mda mb 231 cells  (Qiagen)


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    QIAzol Lysis Reagent
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    For efficient lysis of fatty and standard tissues before RNA isolation Kit contents Qiagen QIAzol Lysis Reagent 200mL Silica Membrane Technology High Yield For Efficient Lysis of Fatty and Standard Tissues Before RNA Isolation Ideal for Downstream Application Benefits Optimized lysis conditions to purify RNA for gene expression analysis High yields of RNA from fatty tissues Easy to follow protocol for lysis and homogenization Integration with RNeasy cleanup to prevent phenol carryover Compatibility with a variety of tissue typ
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    Qiagen mda mb 231 cells
    QIAzol Lysis Reagent
    For efficient lysis of fatty and standard tissues before RNA isolation Kit contents Qiagen QIAzol Lysis Reagent 200mL Silica Membrane Technology High Yield For Efficient Lysis of Fatty and Standard Tissues Before RNA Isolation Ideal for Downstream Application Benefits Optimized lysis conditions to purify RNA for gene expression analysis High yields of RNA from fatty tissues Easy to follow protocol for lysis and homogenization Integration with RNeasy cleanup to prevent phenol carryover Compatibility with a variety of tissue typ
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    Images

    1) Product Images from "Activated kinase screening identifies the IKBKE oncogene as a positive regulator of autophagy"

    Article Title: Activated kinase screening identifies the IKBKE oncogene as a positive regulator of autophagy

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1517855

    IKBKE is required for autophagy induced by the ERBB2 breast oncogene. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon pharmacological inhibition of IKBKE activity by CYT387 (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P
    Figure Legend Snippet: IKBKE is required for autophagy induced by the ERBB2 breast oncogene. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon pharmacological inhibition of IKBKE activity by CYT387 (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P

    Techniques Used: Confocal Microscopy, Multiple Displacement Amplification, Expressing, Immunofluorescence, Transfection, Negative Control, Staining, Software, Inhibition, Activity Assay

    The catalytic activity of IKBKE is required for its ability to control autophagy. (a) WB analysis to evaluate autophagic flux of MDA-MB-231 cells stably expressing IKBKE (WT) and IKBKE kinase dead (KD). Cells stably expressing an empty vector were used as a negative control. Where indicated, 4-h treatment with 400 nM BAF was performed. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels (used as loading control), is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Confocal microscopy analysis of MDA-MB-231 cells expressing IKBKE (WT) and IKBKE (KD), to evaluate autophagic flux. Cells were subjected to immunofluorescence analysis. Where indicated, 4-h treatment with 400 nM BAF was performed. In these representative images, LC3B is visualized in green, IKBKE (WT or KD) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. (c) Time course experiment to analyze the autophagic flux in MDA-MB-231 treated with the CYT387 drug, by WB analysis. Cells were treated with different concentrations of the drug (2, 5 and 10 μM) and treated or not with 400 nM BAF for 2 h. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels (used as loading control), is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Confocal microscopy analysis of MDA-MB-231 cells treated with CYT387, to evaluate the autophagic flux. Cells were treated with different concentrations of the drug (2, 5 and 10 μM) and treated or not with 400 nM BAF for 2 h. Then, cells were subjected to immunofluorescence analysis. In these representative images, LC3B is visualized in green, endogenous IKBKE in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P
    Figure Legend Snippet: The catalytic activity of IKBKE is required for its ability to control autophagy. (a) WB analysis to evaluate autophagic flux of MDA-MB-231 cells stably expressing IKBKE (WT) and IKBKE kinase dead (KD). Cells stably expressing an empty vector were used as a negative control. Where indicated, 4-h treatment with 400 nM BAF was performed. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels (used as loading control), is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Confocal microscopy analysis of MDA-MB-231 cells expressing IKBKE (WT) and IKBKE (KD), to evaluate autophagic flux. Cells were subjected to immunofluorescence analysis. Where indicated, 4-h treatment with 400 nM BAF was performed. In these representative images, LC3B is visualized in green, IKBKE (WT or KD) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. (c) Time course experiment to analyze the autophagic flux in MDA-MB-231 treated with the CYT387 drug, by WB analysis. Cells were treated with different concentrations of the drug (2, 5 and 10 μM) and treated or not with 400 nM BAF for 2 h. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels (used as loading control), is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Confocal microscopy analysis of MDA-MB-231 cells treated with CYT387, to evaluate the autophagic flux. Cells were treated with different concentrations of the drug (2, 5 and 10 μM) and treated or not with 400 nM BAF for 2 h. Then, cells were subjected to immunofluorescence analysis. In these representative images, LC3B is visualized in green, endogenous IKBKE in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P

