane-associated component 1 (PGRMC1) contributes to the progression of adipocyte hypertrophy in mice. a Tissue weights of perirenal white adipose tissue (WAT), subcutaneous WAT, brown adipose tissue (BAT), and liver in C57BL/6J mice (WT or PGRMC1 adipose tissue-specific knockout (AKO)) fed normal diet (ND) or high-fat diet (HFD) for 16 weeks. ( n = 5–8). b Paraffin sections of mesenteric WAT and subcutaneous WAT from WT and PGRMC1-AKO mice fed ND or HFD stained with hematoxylin and eosin (scale bar: 500 μm). The graph depicts the average adipocyte area (at least 100 cells per mouse) ( n = 5). c The paraffin sections of BAT in WT and PGRMC1-AKO mice fed ND and HFD stained with hematoxylin and eosin (scale bar: 500 μm). d Analyses of mRNA expressions in mesenteric WAT of WT and PGRMC1-AKO mice fed ND or HFD by quantitative PCR (qPCR) ( n = 5–8). The graph shows relative fold change after normalizing with the mRNA level of GAPDH . e The analyses of protein expressions in mesenteric WAT of WT and PGRMC1-AKO mice fed ND or HFD by western blotting using the antibody against PGRMC1, CEBPβ, PPARγ, FABP4, or GAPDH. Data are represented as mean ± S.E. Statistical analysis was performed using ANOVA with Tukey’s T test. * P

    Techniques Used: Mouse Assay, Knock-Out, Staining, Real-time Polymerase Chain Reaction, Western Blot

    Progesterone receptor membrane-associated component 1 (PGRMC1) is required for lipid accumulation during 3T3L1 cells differentiation. a Analyses of protein expressions in differentiated 3T3L1 cells (control, KD#1, or KD#2) by western blotting using antibodies against PGRMC1 or GAPDH. b Oil Red O staining of differentiated 3T3L1. Control or two types of stable PGRMC1-KD 3T3L1 cells (PGRMC1 KD#1 and KD#2) were differentiated and stained with Oil Red O. The microscope images are shown in the upper panels of ( b ). Graphs in the lower panel of ( b ) depict the absorbance at 490 nm by Oil Red O ( n = 8). c Analyses of mRNA expression of PGRMC1, CEBPβ, PPARγ , or FABP4 in 3T3L1 cells at the indicated time periods during differentiation (control, PGRMC1 KD) by quantitative PCR (qPCR) ( n = 10). Analyses of protein expressions in undifferentiated or differentiated 3T3L1 cells (control, KD) by western blotting using antibodies against PGRMC1, CEBPβ, PPARγ, FABP4, or GAPDH. d Analyses of protein expressions in 3T3L1 control cells during differentiation by western blotting using antibodies against PGRMC1, PPARγ, FABP4, or GAPDH. e Analyses of protein expressions in undifferentiated or differentiated 3T3L1 cells (Control, KD) by western blotting using antibodies against PGRMC1, CEBPβ, PPARγ, FABP4, or GAPDH. Data represent mean ± S.E. Statistical analyses were performed using ANOVA with Tukey’s T test. * P
    Figure Legend Snippet: Progesterone receptor membrane-associated component 1 (PGRMC1) is required for lipid accumulation during 3T3L1 cells differentiation. a Analyses of protein expressions in differentiated 3T3L1 cells (control, KD#1, or KD#2) by western blotting using antibodies against PGRMC1 or GAPDH. b Oil Red O staining of differentiated 3T3L1. Control or two types of stable PGRMC1-KD 3T3L1 cells (PGRMC1 KD#1 and KD#2) were differentiated and stained with Oil Red O. The microscope images are shown in the upper panels of ( b ). Graphs in the lower panel of ( b ) depict the absorbance at 490 nm by Oil Red O ( n = 8). c Analyses of mRNA expression of PGRMC1, CEBPβ, PPARγ , or FABP4 in 3T3L1 cells at the indicated time periods during differentiation (control, PGRMC1 KD) by quantitative PCR (qPCR) ( n = 10). Analyses of protein expressions in undifferentiated or differentiated 3T3L1 cells (control, KD) by western blotting using antibodies against PGRMC1, CEBPβ, PPARγ, FABP4, or GAPDH. d Analyses of protein expressions in 3T3L1 control cells during differentiation by western blotting using antibodies against PGRMC1, PPARγ, FABP4, or GAPDH. e Analyses of protein expressions in undifferentiated or differentiated 3T3L1 cells (Control, KD) by western blotting using antibodies against PGRMC1, CEBPβ, PPARγ, FABP4, or GAPDH. Data represent mean ± S.E. Statistical analyses were performed using ANOVA with Tukey’s T test. * P

    Techniques Used: Western Blot, Staining, Microscopy, Expressing, Real-time Polymerase Chain Reaction

    The progesterone receptor membrane-associated component 1 (PGRMC1) expression is enhanced during 3T3L1 cell differentiation. a Analyses of mRNA expression in 3T3L1 cells treated with 15 μmol l −1 TZD for 2 days by quantitative PCR (qPCR) ( n = 4). The graph shows relative fold change by normalizing with mRNA levels in 3T3L1 cells treated without TZD. b Analyses of protein expressions in 3T3L1 cells treated with 15 μmol l −1 TZD by western blotting using antibodies against PGRMC1, PPARγ, FABP4, or GAPDH. c , d Reporter gene assay of mouse PGRMC1 promoter. The reporter constructs of the PGRMC1 promoter containing PPRE sequences (−1695/+1-PGRMC1-Luc, -345/+1-PGRMC1-Luc), or lacking PPRE site (-327/+1-PGRMC1-Luc) ( c ), or constructs containing a control SV40 promoter, or three repeats of the PPRE or the mutated PPRE (PPRE-mt) upstream of a control SV40 promoter ( d ) were transfected into 3T3L1 cells and were incubated for 2 days after adding 15 μmol l −1 TZD. The graph shows relative luciferase activity by normalizing with luciferase activity in 3T3L1 cells treated without TZD ( n = 4). e , f Analyses of the PGRMC1 expression by treatment with TZD in mice. TZD (5 mg kg −1 ) was intraperitoneally injected in C57BL/6J mice for 3 consecutive days. The mRNA expressions of PGRMC1 , PPARγ , and FABP4 in white adipose tissue (WAT) were analyzed by qPCR ( n = 5). e The protein expressions in perirenal WAT were analyzed by western blotting using antibodies against PGRMC1, PPARγ, FABP4, or GAPDH. f Data represent mean ± S.E. Statistical analysis was performed using Student’s T test ( a , c , d , and e ). * P
    Figure Legend Snippet: The progesterone receptor membrane-associated component 1 (PGRMC1) expression is enhanced during 3T3L1 cell differentiation. a Analyses of mRNA expression in 3T3L1 cells treated with 15 μmol l −1 TZD for 2 days by quantitative PCR (qPCR) ( n = 4). The graph shows relative fold change by normalizing with mRNA levels in 3T3L1 cells treated without TZD. b Analyses of protein expressions in 3T3L1 cells treated with 15 μmol l −1 TZD by western blotting using antibodies against PGRMC1, PPARγ, FABP4, or GAPDH. c , d Reporter gene assay of mouse PGRMC1 promoter. The reporter constructs of the PGRMC1 promoter containing PPRE sequences (−1695/+1-PGRMC1-Luc, -345/+1-PGRMC1-Luc), or lacking PPRE site (-327/+1-PGRMC1-Luc) ( c ), or constructs containing a control SV40 promoter, or three repeats of the PPRE or the mutated PPRE (PPRE-mt) upstream of a control SV40 promoter ( d ) were transfected into 3T3L1 cells and were incubated for 2 days after adding 15 μmol l −1 TZD. The graph shows relative luciferase activity by normalizing with luciferase activity in 3T3L1 cells treated without TZD ( n = 4). e , f Analyses of the PGRMC1 expression by treatment with TZD in mice. TZD (5 mg kg −1 ) was intraperitoneally injected in C57BL/6J mice for 3 consecutive days. The mRNA expressions of PGRMC1 , PPARγ , and FABP4 in white adipose tissue (WAT) were analyzed by qPCR ( n = 5). e The protein expressions in perirenal WAT were analyzed by western blotting using antibodies against PGRMC1, PPARγ, FABP4, or GAPDH. f Data represent mean ± S.E. Statistical analysis was performed using Student’s T test ( a , c , d , and e ). * P

    Techniques Used: Expressing, Cell Differentiation, Real-time Polymerase Chain Reaction, Western Blot, Reporter Gene Assay, Construct, Transfection, Incubation, Luciferase, Activity Assay, Mouse Assay, Injection

    23) Product Images from "Microglial Response to Aspergillus flavus and Candida albicans: Implications in Endophthalmitis"

    Article Title: Microglial Response to Aspergillus flavus and Candida albicans: Implications in Endophthalmitis