    Techniques Used: Activity Assay, Western Blot, Multiple Displacement Amplification, Stable Transfection, Expressing, Plasmid Preparation, Negative Control, Confocal Microscopy, Immunofluorescence, Staining, Software

    A role for autophagy in IKBKE-dependent normal breast epithelial cell transformation and TNBC proliferation. (a) Proliferation assay of MDA-MB-231 cells upon downregulation of endogenous IKBKE, ATG5, ULK1 protein levels by transfection with the indicated siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679, #680 and siRNA for ATG5 and ULK1 ). Cell viability was evaluated 72 h post-transfection by counting cells in triplicate with a Z2 Coulter Counter. Data were processed in Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Same as in (a), but using MDA-MB-468 TNBC cells. (c) Cell count of 1-7HB2 cell stably expressing IKBKE (WT), evaluating cell proliferation, by counting cells in triplicate with a Z2 Coulter Counter, upon downregulation (72 h post-transfection) of endogenous ATG5 and ULK1 with specific siRNA. A scrambled siRNA was used as a negative control. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Proliferation assay of 1-7HB2 cell stably expressing IKBKE (WT) upon pharmacological inhibition of autophagic activity by SAR-405 (10 μM) and Spautin-1 (100 μM). Cell proliferation was evaluated after 72-h treatment by counting cells in triplicate with a Z2 Coulter Counter. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.
    Figure Legend Snippet: A role for autophagy in IKBKE-dependent normal breast epithelial cell transformation and TNBC proliferation. (a) Proliferation assay of MDA-MB-231 cells upon downregulation of endogenous IKBKE, ATG5, ULK1 protein levels by transfection with the indicated siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679, #680 and siRNA for ATG5 and ULK1 ). Cell viability was evaluated 72 h post-transfection by counting cells in triplicate with a Z2 Coulter Counter. Data were processed in Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Same as in (a), but using MDA-MB-468 TNBC cells. (c) Cell count of 1-7HB2 cell stably expressing IKBKE (WT), evaluating cell proliferation, by counting cells in triplicate with a Z2 Coulter Counter, upon downregulation (72 h post-transfection) of endogenous ATG5 and ULK1 with specific siRNA. A scrambled siRNA was used as a negative control. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Proliferation assay of 1-7HB2 cell stably expressing IKBKE (WT) upon pharmacological inhibition of autophagic activity by SAR-405 (10 μM) and Spautin-1 (100 μM). Cell proliferation was evaluated after 72-h treatment by counting cells in triplicate with a Z2 Coulter Counter. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.

    Techniques Used: Transformation Assay, Proliferation Assay, Multiple Displacement Amplification, Transfection, Software, Cell Counting, Stable Transfection, Expressing, Negative Control, Inhibition, Activity Assay

    The IKBKE human oncogene induces autophagy in MDA-MB-231 breast cancer cells. (a) Endogenous IKBKE protein levels were tested in indicated cell lines, by WB analysis. (b) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and 2 unrelated specific siRNA against human IKBKE , #679 and #680). Where indicated, samples were treated with 400 nM BAF or with starvation medium (STV) for 5 h. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels, is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (c) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells by confocal microscopy analysis of MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 24 h. In these representative images, SQSTM1 is visualized in green and DAPI-stained nuclei in blue. SQSTM1-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Autophagic flux was evaluated in in MDA-MB-231 cells transfected with Scr siRNA and siRNA specific for IKBKE , Followed by rescue with IKBKE (WT) overexpression. Where indicated, 4 h treatment with 400 nM BAF was performed. In these representative images, LC3B is visualized in green, IKBKE (WT) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.
    Figure Legend Snippet: The IKBKE human oncogene induces autophagy in MDA-MB-231 breast cancer cells. (a) Endogenous IKBKE protein levels were tested in indicated cell lines, by WB analysis. (b) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and 2 unrelated specific siRNA against human IKBKE , #679 and #680). Where indicated, samples were treated with 400 nM BAF or with starvation medium (STV) for 5 h. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels, is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (c) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells by confocal microscopy analysis of MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 24 h. In these representative images, SQSTM1 is visualized in green and DAPI-stained nuclei in blue. SQSTM1-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Autophagic flux was evaluated in in MDA-MB-231 cells transfected with Scr siRNA and siRNA specific for IKBKE , Followed by rescue with IKBKE (WT) overexpression. Where indicated, 4 h treatment with 400 nM BAF was performed. In these representative images, LC3B is visualized in green, IKBKE (WT) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.

    Techniques Used: Multiple Displacement Amplification, Western Blot, Transfection, Confocal Microscopy, Staining, Software, Over Expression

    IKBKE is required for autophagy induced by the AKT transforming pathway. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon pharmacological inhibition of IKBKE activity by the CYT387 drug (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars, 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P
    Figure Legend Snippet: IKBKE is required for autophagy induced by the AKT transforming pathway. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon pharmacological inhibition of IKBKE activity by the CYT387 drug (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars, 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P

    Techniques Used: Confocal Microscopy, Multiple Displacement Amplification, Expressing, Immunofluorescence, Transfection, Negative Control, Staining, Software, Inhibition, Activity Assay

    2) Product Images from "Poly(I:C) and CpG-ODN combined aerosolization to treat lung metastases and counter the immunosuppressive microenvironment"

    Article Title: Poly(I:C) and CpG-ODN combined aerosolization to treat lung metastases and counter the immunosuppressive microenvironment

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2015.1040214

    Up-modulation of NKG2D ligand expression on dacarbazine-treated B16 melanoma cells. ( A ) MULT1 and RAE1 expression on the cell surface of B16 melanoma cells analyzed by flow cytometry after 24-h culture in complete medium alone (PBS) or supplemented with
    Figure Legend Snippet: Up-modulation of NKG2D ligand expression on dacarbazine-treated B16 melanoma cells. ( A ) MULT1 and RAE1 expression on the cell surface of B16 melanoma cells analyzed by flow cytometry after 24-h culture in complete medium alone (PBS) or supplemented with

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    Antitumor activity of aerosol CpG-ODN/Poly(I:C) combined with dacarbazine (DTIC) on B16 experimental lung metastases. Number of macroscopic B16 melanoma lung metastases in mice untreated (7 mice) or treated with aerosol CpG-ODN/Poly(I:C) (8 mice), DTIC
    Figure Legend Snippet: Antitumor activity of aerosol CpG-ODN/Poly(I:C) combined with dacarbazine (DTIC) on B16 experimental lung metastases. Number of macroscopic B16 melanoma lung metastases in mice untreated (7 mice) or treated with aerosol CpG-ODN/Poly(I:C) (8 mice), DTIC

    Techniques Used: Activity Assay, Mouse Assay

    3) Product Images from "Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47"

    Article Title: Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47

    Journal: European Journal of Human Genetics

    doi: 10.1038/ejhg.2011.150

    ( a ) Expression levels of CT47 mRNA in different SCLC cell lines relative to the expression in human testis. Commercially available human total testis RNA and RNA isolated from SCLC lines were used for cDNA synthesis under identical conditions. CT47 expression levels are normalized to the expression levels measured in the testis. Different SCLC lines have different, but low levels of CT47 expression compared to the testis. Relative abundance of histone modifications and EZH2 at the CT47 promoter ( b ), exon 3 ( c ) and the distal region in SCLC cell lines ( d ). SCLC cell lines show individual variation in the abundance of different histone modifications. Generally, a loss of the repressive chromatin mark H3K9me3 can be observed in SCLCs. H3K27me3 levels are higher in SCLCs at the promoter and exon 3 region than in LCLs, but lower at the distal region. The relative abundance of the PRC2 component EZH2 responsible for generating H2K27me3 is dramatically reduced in SCLCs compared to LCLs at all region studied. ( e ) DNA methylation levels at CpGs located in the CT47 promoter region in LCL and SCLC samples. The methylation level of seven different CpGs, next to the transcriptional start site of CT47 , was determined by bisulfite sequencing and quantified by ESME program. There is a significant difference between the methylation level of LCLs and SCLCs at every CpG tested ( P
    Figure Legend Snippet: ( a ) Expression levels of CT47 mRNA in different SCLC cell lines relative to the expression in human testis. Commercially available human total testis RNA and RNA isolated from SCLC lines were used for cDNA synthesis under identical conditions. CT47 expression levels are normalized to the expression levels measured in the testis. Different SCLC lines have different, but low levels of CT47 expression compared to the testis. Relative abundance of histone modifications and EZH2 at the CT47 promoter ( b ), exon 3 ( c ) and the distal region in SCLC cell lines ( d ). SCLC cell lines show individual variation in the abundance of different histone modifications. Generally, a loss of the repressive chromatin mark H3K9me3 can be observed in SCLCs. H3K27me3 levels are higher in SCLCs at the promoter and exon 3 region than in LCLs, but lower at the distal region. The relative abundance of the PRC2 component EZH2 responsible for generating H2K27me3 is dramatically reduced in SCLCs compared to LCLs at all region studied. ( e ) DNA methylation levels at CpGs located in the CT47 promoter region in LCL and SCLC samples. The methylation level of seven different CpGs, next to the transcriptional start site of CT47 , was determined by bisulfite sequencing and quantified by ESME program. There is a significant difference between the methylation level of LCLs and SCLCs at every CpG tested ( P