    Journal: Journal of Fungi

    doi: 10.3390/jof6030162

    CHME-3 cells were challenged with A. flavus and C. albicans at indicated time points. Postinfection human microglial total RNA was subjected to RT-PCR and qRT-PCR using SYBR green assay for Toll-Like Receptors (TLRs) (1-7 and -9) and β-actin gene was used as internal control and uninfected CHME-3 cells were taken as control. The data were shown as the mean ± Standard error (SE) from three sets of independent experiments and * p
    Figure Legend Snippet: CHME-3 cells were challenged with A. flavus and C. albicans at indicated time points. Postinfection human microglial total RNA was subjected to RT-PCR and qRT-PCR using SYBR green assay for Toll-Like Receptors (TLRs) (1-7 and -9) and β-actin gene was used as internal control and uninfected CHME-3 cells were taken as control. The data were shown as the mean ± Standard error (SE) from three sets of independent experiments and * p

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

    CHME-3 cells were challenged with A. flavus and C. albicans at indicated time points. Postinfection human microglial total RNA was subjected to RT-PCR and qRT-PCR using SYBR green assay with cytokine-specific primes. Representative RT-PCRs of each cytokines were shown. Human microglia with no fungal infection cDNA was used as control. β-actin was used as an endogenous control. Data are from n = 3 independent experiments done in triplicate. * p
    Figure Legend Snippet: CHME-3 cells were challenged with A. flavus and C. albicans at indicated time points. Postinfection human microglial total RNA was subjected to RT-PCR and qRT-PCR using SYBR green assay with cytokine-specific primes. Representative RT-PCRs of each cytokines were shown. Human microglia with no fungal infection cDNA was used as control. β-actin was used as an endogenous control. Data are from n = 3 independent experiments done in triplicate. * p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay, Infection

    24) Product Images from "LncRNA NEAT1 Regulates 5-Fu Sensitivity, Apoptosis and Invasion in Colorectal Cancer Through the MiR-150-5p/CPSF4 Axis"

    Article Title: LncRNA NEAT1 Regulates 5-Fu Sensitivity, Apoptosis and Invasion in Colorectal Cancer Through the MiR-150-5p/CPSF4 Axis

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S239432

    Effects of CPSF4 overexpression on CRC cell progression. ( A and B ) The mRNA and protein expression levels of CPSF4 were determined by qRT-PCR and WB analysis to evaluate the transfection efficiency of CPSF4 overexpression plasmid in SW480 and HCT116. SW480 and HCT116 cells were co-transfected with si-lnc-NEAT1 and CPSF4 overexpression plasmid. ( C ) The IC 50 values of SW480 and HCT116 cells were detected by MTT assay. ( D ) The apoptosis rates of SW480 and HCT116 cells were measured by flow cytometry. ( E ) Transwell assay was employed to assess the number of invaded SW480 and HCT116 cells. ( F and G ) The proteins levels of P-gp and GST-π in SW480 and HCT116 cells were determined by WB analysis. ( A and B ) One-way ANOVA; ( C – G ) two-way ANOVA.* P
    Figure Legend Snippet: Effects of CPSF4 overexpression on CRC cell progression. ( A and B ) The mRNA and protein expression levels of CPSF4 were determined by qRT-PCR and WB analysis to evaluate the transfection efficiency of CPSF4 overexpression plasmid in SW480 and HCT116. SW480 and HCT116 cells were co-transfected with si-lnc-NEAT1 and CPSF4 overexpression plasmid. ( C ) The IC 50 values of SW480 and HCT116 cells were detected by MTT assay. ( D ) The apoptosis rates of SW480 and HCT116 cells were measured by flow cytometry. ( E ) Transwell assay was employed to assess the number of invaded SW480 and HCT116 cells. ( F and G ) The proteins levels of P-gp and GST-π in SW480 and HCT116 cells were determined by WB analysis. ( A and B ) One-way ANOVA; ( C – G ) two-way ANOVA.* P

    Techniques Used: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, MTT Assay, Flow Cytometry, Transwell Assay

    The expression levels of lnc-NEAT1 and miR-150-5p in tissues and cells. ( A ) The expression of lnc-NEAT1 in CRC tissues (CRC) and adjacent normal tissues (Normal) was measured by qRT-PCR. ( B ) Lnc-NEAT1 expression in CRC cell lines (SW480 and HCT116) and NCM460 cells was detected by qRT-PCR. ( C ) QRT-PCR was used to assess the expression of miR-150-5p in CRC and Normal. ( D ) The expression of miR-150-5p in SW480, HCT116 and NCM460 cells was tested by qRT-PCR. ( E ) The correlation analysis between lnc-NEAT1 and miR-150-5p expression was determined by Pearson correlation analysis. ( A and C ) Student’s t -test; ( B and D ) one-way ANOVA. * P
    Figure Legend Snippet: The expression levels of lnc-NEAT1 and miR-150-5p in tissues and cells. ( A ) The expression of lnc-NEAT1 in CRC tissues (CRC) and adjacent normal tissues (Normal) was measured by qRT-PCR. ( B ) Lnc-NEAT1 expression in CRC cell lines (SW480 and HCT116) and NCM460 cells was detected by qRT-PCR. ( C ) QRT-PCR was used to assess the expression of miR-150-5p in CRC and Normal. ( D ) The expression of miR-150-5p in SW480, HCT116 and NCM460 cells was tested by qRT-PCR. ( E ) The correlation analysis between lnc-NEAT1 and miR-150-5p expression was determined by Pearson correlation analysis. ( A and C ) Student’s t -test; ( B and D ) one-way ANOVA. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    MiR-150-5p could directly target CPSF4. ( A ) CPSF4 3ʹUTR containing the miR-150-5p binding sites and mutant binding sites were shown. ( B ) Dual-luciferase reporter assay was performed to detect the luciferase activity of CPSF4 3ʹUTR-WT/MUT in SW480 and HCT116 cells. ( C and D ) The mRNA and protein expression levels of CPSF4 in CRC tissues (CRC) and adjacent normal tissues (Normal) were measured by qRT-PCR and WB analysis. ( E ) The correlation analysis between lnc-NEAT1 and CPSF4 expression was assessed by Pearson correlation analysis. ( F and G ) QRT-PCR and WB analysis were used to determine the mRNA and protein expression levels of CPSF4 in NCM460 and CRC cell lines (SW480 and HCT116). ( H ) The expression of miR-150-5p was determined by qRT-PCR to evaluate the transfection efficiency of miR-150-5p mimics in SW480 and HCT116 cells. SW480 and HCT116 cells were transfected with anti-miR-150-5p or miR-150-5p mimics. ( I and J ) The protein level of CPSF4 was evaluated by WB analysis in SW480 and HCT116 cells. ( B and F – H ) One-way ANOVA; ( C and D ) Student’s t -test; ( I and J ) two-way ANOVA. * P
    Figure Legend Snippet: MiR-150-5p could directly target CPSF4. ( A ) CPSF4 3ʹUTR containing the miR-150-5p binding sites and mutant binding sites were shown. ( B ) Dual-luciferase reporter assay was performed to detect the luciferase activity of CPSF4 3ʹUTR-WT/MUT in SW480 and HCT116 cells. ( C and D ) The mRNA and protein expression levels of CPSF4 in CRC tissues (CRC) and adjacent normal tissues (Normal) were measured by qRT-PCR and WB analysis. ( E ) The correlation analysis between lnc-NEAT1 and CPSF4 expression was assessed by Pearson correlation analysis. ( F and G ) QRT-PCR and WB analysis were used to determine the mRNA and protein expression levels of CPSF4 in NCM460 and CRC cell lines (SW480 and HCT116). ( H ) The expression of miR-150-5p was determined by qRT-PCR to evaluate the transfection efficiency of miR-150-5p mimics in SW480 and HCT116 cells. SW480 and HCT116 cells were transfected with anti-miR-150-5p or miR-150-5p mimics. ( I and J ) The protein level of CPSF4 was evaluated by WB analysis in SW480 and HCT116 cells. ( B and F – H ) One-way ANOVA; ( C and D ) Student’s t -test; ( I and J ) two-way ANOVA. * P

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

    Lnc-NEAT1 could sponge miR-150-5p. ( A ) The binding sites and mutant binding sites between lnc-NEAT1 and miR-150-5p were shown. ( B ) Dual-luciferase reporter assay was used to detect the luciferase activity of lnc-NEAT1-WT/MUT in SW480 and HCT116 cells. ( C ) The expression of lnc-NEAT1 was determined by qRT-PCR to evaluate the transfection efficiency of lnc-NEAT1 overexpression plasmid in SW480 and HCT116 cells. ( D ) The expression of miR-150-5p was measured by qRT-PCR to assess the effect of lnc-NEAT1 expression on miR-150-5p expression. ( E ) QRT-PCR was used to test the expression of miR-150-5p to evaluate the transfection efficiency of anti-miR-150-5p in SW480 and HCT116 cells. SW480 and HCT116 cells were co-transfected with si-lnc-NEAT1 and anti-miR-150-5p. ( F ) The IC 50 values of SW480 and HCT116 cells were measured by MTT assay. ( G ) The apoptosis rates of SW480 and HCT116 cells were determined by flow cytometry. ( H ) Transwell assay was used to detect the number of invaded SW480 and HCT116 cells. ( I and J ) WB analysis was used to test the protein levels of P-gp and GST-π in SW480 and HCT116 cells. ( B, C and E ) one-way ANOVA; ( D and F – G ) two-way ANOVA.* P
    Figure Legend Snippet: Lnc-NEAT1 could sponge miR-150-5p. ( A ) The binding sites and mutant binding sites between lnc-NEAT1 and miR-150-5p were shown. ( B ) Dual-luciferase reporter assay was used to detect the luciferase activity of lnc-NEAT1-WT/MUT in SW480 and HCT116 cells. ( C ) The expression of lnc-NEAT1 was determined by qRT-PCR to evaluate the transfection efficiency of lnc-NEAT1 overexpression plasmid in SW480 and HCT116 cells. ( D ) The expression of miR-150-5p was measured by qRT-PCR to assess the effect of lnc-NEAT1 expression on miR-150-5p expression. ( E ) QRT-PCR was used to test the expression of miR-150-5p to evaluate the transfection efficiency of anti-miR-150-5p in SW480 and HCT116 cells. SW480 and HCT116 cells were co-transfected with si-lnc-NEAT1 and anti-miR-150-5p. ( F ) The IC 50 values of SW480 and HCT116 cells were measured by MTT assay. ( G ) The apoptosis rates of SW480 and HCT116 cells were determined by flow cytometry. ( H ) Transwell assay was used to detect the number of invaded SW480 and HCT116 cells. ( I and J ) WB analysis was used to test the protein levels of P-gp and GST-π in SW480 and HCT116 cells. ( B, C and E ) one-way ANOVA; ( D and F – G ) two-way ANOVA.* P

    Techniques Used: Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Expressing, Quantitative RT-PCR, Transfection, Over Expression, Plasmid Preparation, MTT Assay, Flow Cytometry, Transwell Assay, Western Blot

    Effects of silenced-lnc-NEAT1 on CRC tumor growth in vivo. ( A ) Tumor volume was calculated with length × width 2 /2 method at the indicated time point. ( B ) Tumor weight was measured in mice xenograft models. ( C and D ) The expression levels of lnc-NEAT1 and miR-150-5p in the tumors were detected by qRT-PCR. ( E ) WB analysis was used to determine the protein level of CPSF4 in the tumors. Two-way ANOVA. * P
    Figure Legend Snippet: Effects of silenced-lnc-NEAT1 on CRC tumor growth in vivo. ( A ) Tumor volume was calculated with length × width 2 /2 method at the indicated time point. ( B ) Tumor weight was measured in mice xenograft models. ( C and D ) The expression levels of lnc-NEAT1 and miR-150-5p in the tumors were detected by qRT-PCR. ( E ) WB analysis was used to determine the protein level of CPSF4 in the tumors. Two-way ANOVA. * P

    Techniques Used: In Vivo, Mouse Assay, Expressing, Quantitative RT-PCR, Western Blot

    Effects of lnc-NEAT1 knockdown on CRC cell progression. SW480 and HCT116 cells were transfected with si-lnc-NEAT1 or si-NC. ( A ) The expression of lnc-NEAT1 was determined by qRT-PCR to evaluate transfection efficiency. ( B and C ) The IC 50 values of SW480 and HCT116 cells were assessed by MTT assay. ( D ) Flow cytometry was used to measure the apoptosis rates of SW480 and HCT116 cells. ( E ) Transwell assay was performed to test the number of invaded SW480 and HCT116 cells. ( F ) The proteins levels of P-gp and GST-π in SW480 and HCT116 cells were detected by WB analysis. One-way ANOVA. * P
    Figure Legend Snippet: Effects of lnc-NEAT1 knockdown on CRC cell progression. SW480 and HCT116 cells were transfected with si-lnc-NEAT1 or si-NC. ( A ) The expression of lnc-NEAT1 was determined by qRT-PCR to evaluate transfection efficiency. ( B and C ) The IC 50 values of SW480 and HCT116 cells were assessed by MTT assay. ( D ) Flow cytometry was used to measure the apoptosis rates of SW480 and HCT116 cells. ( E ) Transwell assay was performed to test the number of invaded SW480 and HCT116 cells. ( F ) The proteins levels of P-gp and GST-π in SW480 and HCT116 cells were detected by WB analysis. One-way ANOVA. * P

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

    25) Product Images from "IL10-modified Human Mesenchymal Stem Cells inhibit Pancreatic Cancer growth through Angiogenesis Inhibition"

    Article Title: IL10-modified Human Mesenchymal Stem Cells inhibit Pancreatic Cancer growth through Angiogenesis Inhibition

    Journal: Journal of Cancer

    doi: 10.7150/jca.38062

    Stable expression of IL10 gene in BMSC following transfection with IL10-bering plasmid and inhibitory effect of MSC-IL10 on inflammatory cytokines in MSC. MSC modified with IL10 gene photographed under light microscope (×200). MSC modified with IL10 gene photographed under fluorescent microscope (×200). RT-PCR analysis for IL10 from MSC-IL10 as well as MSC and MSC-vector transfected groups. Western blot analysis for IL10 from MSC-IL10 as well as MSC and MSC-vector transfected groups. A significant decrease in IL6 was observed in the supernatant of MSC-IL10 group in comparison to the other groups (***P
    Figure Legend Snippet: Stable expression of IL10 gene in BMSC following transfection with IL10-bering plasmid and inhibitory effect of MSC-IL10 on inflammatory cytokines in MSC. MSC modified with IL10 gene photographed under light microscope (×200). MSC modified with IL10 gene photographed under fluorescent microscope (×200). RT-PCR analysis for IL10 from MSC-IL10 as well as MSC and MSC-vector transfected groups. Western blot analysis for IL10 from MSC-IL10 as well as MSC and MSC-vector transfected groups. A significant decrease in IL6 was observed in the supernatant of MSC-IL10 group in comparison to the other groups (***P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Modification, Light Microscopy, Microscopy, Reverse Transcription Polymerase Chain Reaction, Western Blot

    26) Product Images from "Long non‐coding RNA FTH1P3 activates paclitaxel resistance in breast cancer through miR‐206/ ABCB1. Long non‐coding RNA FTH1P3 activates paclitaxel resistance in breast cancer through miR‐206/ABCB1"

    Article Title: Long non‐coding RNA FTH1P3 activates paclitaxel resistance in breast cancer through miR‐206/ ABCB1. Long non‐coding RNA FTH1P3 activates paclitaxel resistance in breast cancer through miR‐206/ABCB1

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13679

    Lnc RNA FTH 1P3 silencing suppressed the tumour growth of paclitaxel‐resistant breast cancer cells and ABCB 1 protein in vivo. A, Images of tumour neoplasm in xenograft mice samples injected with MCF ‐7/ PTX transfected with sh‐ FTH 1P3 or empty vector. B, Tumour volume of neoplasm measured every 3 days after subcutaneous injection. C, Tumour weight of neoplasm excised from mice after they were killed. D, Images of Western blot band of ABCB 1 protein. E, Quantitative value of ABCB 1 protein expression. F, RT ‐ PCR showed the expression of miR‐206 in FTH 1P3 knockdown group compared to empty vector group. Data were expressed as mean ± SD . * P
    Figure Legend Snippet: Lnc RNA FTH 1P3 silencing suppressed the tumour growth of paclitaxel‐resistant breast cancer cells and ABCB 1 protein in vivo. A, Images of tumour neoplasm in xenograft mice samples injected with MCF ‐7/ PTX transfected with sh‐ FTH 1P3 or empty vector. B, Tumour volume of neoplasm measured every 3 days after subcutaneous injection. C, Tumour weight of neoplasm excised from mice after they were killed. D, Images of Western blot band of ABCB 1 protein. E, Quantitative value of ABCB 1 protein expression. F, RT ‐ PCR showed the expression of miR‐206 in FTH 1P3 knockdown group compared to empty vector group. Data were expressed as mean ± SD . * P

    Techniques Used: In Vivo, Mouse Assay, Injection, Transfection, Plasmid Preparation, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