    Techniques Used: Expressing, Isolation, DNA Methylation Assay, Methylation, Methylation Sequencing

    4) Product Images from "Dendrobium moniliforme Exerts Inhibitory Effects on Both Receptor Activator of Nuclear Factor Kappa-B Ligand-Mediated Osteoclast Differentiation in Vitro and Lipopolysaccharide-Induced Bone Erosion in Vivo"

    Article Title: Dendrobium moniliforme Exerts Inhibitory Effects on Both Receptor Activator of Nuclear Factor Kappa-B Ligand-Mediated Osteoclast Differentiation in Vitro and Lipopolysaccharide-Induced Bone Erosion in Vivo

    Journal: Molecules

    doi: 10.3390/molecules21030295

    DM suppresses the expressions of c-Fos, NFATc1, and other osteoclast marker genes. ( A ) BMMs were stimulated with RANKL (50 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of DM (50 ng/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent; mRNA expression levels of c-Fos and NFATc1 were evaluated using quantitative real-time RT-PCR. *** p
    Figure Legend Snippet: DM suppresses the expressions of c-Fos, NFATc1, and other osteoclast marker genes. ( A ) BMMs were stimulated with RANKL (50 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of DM (50 ng/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent; mRNA expression levels of c-Fos and NFATc1 were evaluated using quantitative real-time RT-PCR. *** p

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

    5) Product Images from "Nexilin/NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP"

    Article Title: Nexilin/NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31328-2

    Knockdown of Nexilin/ NEXN reduces actin polymerization and SMC marker expression. NEXN was silenced using a short hairpin adenoviral construct (sh-NEXN) and the contents of NEXN and a number of SMC differentiation markers were examined at the mRNA (panel A, N = 6–9) and protein levels (panels B and C, N = 12). Panel D shows that depolymerization of actin using LatB reduces SRF expression in both human bladder and coronary artery SMCs (HBSMCs, N = 8; HCASMCs, N = 9). Panel E shows phalloidin staining of actin filaments in control cell (Null) and after NEXN silencing (sh-NEXN). The scale bar represents 50 μm. Panel F shows a sedimentation assay to determine filamentous (F-) and globular (G-) actin in control cells (null) and after silencing of NEXN . Panel G shows the normalized F- to G-actin ratio in bladder and coronary artery SMCs (HBSMCs, N = 12; HCASMCs, N = 6). Panel H shows that polymerization of actin using jasplakinolide (Jasp) increases MYH11 in HBSMCs in control conditions and after NEXN silencing (N = 6). Panel I shows cell density at different times following the creation of a cell-free area in the culture dish (N = 9–10). Panel J shows the speed of cell movement within the cell-free area (N = 19 and 22 motile cells in Null and sh- NEXN , respectively). Panel K shows a cell viability assay comparing control and NEXN -silenced cells (N = 12 throughout). The effect of NEXN silencing on cell migration was confirmed using an independent siRNA in L (N = 9). The associated repression of the NEXN protein is shown in ( M ) (N = 6). The schematic illustration in panel N summarizes our findings regarding the transcriptional control of NEXN and its impact on actin polymerization and cell motility. Several aspects of this model, including the involvement of a ( G ) protein-coupled receptor in the S1P effect, were not directly tested here, and they are thus drawn in grey.
    Figure Legend Snippet: Knockdown of Nexilin/ NEXN reduces actin polymerization and SMC marker expression. NEXN was silenced using a short hairpin adenoviral construct (sh-NEXN) and the contents of NEXN and a number of SMC differentiation markers were examined at the mRNA (panel A, N = 6–9) and protein levels (panels B and C, N = 12). Panel D shows that depolymerization of actin using LatB reduces SRF expression in both human bladder and coronary artery SMCs (HBSMCs, N = 8; HCASMCs, N = 9). Panel E shows phalloidin staining of actin filaments in control cell (Null) and after NEXN silencing (sh-NEXN). The scale bar represents 50 μm. Panel F shows a sedimentation assay to determine filamentous (F-) and globular (G-) actin in control cells (null) and after silencing of NEXN . Panel G shows the normalized F- to G-actin ratio in bladder and coronary artery SMCs (HBSMCs, N = 12; HCASMCs, N = 6). Panel H shows that polymerization of actin using jasplakinolide (Jasp) increases MYH11 in HBSMCs in control conditions and after NEXN silencing (N = 6). Panel I shows cell density at different times following the creation of a cell-free area in the culture dish (N = 9–10). Panel J shows the speed of cell movement within the cell-free area (N = 19 and 22 motile cells in Null and sh- NEXN , respectively). Panel K shows a cell viability assay comparing control and NEXN -silenced cells (N = 12 throughout). The effect of NEXN silencing on cell migration was confirmed using an independent siRNA in L (N = 9). The associated repression of the NEXN protein is shown in ( M ) (N = 6). The schematic illustration in panel N summarizes our findings regarding the transcriptional control of NEXN and its impact on actin polymerization and cell motility. Several aspects of this model, including the involvement of a ( G ) protein-coupled receptor in the S1P effect, were not directly tested here, and they are thus drawn in grey.