    Lnc RNA FTH 1P3 activated the paclitaxel sensitivity of breast cancer cells and induced the G2/M phase arrest. A, Synthesized small interfering RNA (si RNA ) targeting FTH 1P3 was transfected into MCF ‐7/ PTX and MDA ‐ MB ‐231/ PTX cells. FTH 1P3 expression was measured using RT ‐ PCR . B, RT ‐ PCR showed the FTH 1P3 expression in MCF ‐7/ PTX and MDA ‐ MB ‐231/ PTX cells transfected with plasmids containing FTH 1P3 sequence. C, The 50% inhibitory concentration ( IC 50) value of paclitaxel in MCF ‐7/ PTX cells transfected with si‐ FTH 1P3 (1.87 μmol/L) or si‐ NC (4.21 μmol/L). D, The IC 50 value of paclitaxel in MDA ‐ MB ‐231/ PTX cells transfected with si‐ FTH 1P3 (1.23 μmol/L) or si‐ NC (3.07 μmol/L). E, The IC 50 value of paclitaxel in MCF ‐7/ PTX cells transfected with pc DNA ‐ FTH 1P3 (12.83 μmol/L) or pc DNA ‐ NC (4.39 μmol/L). F, The IC 50 value of paclitaxel in MDA ‐ MB ‐231/ PTX cells transfected with pc DNA ‐ FTH 1P3 (9.86 μmol/L) or pc DNA ‐ NC (3.23 μmol/L). G, Images of flow cytometry assay. H, The cell distribution in MCF ‐7/ PTX and MDA ‐ MB ‐231/ PTX cells. Data were expressed as mean ± SD . * P
    Figure Legend Snippet: Lnc RNA FTH 1P3 activated the paclitaxel sensitivity of breast cancer cells and induced the G2/M phase arrest. A, Synthesized small interfering RNA (si RNA ) targeting FTH 1P3 was transfected into MCF ‐7/ PTX and MDA ‐ MB ‐231/ PTX cells. FTH 1P3 expression was measured using RT ‐ PCR . B, RT ‐ PCR showed the FTH 1P3 expression in MCF ‐7/ PTX and MDA ‐ MB ‐231/ PTX cells transfected with plasmids containing FTH 1P3 sequence. C, The 50% inhibitory concentration ( IC 50) value of paclitaxel in MCF ‐7/ PTX cells transfected with si‐ FTH 1P3 (1.87 μmol/L) or si‐ NC (4.21 μmol/L). D, The IC 50 value of paclitaxel in MDA ‐ MB ‐231/ PTX cells transfected with si‐ FTH 1P3 (1.23 μmol/L) or si‐ NC (3.07 μmol/L). E, The IC 50 value of paclitaxel in MCF ‐7/ PTX cells transfected with pc DNA ‐ FTH 1P3 (12.83 μmol/L) or pc DNA ‐ NC (4.39 μmol/L). F, The IC 50 value of paclitaxel in MDA ‐ MB ‐231/ PTX cells transfected with pc DNA ‐ FTH 1P3 (9.86 μmol/L) or pc DNA ‐ NC (3.23 μmol/L). G, Images of flow cytometry assay. H, The cell distribution in MCF ‐7/ PTX and MDA ‐ MB ‐231/ PTX cells. Data were expressed as mean ± SD . * P

    Techniques Used: Synthesized, Small Interfering RNA, Transfection, Multiple Displacement Amplification, Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Concentration Assay, Flow Cytometry, Cytometry

    Lnc RNA FTH 1P3 was up‐regulated in paclitaxel‐resistant breast cancer tissue and cells. A, RT ‐ PCR showed the lnc RNA FTH 1P3 expression level in 30 cases of breast cancer samples, including 15 cases of paclitaxel‐sensitive samples and 15 cases of paclitaxel‐resistant samples. B, RT ‐ PCR showed the lnc RNA FTH 1P3 expression levels in paclitaxel‐resistant breast cancer cell lines ( MCF ‐7/ PTX , MDA ‐ MB ‐231/ PTX ) and parental cell lines ( MCF ‐7, MDA ‐ MB ‐231). C, FTH 1P3 expression in MCF ‐7 cells treated with different doses of paclitaxel. D, FTH 1P3 expression in MDA ‐ MB ‐231 cells treated with different doses of paclitaxel. Data were expressed as mean ± SD . * P
    Figure Legend Snippet: Lnc RNA FTH 1P3 was up‐regulated in paclitaxel‐resistant breast cancer tissue and cells. A, RT ‐ PCR showed the lnc RNA FTH 1P3 expression level in 30 cases of breast cancer samples, including 15 cases of paclitaxel‐sensitive samples and 15 cases of paclitaxel‐resistant samples. B, RT ‐ PCR showed the lnc RNA FTH 1P3 expression levels in paclitaxel‐resistant breast cancer cell lines ( MCF ‐7/ PTX , MDA ‐ MB ‐231/ PTX ) and parental cell lines ( MCF ‐7, MDA ‐ MB ‐231). C, FTH 1P3 expression in MCF ‐7 cells treated with different doses of paclitaxel. D, FTH 1P3 expression in MDA ‐ MB ‐231 cells treated with different doses of paclitaxel. Data were expressed as mean ± SD . * P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Multiple Displacement Amplification

    27) Product Images from "miR-17-5p Regulates Endocytic Trafficking through Targeting TBC1D2/Armus"

    Article Title: miR-17-5p Regulates Endocytic Trafficking through Targeting TBC1D2/Armus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052555

    TBC1D2 and LDLR are directly targeted by miR-17. ( A ) miR-17 targets 3′UTRs of TBC1D2 and LDLR as determined by a luciferase reporter assay. HeLa cells were co-transfected with the reporters containing wild-type 3′UTRs of LDLR and TBC1D2 and mutated 3′UTR of TBC1D2, Pre-miR-17 and Anti-miR-17. Luciferase activity was measured 24 h following co-transfection. The activity of luciferase for each experiment was normalized to the activity of the control samples, co-transfected with the respective reporter vector and control Anti-miR or Pre-miR (see Methods ). The bars show mean fold changes of luciferase activity and the error bars show s.e.m. derived from 3 independent experiments. Over-expression of miR-17 decreased the expression level of LDLR ( B ) and TBC1D2 ( C ) mRNAs as determined by qRT-PCR. The relative expression levels of TBC1D2 and LDLR after treatment with siRNAs and Pre-miR-17 were normalized to the expression level when control siRNAs and control Pre-miR were transfected (see Methods ). Bars show mean of the relative mRNA expression levels and error bars show s.e.m. derived from 2 independent experiments. *, p≤0,05; **, p≤0,01; ***, p≤0,001.
    Figure Legend Snippet: TBC1D2 and LDLR are directly targeted by miR-17. ( A ) miR-17 targets 3′UTRs of TBC1D2 and LDLR as determined by a luciferase reporter assay. HeLa cells were co-transfected with the reporters containing wild-type 3′UTRs of LDLR and TBC1D2 and mutated 3′UTR of TBC1D2, Pre-miR-17 and Anti-miR-17. Luciferase activity was measured 24 h following co-transfection. The activity of luciferase for each experiment was normalized to the activity of the control samples, co-transfected with the respective reporter vector and control Anti-miR or Pre-miR (see Methods ). The bars show mean fold changes of luciferase activity and the error bars show s.e.m. derived from 3 independent experiments. Over-expression of miR-17 decreased the expression level of LDLR ( B ) and TBC1D2 ( C ) mRNAs as determined by qRT-PCR. The relative expression levels of TBC1D2 and LDLR after treatment with siRNAs and Pre-miR-17 were normalized to the expression level when control siRNAs and control Pre-miR were transfected (see Methods ). Bars show mean of the relative mRNA expression levels and error bars show s.e.m. derived from 2 independent experiments. *, p≤0,05; **, p≤0,01; ***, p≤0,001.

    Techniques Used: Luciferase, Reporter Assay, Transfection, Activity Assay, Cotransfection, Plasmid Preparation, Derivative Assay, Over Expression, Expressing, Quantitative RT-PCR

    28) Product Images from "Pulmonary Delivery of Nanoparticle-Bound Toll-like Receptor 9 Agonist for the Treatment of Metastatic Lung Cancer"

    Article Title: Pulmonary Delivery of Nanoparticle-Bound Toll-like Receptor 9 Agonist for the Treatment of Metastatic Lung Cancer

    Journal: ACS nano

    doi: 10.1021/acsnano.0c02207

    Evaluation of lung cytokines. qRT-pCR for mRNA relative expression of IL12p40, IFN γ , TNF α , Fpr2, CXCL10, and Fpr2 by mouse right lungs homogenized on day 7, 24 h after receiving either no treatment or 2 treatments of soluble CpG or PRINT-CpG. Data are normalized to Gapdh mRNA and graphed as fold change over non-tumor-bearing lungs. Statistics performed by 1-way ANOVA with Tukey’s multiple comparisons test, * p
    Figure Legend Snippet: Evaluation of lung cytokines. qRT-pCR for mRNA relative expression of IL12p40, IFN γ , TNF α , Fpr2, CXCL10, and Fpr2 by mouse right lungs homogenized on day 7, 24 h after receiving either no treatment or 2 treatments of soluble CpG or PRINT-CpG. Data are normalized to Gapdh mRNA and graphed as fold change over non-tumor-bearing lungs. Statistics performed by 1-way ANOVA with Tukey’s multiple comparisons test, * p

    Techniques Used: Quantitative RT-PCR, Expressing

    Biological function of PRINT-CpG. ELISA evaluation of proinflammatory cytokine production for PRINT-CpG or CpG incubated in ALF or serum for various time points and then incubated with BMDCs (A). qRT-pCR for mRNA relative expression of cytokines in mouse lungs homogenized at various time points after either receiving no treatment, soluble CpG, or PRINT-CpG (B); data are normalized to Gapdh mRNA and graphed as fold change over nontreated lungs. Statistics performed by 2-way ANOVA with Tukey’s multiple comparisons test of dCT values, * p
    Figure Legend Snippet: Biological function of PRINT-CpG. ELISA evaluation of proinflammatory cytokine production for PRINT-CpG or CpG incubated in ALF or serum for various time points and then incubated with BMDCs (A). qRT-pCR for mRNA relative expression of cytokines in mouse lungs homogenized at various time points after either receiving no treatment, soluble CpG, or PRINT-CpG (B); data are normalized to Gapdh mRNA and graphed as fold change over nontreated lungs. Statistics performed by 2-way ANOVA with Tukey’s multiple comparisons test of dCT values, * p

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

    29) Product Images from "Differential Mucin Expression by Respiratory Syncytial Virus and Human Metapneumovirus Infection in Human Epithelial Cells"

    Article Title: Differential Mucin Expression by Respiratory Syncytial Virus and Human Metapneumovirus Infection in Human Epithelial Cells

    Journal: Mediators of Inflammation

    doi: 10.1155/2015/347292

    Secreted mucins expression in A549 by hMPV and RSV infection. Cells were infected with hMPV or RSV at an MOI of 3 for 12, 24, 48, or 72 h. RNA was isolated, and the expression of MUC genes was determined by qRT-PCR. Data shown in the graphs are the mean of 5 independent experiments. Statistical significant differences are indicated. ∗ P
    Figure Legend Snippet: Secreted mucins expression in A549 by hMPV and RSV infection. Cells were infected with hMPV or RSV at an MOI of 3 for 12, 24, 48, or 72 h. RNA was isolated, and the expression of MUC genes was determined by qRT-PCR. Data shown in the graphs are the mean of 5 independent experiments. Statistical significant differences are indicated. ∗ P

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

    Cell-adhered mucins expression in A549 by hMPV and RSV infection. Cells were infected with hMPV or RSV at an MOI of 3 for 12, 24, 48, or 72 h. RNA was isolated, and the expression of MUC genes was determined by qRT-PCR. Data shown in the graphs are the mean of 5 independent experiments. Statistical significant differences are indicated. ∗ P
    Figure Legend Snippet: Cell-adhered mucins expression in A549 by hMPV and RSV infection. Cells were infected with hMPV or RSV at an MOI of 3 for 12, 24, 48, or 72 h. RNA was isolated, and the expression of MUC genes was determined by qRT-PCR. Data shown in the graphs are the mean of 5 independent experiments. Statistical significant differences are indicated. ∗ P

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

    30) Product Images from "Low-Intensity Pulsed Ultrasound Accelerates Tooth Movement via Activation of the BMP-2 Signaling Pathway"

    Article Title: Low-Intensity Pulsed Ultrasound Accelerates Tooth Movement via Activation of the BMP-2 Signaling Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068926

    LIPUS stimulation increased BMP-2 expression mediated by Runx2. (A). hPDL cells were incubated with HGF for 24 h, and BMP-2 mRNA was examined by qRT-PCR. (B and C) hPDL cells were incubated with HGF for 48 h, and BMP-2 protein amounts were detected by Western blotting. The data shows that HGF significantly increased BMP-2 expression. (D). Cells were transfected with Runx2 siRNA for 24 h followed by stimulation with LIPUS for 5 days, and BMP-2 mRNA expression was examined by qRT-PCR. (E and F) hPDL cells were transfected with Runx2 siRNA for 5 days, and BMP-2 protein expression was examined by Western blot. Transfection of cells with Runx2 siRNA reduced LIPUS-increased BMP-2 expression. * P
    Figure Legend Snippet: LIPUS stimulation increased BMP-2 expression mediated by Runx2. (A). hPDL cells were incubated with HGF for 24 h, and BMP-2 mRNA was examined by qRT-PCR. (B and C) hPDL cells were incubated with HGF for 48 h, and BMP-2 protein amounts were detected by Western blotting. The data shows that HGF significantly increased BMP-2 expression. (D). Cells were transfected with Runx2 siRNA for 24 h followed by stimulation with LIPUS for 5 days, and BMP-2 mRNA expression was examined by qRT-PCR. (E and F) hPDL cells were transfected with Runx2 siRNA for 5 days, and BMP-2 protein expression was examined by Western blot. Transfection of cells with Runx2 siRNA reduced LIPUS-increased BMP-2 expression. * P

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

    LIPUS stimulation enhanced the BMP-2 signaling pathway gene expression in vitro and in vivo . (A). hPDL cells were cultured in the presence and absence of daily LIPUS stimulation. BMP-2 mRNA expression was determined using qRT-PCR. (B). BMP-2 protein (ROW 1; 45 kDa) can be detected in the control and LIPUS groups (1: CON; 2: LIPUS). The LIPUS groups showed higher expression of BMP-2 from day 5. (C). Quantification of BMP-2 protein expression in the LIPUS stimulation group was greater than that in the control group on days 5, 7, and 14. *indicates P
    Figure Legend Snippet: LIPUS stimulation enhanced the BMP-2 signaling pathway gene expression in vitro and in vivo . (A). hPDL cells were cultured in the presence and absence of daily LIPUS stimulation. BMP-2 mRNA expression was determined using qRT-PCR. (B). BMP-2 protein (ROW 1; 45 kDa) can be detected in the control and LIPUS groups (1: CON; 2: LIPUS). The LIPUS groups showed higher expression of BMP-2 from day 5. (C). Quantification of BMP-2 protein expression in the LIPUS stimulation group was greater than that in the control group on days 5, 7, and 14. *indicates P

    Techniques Used: Expressing, In Vitro, In Vivo, Cell Culture, Quantitative RT-PCR

    31) Product Images from "Transcriptome Analysis of Ullrich Congenital Muscular Dystrophy Fibroblasts Reveals a Disease Extracellular Matrix Signature and Key Molecular Regulators"

    Article Title: Transcriptome Analysis of Ullrich Congenital Muscular Dystrophy Fibroblasts Reveals a Disease Extracellular Matrix Signature and Key Molecular Regulators

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145107

    miRNAs expression analysis. Real-time PCR was used to measure relative expression of miR-181a and miR-30c in skeletal muscle (A) and fibroblasts (B) from UCMD patients and in serum samples from UCMD, BM and DMD patients for miR-181a (C) and miR-30c (D). miRNA expression level was normalized against U6 miRNA. Results were calculated relative to control samples and are represented as mean and standard error.
    Figure Legend Snippet: miRNAs expression analysis. Real-time PCR was used to measure relative expression of miR-181a and miR-30c in skeletal muscle (A) and fibroblasts (B) from UCMD patients and in serum samples from UCMD, BM and DMD patients for miR-181a (C) and miR-30c (D). miRNA expression level was normalized against U6 miRNA. Results were calculated relative to control samples and are represented as mean and standard error.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    32) Product Images from "RhMYB108, an R2R3-MYB transcription factor, is involved in ethylene- and JA-induced petal senescence in rose plants"

    Article Title: RhMYB108, an R2R3-MYB transcription factor, is involved in ethylene- and JA-induced petal senescence in rose plants

    Journal: Horticulture Research

    doi: 10.1038/s41438-019-0221-8

    RhMYB108 directly binds to the promoter of RhNAC053, RhNAC092 and RhSAG113 . a qRT-PCR analysis of SAG expression in RhMYB108- silenced petals. b Interaction of RhMYB108 protein with the SAG promoter regions, as revealed using yeast one-hybrid assays. Interactions were determined based on yeast cell growth and were confirmed by the color indication of X-β-gal on SD/-Trp/-Ura medium plates. c , d Images of firefly luciferase fluorescence signals and relative reporter activity (LUC/REN) in N. benthamiana leaves. RhMYB108 protein was separately coexpressed with pNAC053::LUC , pNAC092::LUC and pSAG113::LUC in tobacco leaves. After 48 h of Agrobacterium tumefaciens infiltration, live LUC images and relative LUC activity (LUC/REN) in tobacco leaves were assayed. The error bars represent the means of six biological replicates, and the asterisks indicate statistically significant differences according to Student’s t test (* P
    Figure Legend Snippet: RhMYB108 directly binds to the promoter of RhNAC053, RhNAC092 and RhSAG113 . a qRT-PCR analysis of SAG expression in RhMYB108- silenced petals. b Interaction of RhMYB108 protein with the SAG promoter regions, as revealed using yeast one-hybrid assays. Interactions were determined based on yeast cell growth and were confirmed by the color indication of X-β-gal on SD/-Trp/-Ura medium plates. c , d Images of firefly luciferase fluorescence signals and relative reporter activity (LUC/REN) in N. benthamiana leaves. RhMYB108 protein was separately coexpressed with pNAC053::LUC , pNAC092::LUC and pSAG113::LUC in tobacco leaves. After 48 h of Agrobacterium tumefaciens infiltration, live LUC images and relative LUC activity (LUC/REN) in tobacco leaves were assayed. The error bars represent the means of six biological replicates, and the asterisks indicate statistically significant differences according to Student’s t test (* P

    Techniques Used: Quantitative RT-PCR, Expressing, Luciferase, Fluorescence, Activity Assay

    qRT-PCR analysis of the expression level of RhMYB108 . a Expression profiles of RhMYB108 in the roots, stems, leaves, and petals of rose plants. b , c , d , e , f Expression profiles of RhMYB108 in the petals, receptacles, sepals, stamens, and pistils at various stages of flower opening. g Expression of RhMYB108 in response to exogenous hormones. ETH, 10 μL/L ethylene; JA, 100 μM MeJA; 1-MCP, 2 μL/L. The petals at stage 2 were used as materials in g . RhUBI2 was used as an internal control. Three independent biological repeats were tested, and the data are presented as the means ± SDs. The letters indicate significant differences according to Duncan’s multiple range test ( P
    Figure Legend Snippet: qRT-PCR analysis of the expression level of RhMYB108 . a Expression profiles of RhMYB108 in the roots, stems, leaves, and petals of rose plants. b , c , d , e , f Expression profiles of RhMYB108 in the petals, receptacles, sepals, stamens, and pistils at various stages of flower opening. g Expression of RhMYB108 in response to exogenous hormones. ETH, 10 μL/L ethylene; JA, 100 μM MeJA; 1-MCP, 2 μL/L. The petals at stage 2 were used as materials in g . RhUBI2 was used as an internal control. Three independent biological repeats were tested, and the data are presented as the means ± SDs. The letters indicate significant differences according to Duncan’s multiple range test ( P