    Techniques Used: Marker, Expressing, Construct, Staining, Sedimentation, Viability Assay, Migration

    NEXN correlates with gene products that control and respond to changes in actin polymerization and Nexilin is reduced by depolymerization of actin. Correlations of NEXN versus all other RNAs in the top-ten NEXN expressing tissues were examined (data from GTExPortal). The sum of correlation coefficients for individual RNAs across tissues was calculated (R sum ) and the positive extreme of this distribution was plotted ( A ). Actin controlling and responding gene products represented in the extreme are highlighted in blue colors. Examples of NEXN correlations in the human coronary artery (N = 133) are shown in panels B through ( D ). P-values and Spearman Rho values are given in the respective panels. Panels E and F show mRNA data for NEXN in cultured human bladder (HBSMCs, N = 8) and coronary artery (HCASMCs, N = 9) SMCs after treatment with Latrunculin B (LatB). Panels H and I show protein data for Nexilin/ NEXN in the presence and absence of LatB (HBSMCs, 300 nM, N = 12; HCASMCs, 100 nM, N = 10). The top micrographs in panel J shows confocal imaging of YAP (red) on the left, and YAP (green) and CAV1 (red) on the right. The bottom row shows YAP (red) and Nexilin (green). The high magnification overlay at the bottom right shows partial colocalization of YAP and Nexilin at the cell membrane in yellow. All micrographs are from cross-sectioned HBSMCs and white scale bars represent 5 μm throughout.
    Figure Legend Snippet: NEXN correlates with gene products that control and respond to changes in actin polymerization and Nexilin is reduced by depolymerization of actin. Correlations of NEXN versus all other RNAs in the top-ten NEXN expressing tissues were examined (data from GTExPortal). The sum of correlation coefficients for individual RNAs across tissues was calculated (R sum ) and the positive extreme of this distribution was plotted ( A ). Actin controlling and responding gene products represented in the extreme are highlighted in blue colors. Examples of NEXN correlations in the human coronary artery (N = 133) are shown in panels B through ( D ). P-values and Spearman Rho values are given in the respective panels. Panels E and F show mRNA data for NEXN in cultured human bladder (HBSMCs, N = 8) and coronary artery (HCASMCs, N = 9) SMCs after treatment with Latrunculin B (LatB). Panels H and I show protein data for Nexilin/ NEXN in the presence and absence of LatB (HBSMCs, 300 nM, N = 12; HCASMCs, 100 nM, N = 10). The top micrographs in panel J shows confocal imaging of YAP (red) on the left, and YAP (green) and CAV1 (red) on the right. The bottom row shows YAP (red) and Nexilin (green). The high magnification overlay at the bottom right shows partial colocalization of YAP and Nexilin at the cell membrane in yellow. All micrographs are from cross-sectioned HBSMCs and white scale bars represent 5 μm throughout.