    Techniques Used: Quantitative RT-PCR, Expressing

    33) Product Images from "Cloning and Expression of a Cytosolic HSP90 Gene in Chlorella vulgaris"

    Article Title: Cloning and Expression of a Cytosolic HSP90 Gene in Chlorella vulgaris

    Journal: BioMed Research International

    doi: 10.1155/2014/487050

    CvHSP90 mRNA expression levels in different heat shock times (0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, and 12 h) at 35°C were analyzed by real-time quantitative RT-PCR. CvHSP90 mRNA expression was normalized to the control group, and β -actin gene was used as internal control to calibrate the cDNA template for all the samples. Each bar represents the mean value from five determinations with standard error. Significant differences across control were indicated with an asterisk at P
    Figure Legend Snippet: CvHSP90 mRNA expression levels in different heat shock times (0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, and 12 h) at 35°C were analyzed by real-time quantitative RT-PCR. CvHSP90 mRNA expression was normalized to the control group, and β -actin gene was used as internal control to calibrate the cDNA template for all the samples. Each bar represents the mean value from five determinations with standard error. Significant differences across control were indicated with an asterisk at P

    Techniques Used: Expressing, Quantitative RT-PCR

    CvHSP90 mRNA expression levels relative to β -actin mRNA levels under stress of different salt concentrations analyzed by real-time quantitative RT-PCR. The β -actin gene was used as an internal control to calibrate the cDNA template for all the samples. Vertical bars represented the mean ± SE ( N = 5). Significant differences across control were indicated with an asterisk at P
    Figure Legend Snippet: CvHSP90 mRNA expression levels relative to β -actin mRNA levels under stress of different salt concentrations analyzed by real-time quantitative RT-PCR. The β -actin gene was used as an internal control to calibrate the cDNA template for all the samples. Vertical bars represented the mean ± SE ( N = 5). Significant differences across control were indicated with an asterisk at P

    Techniques Used: Expressing, Quantitative RT-PCR

    34) Product Images from "Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression"

    Article Title: Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124865

    MT3 affects the expression of MMP3 in breast cancer MDA-MB-231 cells. Expression of MT3 mRNA (A) and MMP3 mRNA (B) in MDA-MB-231 cells treated with scrambled siRNA (MDA-MB-231.CTRL) and MDA-MB-231 cells treated with siRNA directed against MT3 mRNA (MDA-MB-231.siRNA7). Real-time PCR was used to analyze MT3 mRNA and MMP3 mRNA. Relative expression (RQ) of MT3 and MMP3 genes was normalized against expression of ACTB gene and MDA-MB-231.CTRL cells were assigned as a calibrator sample. Results are expressed as mean ± SD.
    Figure Legend Snippet: MT3 affects the expression of MMP3 in breast cancer MDA-MB-231 cells. Expression of MT3 mRNA (A) and MMP3 mRNA (B) in MDA-MB-231 cells treated with scrambled siRNA (MDA-MB-231.CTRL) and MDA-MB-231 cells treated with siRNA directed against MT3 mRNA (MDA-MB-231.siRNA7). Real-time PCR was used to analyze MT3 mRNA and MMP3 mRNA. Relative expression (RQ) of MT3 and MMP3 genes was normalized against expression of ACTB gene and MDA-MB-231.CTRL cells were assigned as a calibrator sample. Results are expressed as mean ± SD.

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

    Expression of MMPs and TIMP1 in control BO2/LUC/PURO cells and BO2/MT3/LUC/PURO cells overexpressing MT3. (A) MMP1, (B) MMP2, (C) MMP3, (D) MMP9 and (E) TIMP1 mRNAs Real-time PCR was used to analyze MT3 mRNA. Relative expression (RQ) of MT3 gene was normalized against expression of ACTB gene and BO2/LUC/PURO cells were assigned as a calibrator sample. Results are expressed as mean ± SD. (F) MMP3 protein level in culture media of control BO2/LUC/PURO cells and BO2/MT3/LUC/PURO cells overexpressing MT3. The Quantikine Human Total MMPs Immunoassay (R D Systems, Minneapolis, MN USA) was used to detect MMP3. Mann–Whitney test, *p
    Figure Legend Snippet: Expression of MMPs and TIMP1 in control BO2/LUC/PURO cells and BO2/MT3/LUC/PURO cells overexpressing MT3. (A) MMP1, (B) MMP2, (C) MMP3, (D) MMP9 and (E) TIMP1 mRNAs Real-time PCR was used to analyze MT3 mRNA. Relative expression (RQ) of MT3 gene was normalized against expression of ACTB gene and BO2/LUC/PURO cells were assigned as a calibrator sample. Results are expressed as mean ± SD. (F) MMP3 protein level in culture media of control BO2/LUC/PURO cells and BO2/MT3/LUC/PURO cells overexpressing MT3. The Quantikine Human Total MMPs Immunoassay (R D Systems, Minneapolis, MN USA) was used to detect MMP3. Mann–Whitney test, *p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    MMP3 is responsible for the increased invasiveness of MT3-overexpressing breast cancer cells. (A) Expression of MMP3 mRNA in control MDA-MB-231/BO2 cells transfected with scrambled siRNA (BO2.MT3.CTRL) and MDA-MB-231/BO2 cells treated with siRNA directed against MMP3 mRNA (BO2.MT3.siRNA8). Real-time PCR was used to analyze MT3 mRNA. Relative expression (RQ) of MMP3 gene was normalized against expression of ACTB gene and BO2.MT3.CTRL cells was assigned as a calibrator sample. Results are expressed as mean ± SD. (B) Western blot analysis using anti-MMP3 monoclonal murine antibody on whole cell lysates of control MDA-MB-231/BO2 cells transfected with scrambled siRNA (BO2.MT3.CTRL) and MDA-MB-231 cells treated with siRNA directed against MMP3 mRNA (BO2.MT3.siRNA8). Cell lysates equivalent to 30 μg of protein were separated by SDS-PAGE under reducing conditions on a 12% gel and electrophoretically transferred onto a nitrocellulose membrane. β-Actin served as an internal control . (C) Invasiveness of control MDA-MB-231/BO2 cells transfected with scrambled siRNA (BO2.MT3.CTRL) and MDA-MB-231 cells treated with siRNA directed against MMP3 mRNA (BO2.MT3.siRNA8). Data are presented as mean ± SD. ****p
    Figure Legend Snippet: MMP3 is responsible for the increased invasiveness of MT3-overexpressing breast cancer cells. (A) Expression of MMP3 mRNA in control MDA-MB-231/BO2 cells transfected with scrambled siRNA (BO2.MT3.CTRL) and MDA-MB-231/BO2 cells treated with siRNA directed against MMP3 mRNA (BO2.MT3.siRNA8). Real-time PCR was used to analyze MT3 mRNA. Relative expression (RQ) of MMP3 gene was normalized against expression of ACTB gene and BO2.MT3.CTRL cells was assigned as a calibrator sample. Results are expressed as mean ± SD. (B) Western blot analysis using anti-MMP3 monoclonal murine antibody on whole cell lysates of control MDA-MB-231/BO2 cells transfected with scrambled siRNA (BO2.MT3.CTRL) and MDA-MB-231 cells treated with siRNA directed against MMP3 mRNA (BO2.MT3.siRNA8). Cell lysates equivalent to 30 μg of protein were separated by SDS-PAGE under reducing conditions on a 12% gel and electrophoretically transferred onto a nitrocellulose membrane. β-Actin served as an internal control . (C) Invasiveness of control MDA-MB-231/BO2 cells transfected with scrambled siRNA (BO2.MT3.CTRL) and MDA-MB-231 cells treated with siRNA directed against MMP3 mRNA (BO2.MT3.siRNA8). Data are presented as mean ± SD. ****p

    Techniques Used: Expressing, Multiple Displacement Amplification, Transfection, Real-time Polymerase Chain Reaction, Western Blot, SDS Page

    35) Product Images from "Replication Study: BET bromodomain inhibition as a therapeutic strategy to target c-Myc"

    Article Title: Replication Study: BET bromodomain inhibition as a therapeutic strategy to target c-Myc