    Techniques Used: Expressing, Cell Culture, Imaging

    6) Product Images from "Increased DUX4 expression during muscle differentiation correlates with decreased SMCHD1 protein levels at D4Z4 "

    Article Title: Increased DUX4 expression during muscle differentiation correlates with decreased SMCHD1 protein levels at D4Z4

    Journal: Epigenetics

    doi: 10.1080/15592294.2015.1113798

    DUX4 activation during myogenic differentiation coincides with SMCHD1 reduction. ( A) Immunofluorescence microscopy analysis confirmed the typical DUX4 protein expression pattern in myosin positive, multinucleated myotubes derived from FSHD1 and FSHD2 individuals. Images were taken using a 200x magnification, scale bars are displayed. ( B ) Quantitative mRNA analysis in control ( C ), FSHD1 (F1), and FSHD2 (F2) primary myoblasts (B) and myotubes (T) showing increased levels of DUX4 expression upon differentiation. GAPDH and GUSB were used as reference genes, n indicates number of samples, error bars display SD, and significance was calculated using a 2-tailed Student t-test. ( C ) Quantitative mRNA analysis showed robust ZSCAN4 activation upon myogenic differentiation in FSHD myotubes. GAPDH and GUSB were used as reference genes, n indicates number of samples, error bars display SD, and significance was calculated using a 2-tailed Student t-test. ( D ) Western blot analysis showing reduced levels of SMCHD1 upon muscle cell differentiation in a primary muscle cell culture derived from a control individual. Myoblasts (B) were differentiated into myotubes (T) for 48 h. H3 serves as a control for equal protein loading, α-ACTIN serves as a control for the induction of myogenic differentiation. ( E ) Western blot analysis of a control derived primary fibroblast undergoing forced myogenesis by ectopic MyoD expression for 48, 72, or 94 h showed a decrease in SMCHD1 protein expression. TUBULIN serves as a loading control. ( F ) Duplicate RT-PCR analysis of DUX4 expression upon ectopic MyoD expression in a Control 1, FSHD1 1 or FSHD2 1 fibroblast (Table S2) revealed DUX4 activation in patient cells exclusively. Ectopic GFP expression was used as a control and GUSB serves as an internal PCR control. ( G , H ) Normalized ChIP qPCR analysis of SMCHD1 binding at D4Z4 in isogenic fibroblasts (F), myoblasts (B), and myotubes (T) derived from the same control individual (panel G) and 3 independent control derived myoblast – myotube pairs showed the highest SMCHD1 abundance in myoblasts with a further decrease during myogenesis. Error bars display SD in panel G and H and significance was calculated using a 2-tailed Student's test. NS = not significant; * = P
    Figure Legend Snippet: DUX4 activation during myogenic differentiation coincides with SMCHD1 reduction. ( A) Immunofluorescence microscopy analysis confirmed the typical DUX4 protein expression pattern in myosin positive, multinucleated myotubes derived from FSHD1 and FSHD2 individuals. Images were taken using a 200x magnification, scale bars are displayed. ( B ) Quantitative mRNA analysis in control ( C ), FSHD1 (F1), and FSHD2 (F2) primary myoblasts (B) and myotubes (T) showing increased levels of DUX4 expression upon differentiation. GAPDH and GUSB were used as reference genes, n indicates number of samples, error bars display SD, and significance was calculated using a 2-tailed Student t-test. ( C ) Quantitative mRNA analysis showed robust ZSCAN4 activation upon myogenic differentiation in FSHD myotubes. GAPDH and GUSB were used as reference genes, n indicates number of samples, error bars display SD, and significance was calculated using a 2-tailed Student t-test. ( D ) Western blot analysis showing reduced levels of SMCHD1 upon muscle cell differentiation in a primary muscle cell culture derived from a control individual. Myoblasts (B) were differentiated into myotubes (T) for 48 h. H3 serves as a control for equal protein loading, α-ACTIN serves as a control for the induction of myogenic differentiation. ( E ) Western blot analysis of a control derived primary fibroblast undergoing forced myogenesis by ectopic MyoD expression for 48, 72, or 94 h showed a decrease in SMCHD1 protein expression. TUBULIN serves as a loading control. ( F ) Duplicate RT-PCR analysis of DUX4 expression upon ectopic MyoD expression in a Control 1, FSHD1 1 or FSHD2 1 fibroblast (Table S2) revealed DUX4 activation in patient cells exclusively. Ectopic GFP expression was used as a control and GUSB serves as an internal PCR control. ( G , H ) Normalized ChIP qPCR analysis of SMCHD1 binding at D4Z4 in isogenic fibroblasts (F), myoblasts (B), and myotubes (T) derived from the same control individual (panel G) and 3 independent control derived myoblast – myotube pairs showed the highest SMCHD1 abundance in myoblasts with a further decrease during myogenesis. Error bars display SD in panel G and H and significance was calculated using a 2-tailed Student's test. NS = not significant; * = P