    Journal: eLife

    doi: 10.7554/eLife.21253

    MYC expression in JQ1-treated MM.1S-luc cells. MM.1S-luc cells were treated with 500 nM (+)-JQ1, 500 nM (−)-JQ1, or an equivalent volume of DMSO. Total RNA was isolated at 0 hr, 1 hr, and 8 hr after treatment and qRT-PCR analysis was performed to detect MYC and GAPDH levels. Relative expression ( MYC/GAPDH ) is presented for each time point and condition normalized to (+)-JQ1 treated cells at 0 hr. Means reported and error bars represent s.d. from five independent biological repeats. Mixed-design analysis of variance (ANOVA) with time (0 hr, 1 hr, and 8 hr) as the within-subjects factor and treatment ((+)-JQ1, (−)-JQ1, or vehicle) as the between-subjects factor; interaction effect: F (4,24) = 268.9, p =1.49x10 −19 , treatment main effect: F (2,12) = 393.5, p =1.15x10 −11 , time main effect: F (2, 24) = 368.0, p =9.84x10 −19 . Planned paired t -test of MM.1S-luc cells harvested 8 hr after (+)-JQ1 treatment compared to cells 0 hr after (+)-JQ1 treatment; t (4) = 38.92, uncorrected p =2.60x10 −6 , a priori Bonferroni adjusted significance threshold = 0.025; (Bonferroni corrected p =5.21x10 −6 ). Planned paired t -test of MM.1S-luc cells harvested 1 hr after (+)-JQ1 treatment compared to cells 0 hr after (+)-JQ1 treatment; t (4) = 25.10, uncorrected p =1.50x10 −5 , a priori Bonferroni adjusted significance threshold = 0.025; (Bonferroni corrected p =2.99x10 −5 ). Additional details for this experiment can be found at https://osf.io/9swnx/ . DOI: http://dx.doi.org/10.7554/eLife.21253.002
    Figure Legend Snippet: MYC expression in JQ1-treated MM.1S-luc cells. MM.1S-luc cells were treated with 500 nM (+)-JQ1, 500 nM (−)-JQ1, or an equivalent volume of DMSO. Total RNA was isolated at 0 hr, 1 hr, and 8 hr after treatment and qRT-PCR analysis was performed to detect MYC and GAPDH levels. Relative expression ( MYC/GAPDH ) is presented for each time point and condition normalized to (+)-JQ1 treated cells at 0 hr. Means reported and error bars represent s.d. from five independent biological repeats. Mixed-design analysis of variance (ANOVA) with time (0 hr, 1 hr, and 8 hr) as the within-subjects factor and treatment ((+)-JQ1, (−)-JQ1, or vehicle) as the between-subjects factor; interaction effect: F (4,24) = 268.9, p =1.49x10 −19 , treatment main effect: F (2,12) = 393.5, p =1.15x10 −11 , time main effect: F (2, 24) = 368.0, p =9.84x10 −19 . Planned paired t -test of MM.1S-luc cells harvested 8 hr after (+)-JQ1 treatment compared to cells 0 hr after (+)-JQ1 treatment; t (4) = 38.92, uncorrected p =2.60x10 −6 , a priori Bonferroni adjusted significance threshold = 0.025; (Bonferroni corrected p =5.21x10 −6 ). Planned paired t -test of MM.1S-luc cells harvested 1 hr after (+)-JQ1 treatment compared to cells 0 hr after (+)-JQ1 treatment; t (4) = 25.10, uncorrected p =1.50x10 −5 , a priori Bonferroni adjusted significance threshold = 0.025; (Bonferroni corrected p =2.99x10 −5 ). Additional details for this experiment can be found at https://osf.io/9swnx/ . DOI: http://dx.doi.org/10.7554/eLife.21253.002

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR

    36) Product Images from "Potential Role of Activating Transcription Factor 5 during Osteogenesis"

    Article Title: Potential Role of Activating Transcription Factor 5 during Osteogenesis

    Journal: Stem Cells International

    doi: 10.1155/2016/5282185

    (a) Alizarin Red S staining of hADSCs during osteogenic differentiation. Mineralization of the extracellular matrix with the presence of calcium precipitates was visualized by staining with Alizarin Red S at days 8, 16, and 24. (b) Gene expression levels during osteogenic differentiation of hADSCs. Relative quantification of mRNA level assessed by qRT-PCR for RUNX2, OSTERIX, and ALPL after 3, 11, 16, 21, and 24 days of osteogenic differentiation. Proliferation group is used as reference for quantitation.
    Figure Legend Snippet: (a) Alizarin Red S staining of hADSCs during osteogenic differentiation. Mineralization of the extracellular matrix with the presence of calcium precipitates was visualized by staining with Alizarin Red S at days 8, 16, and 24. (b) Gene expression levels during osteogenic differentiation of hADSCs. Relative quantification of mRNA level assessed by qRT-PCR for RUNX2, OSTERIX, and ALPL after 3, 11, 16, 21, and 24 days of osteogenic differentiation. Proliferation group is used as reference for quantitation.

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

    37) Product Images from "Engineering Xeno-Free Microcarriers with Recombinant Vitronectin, Albumin and UV Irradiation for Human Pluripotent Stem Cell Bioprocessing"

    Article Title: Engineering Xeno-Free Microcarriers with Recombinant Vitronectin, Albumin and UV Irradiation for Human Pluripotent Stem Cell Bioprocessing

    Journal: ACS biomaterials science & engineering

    doi: 10.1021/acsbiomaterials.6b00253

    Embryoid body (EB) formation and multilineage differentiation for hPSCs expanded in VN+HSA+UV microcarrier cultures for 5 continuous passages. (A) Cells within EBs displayed markers of DE, MS and NE analyzed by qPCR. Expression was normalized to that of undifferentiated cells with ACTB as the housekeeping gene. Quantitative PCR analysis of hPSCs expanded for 5 passages in microcarrier cultures and subjected to directed differentiation toward (B) DE, (C) MS and (D) NE and the corresponding immunocytochemistry results (E–G). The expression results in (B–D) was normalized to that of hPSCs cultured in basal media without the differentiation factors.
    Figure Legend Snippet: Embryoid body (EB) formation and multilineage differentiation for hPSCs expanded in VN+HSA+UV microcarrier cultures for 5 continuous passages. (A) Cells within EBs displayed markers of DE, MS and NE analyzed by qPCR. Expression was normalized to that of undifferentiated cells with ACTB as the housekeeping gene. Quantitative PCR analysis of hPSCs expanded for 5 passages in microcarrier cultures and subjected to directed differentiation toward (B) DE, (C) MS and (D) NE and the corresponding immunocytochemistry results (E–G). The expression results in (B–D) was normalized to that of hPSCs cultured in basal media without the differentiation factors.

    Techniques Used: Mass Spectrometry, Real-time Polymerase Chain Reaction, Expressing, Immunocytochemistry, Cell Culture

    38) Product Images from "Identification of microRNA-regulated pathways using an integration of microRNA-mRNA microarray and bioinformatics analysis in CD34+ cells of myelodysplastic syndromes"

    Article Title: Identification of microRNA-regulated pathways using an integration of microRNA-mRNA microarray and bioinformatics analysis in CD34+ cells of myelodysplastic syndromes

    Journal: Scientific Reports

    doi: 10.1038/srep32232

    Validation of screened miRNAs and mRNAs using qRT-PCR. The expression of miR-17-3p ( A ) and miR-19a-3p ( B ) were significantly higher in the high-grade and low-grade MDS groups than those in the normal control group (all P
    Figure Legend Snippet: Validation of screened miRNAs and mRNAs using qRT-PCR. The expression of miR-17-3p ( A ) and miR-19a-3p ( B ) were significantly higher in the high-grade and low-grade MDS groups than those in the normal control group (all P

    Techniques Used: Quantitative RT-PCR, Expressing

    39) Product Images from "Ribose Accelerates Gut Motility and Suppresses Mouse Body Weight Gaining"

    Article Title: Ribose Accelerates Gut Motility and Suppresses Mouse Body Weight Gaining

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.13635

    Expression pattern of Rbks gene in mice. (A) Quantitative real-time (qRT) PCR revealed a high RNA level of Rbks gene in kidney, liver, testis, small intestine (duodenum, jejunum and ileum) and cecum of wild type mice. Primers for qRT PCR located on exon5 and exon6. (B) The expression pattern of Rbks gene was also revealed by whole mount X-gal staining with Rbks galeo/galeo mice tissues. The wild type litter mates served as controls. Black bar: 1cm. (C) The whole mount staining intestines were further processed for paraffin sections, and the cellular β-galactosidase signals were identified in intestinal brush boarders. Black bar: 100μm.
    Figure Legend Snippet: Expression pattern of Rbks gene in mice. (A) Quantitative real-time (qRT) PCR revealed a high RNA level of Rbks gene in kidney, liver, testis, small intestine (duodenum, jejunum and ileum) and cecum of wild type mice. Primers for qRT PCR located on exon5 and exon6. (B) The expression pattern of Rbks gene was also revealed by whole mount X-gal staining with Rbks galeo/galeo mice tissues. The wild type litter mates served as controls. Black bar: 1cm. (C) The whole mount staining intestines were further processed for paraffin sections, and the cellular β-galactosidase signals were identified in intestinal brush boarders. Black bar: 100μm.