    Techniques Used: Polyacrylamide Gel Electrophoresis, Activation Assay, Immunofluorescence, Microscopy, Expressing, Derivative Assay, Western Blot, Cell Differentiation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    SMCHD1 depletion at D4Z4 leads to increased H3K27me3 and PRC2 binding. ( A ) ChIP-qPCR analysis showed a statistically significant reduction of SMCHD1 at qD4Z4 upon its lentiviral knockdown in control myotubes with 2 independent shRNA constructs in 2 independent control myotube cultures. ( B ) ChIP-qPCR analysis of H3K27me3 at qD4Z4 upon SMCHD1 depletion showed a significant increase at qD4Z4. Enrichment values were normalized to H3 enrichment values. ( C ) Normalized ChIP-qPCR analysis of SUZ12 upon SMCHD1 depletion showed a statistically significant increase at qD4Z4 in 2 independent experiments performed on 2 control myoblast cultures. n indicates sample size , error bars indicate SD, and significance was tested with Student t-test. ( D ) ChIP-qPCR analysis of H3K27me3 at qD4Z4 showed a significant increase in FSHD2 myotubes compared to controls. Enrichment values were normalized to H3 enrichment values. ( E ) ChIP-qPCR analysis of SUZ12abundance showed a significant increase in FSHD2 myotubes at qD4Z4. On panel A, B, D, and E, n indicates sample size, error bars display SD, and significance was tested using a one way-ANOVA followed by Bonferroni multiple comparison test. *= P
    Figure Legend Snippet: SMCHD1 depletion at D4Z4 leads to increased H3K27me3 and PRC2 binding. ( A ) ChIP-qPCR analysis showed a statistically significant reduction of SMCHD1 at qD4Z4 upon its lentiviral knockdown in control myotubes with 2 independent shRNA constructs in 2 independent control myotube cultures. ( B ) ChIP-qPCR analysis of H3K27me3 at qD4Z4 upon SMCHD1 depletion showed a significant increase at qD4Z4. Enrichment values were normalized to H3 enrichment values. ( C ) Normalized ChIP-qPCR analysis of SUZ12 upon SMCHD1 depletion showed a statistically significant increase at qD4Z4 in 2 independent experiments performed on 2 control myoblast cultures. n indicates sample size , error bars indicate SD, and significance was tested with Student t-test. ( D ) ChIP-qPCR analysis of H3K27me3 at qD4Z4 showed a significant increase in FSHD2 myotubes compared to controls. Enrichment values were normalized to H3 enrichment values. ( E ) ChIP-qPCR analysis of SUZ12abundance showed a significant increase in FSHD2 myotubes at qD4Z4. On panel A, B, D, and E, n indicates sample size, error bars display SD, and significance was tested using a one way-ANOVA followed by Bonferroni multiple comparison test. *= P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, shRNA, Construct

    Treatment of FSHD2 myotube cultures with EZH2 inhibitor GSK126 increases DUX4 levels. ( A ) Western blot analysis of control, FSHD1, and FSHD2 myotube samples after treatment with 0, 1, 2, and 4 μM GSK126. Blots were probed with H3K27me3 antibody and H3 antibody as loading control. Shown are the reduced ratios of H3K27me3:H3 signal intensities in samples treated with GSK126 compared to the untreated sample as a result of EZH2 inhibition. ( B ) qRT-PCR analysis of DUX4 expression in FSHD1 and FSHD2 myotubes after GSK126 treatment shows that relative DUX4 transcript levels are significantly increased in FSHD2 samples treated with 2 μM GSK126 but