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

    40) Product Images from "A Novel Function of Human Pumilio Proteins in Cytoplasmic Sensing of Viral Infection"

    Article Title: A Novel Function of Human Pumilio Proteins in Cytoplasmic Sensing of Viral Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004417

    Knockdown of PUM1 and PUM2 downregulates NDV-induced gene activation. (A–C) HEK293T cells were transfected with control siRNA (siN.C.) or siRNA targeting human PUM1 or PUM2 for 48 h. Knockdown efficiency was confirmed by immunoblotting with anti-PUM1, anti-PUM2 and anti-β-actin antibodies (A). The cells were infected with NDV for 9 h, and IFNB (B) and CXCL10 (C) mRNA levels were determined by quantitative RT-PCR. (D and E) HEK293T cells were transfected with control siRNA or siRNA targeting PUM1 or PUM2 for 48 h. The cells were infected with NDV for 24 h. The culture media were collected and subjected to IFN-β ELISA (D). Total cellular RNA was extracted and subjected to qRT-PCR for NDV RNA (E). Data are from one representative of at least two independent experiments; means and S.D. of duplicate experiments are shown (*p
    Figure Legend Snippet: Knockdown of PUM1 and PUM2 downregulates NDV-induced gene activation. (A–C) HEK293T cells were transfected with control siRNA (siN.C.) or siRNA targeting human PUM1 or PUM2 for 48 h. Knockdown efficiency was confirmed by immunoblotting with anti-PUM1, anti-PUM2 and anti-β-actin antibodies (A). The cells were infected with NDV for 9 h, and IFNB (B) and CXCL10 (C) mRNA levels were determined by quantitative RT-PCR. (D and E) HEK293T cells were transfected with control siRNA or siRNA targeting PUM1 or PUM2 for 48 h. The cells were infected with NDV for 24 h. The culture media were collected and subjected to IFN-β ELISA (D). Total cellular RNA was extracted and subjected to qRT-PCR for NDV RNA (E). Data are from one representative of at least two independent experiments; means and S.D. of duplicate experiments are shown (*p

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

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    Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) <t>qRT-PCR</t> indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p
    Quantitative Real Time Reverse Transcription Polymerase Chain Reaction Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative real time pcr qrt pcr
    LINC01296 knockdown suppressed the migration of bladder cancer cells. Notes: ( A , B ) Transwell assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( C , D ) Wound healing assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( E ) <t>qRT-</t> <t>PCR</t> analyses were used to measure mRNA levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. ( F ) Western blot assays were used to measure protein levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P
    Quantitative Real Time Pcr Qrt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1655 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative real time polymerase chain reaction qrt pcr total rna
    Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by <t>qRT-PCR;</t> b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P
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    Image Search Results


    Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) qRT-PCR indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p

    Journal: OncoTargets and therapy

    Article Title: Nuclear factor-κB p65 regulates glutaminase 1 expression in human hepatocellular carcinoma

    doi: 10.2147/OTT.S167408

    Figure Lengend Snippet: Knockdown or suppression of p65 reduces GLS1 expression in HCC cells. ( A ) Western blot shows the knockdown of c-Myc without interfering with GLS1 expression in HepG2 and Hep3B cells. ( B ) Western blot shows that p65 knockdown reduces GLS1 protein levels in both HepG2 and Hep3B cells. ( C ) qRT-PCR indicates that p65 knockdown reduces GLS1 mRNA levels in both HepG2 and Hep3B cells. ** p

    Article Snippet: Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) Upon relative treatment, cells were lysed, and total RNA was isolated using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR

    LINC01296 knockdown suppressed the migration of bladder cancer cells. Notes: ( A , B ) Transwell assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( C , D ) Wound healing assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( E ) qRT- PCR analyses were used to measure mRNA levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. ( F ) Western blot assays were used to measure protein levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P

    Journal: OncoTargets and therapy

    Article Title: Long noncoding RNA LINC01296 promotes cancer-cell proliferation and metastasis in urothelial carcinoma of the bladder

    doi: 10.2147/OTT.S192809

    Figure Lengend Snippet: LINC01296 knockdown suppressed the migration of bladder cancer cells. Notes: ( A , B ) Transwell assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( C , D ) Wound healing assays were used to measure the migration ability of T24 and 5637 cells transfected with si-LINC01296 or si-NC. ( E ) qRT- PCR analyses were used to measure mRNA levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. ( F ) Western blot assays were used to measure protein levels of E-cadherin and N-cadherin following the treatment of bladder cells with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500 (Thermo Fisher Scientific) instrument using a KAPA SYBR Green FAST qPCR Kit (KAPA, Wilmington, MA, US) according to the manufacturer’s instructions.

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

    LINC01296 knockdown inhibited the proliferation of bladder cancer cells. Notes: ( A ) Relative LINC01296 expressions in human bladder cancer cell lines (RT4, T24, and 5637). ( B , C ) MTT assays were performed to measure the proliferation viability of T24 and 5637 cells after transfection. ( D , E ) qRT-PCR results of LINC01296 expressions following the treatment of T24 and 5637 cells with anti-LINC01296 siRNA. ( F , G ) Colony formation assays were used to measure the number of clone formation of bladder cancer cells after transfection. ( H , I ) Flow cytometry assays were performed to analyze the cell cycle progression of T24 and 5637 cells after they were transfected with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P

    Journal: OncoTargets and therapy

    Article Title: Long noncoding RNA LINC01296 promotes cancer-cell proliferation and metastasis in urothelial carcinoma of the bladder

    doi: 10.2147/OTT.S192809

    Figure Lengend Snippet: LINC01296 knockdown inhibited the proliferation of bladder cancer cells. Notes: ( A ) Relative LINC01296 expressions in human bladder cancer cell lines (RT4, T24, and 5637). ( B , C ) MTT assays were performed to measure the proliferation viability of T24 and 5637 cells after transfection. ( D , E ) qRT-PCR results of LINC01296 expressions following the treatment of T24 and 5637 cells with anti-LINC01296 siRNA. ( F , G ) Colony formation assays were used to measure the number of clone formation of bladder cancer cells after transfection. ( H , I ) Flow cytometry assays were performed to analyze the cell cycle progression of T24 and 5637 cells after they were transfected with anti-LINC01296 siRNA. Data are presented as mean ± SD of three independent experiments. *P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500 (Thermo Fisher Scientific) instrument using a KAPA SYBR Green FAST qPCR Kit (KAPA, Wilmington, MA, US) according to the manufacturer’s instructions.

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

    LINC01296 was upregulated in bladder cancer and the expression level of LINC01296 was correlated with clinicopathological features of bladder cancer patients. Notes: ( A ) Comparison of relative expressions of LINC01296 in cancer and normal tissues using the GEPIA database. ( B ) Comparison of relative expressions of LINC01296 in bladder cancer and adjacent normal tissues using the GEPIA database. ( C ) A microarray containing 37,000 lncRNA and 34,000 mRNA probes was used to screen differentially expressed lncRNAs in four pairs of bladder cancer patients. ( D ) Comparison of relative expressions of LINC01296 in 54 bladder cancer tissues and 24 normal bladder tissues using qRT-PCR. ( E ) Two groups were set up according to the mean expression of LINC01296 in tumor tissues. ( F ) The LINC01296 expression was significantly higher in patients with a higher tumor stage, lymph node metastasis and a higher pathologic grade. * P

    Journal: OncoTargets and therapy

    Article Title: Long noncoding RNA LINC01296 promotes cancer-cell proliferation and metastasis in urothelial carcinoma of the bladder

    doi: 10.2147/OTT.S192809

    Figure Lengend Snippet: LINC01296 was upregulated in bladder cancer and the expression level of LINC01296 was correlated with clinicopathological features of bladder cancer patients. Notes: ( A ) Comparison of relative expressions of LINC01296 in cancer and normal tissues using the GEPIA database. ( B ) Comparison of relative expressions of LINC01296 in bladder cancer and adjacent normal tissues using the GEPIA database. ( C ) A microarray containing 37,000 lncRNA and 34,000 mRNA probes was used to screen differentially expressed lncRNAs in four pairs of bladder cancer patients. ( D ) Comparison of relative expressions of LINC01296 in 54 bladder cancer tissues and 24 normal bladder tissues using qRT-PCR. ( E ) Two groups were set up according to the mean expression of LINC01296 in tumor tissues. ( F ) The LINC01296 expression was significantly higher in patients with a higher tumor stage, lymph node metastasis and a higher pathologic grade. * P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500 (Thermo Fisher Scientific) instrument using a KAPA SYBR Green FAST qPCR Kit (KAPA, Wilmington, MA, US) according to the manufacturer’s instructions.

    Techniques: Expressing, Microarray, Quantitative RT-PCR

    Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by qRT-PCR; b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P

    Journal: Cancer Cell International

    Article Title: miR-96 exerts carcinogenic effect by activating AKT/GSK-3β/β-catenin signaling pathway through targeting inhibition of FOXO1 in hepatocellular carcinoma

    doi: 10.1186/s12935-019-0756-7

    Figure Lengend Snippet: Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by qRT-PCR; b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) Total RNA in tumor tissues and cells was obtained by Trizol kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the the instruction manual.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    FOXO1 was directly suppressed by miR-96. a Prediction of binding sites for FOXO1 and miR-96 by Target Scan; b Luciferase reporter assay; c FOXO1 mRNA expression by qRT-PCR; d FOXO1 protein expression by Western blot. ## P

    Journal: Cancer Cell International

    Article Title: miR-96 exerts carcinogenic effect by activating AKT/GSK-3β/β-catenin signaling pathway through targeting inhibition of FOXO1 in hepatocellular carcinoma

    doi: 10.1186/s12935-019-0756-7

    Figure Lengend Snippet: FOXO1 was directly suppressed by miR-96. a Prediction of binding sites for FOXO1 and miR-96 by Target Scan; b Luciferase reporter assay; c FOXO1 mRNA expression by qRT-PCR; d FOXO1 protein expression by Western blot. ## P

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) Total RNA in tumor tissues and cells was obtained by Trizol kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the the instruction manual.

    Techniques: Binding Assay, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot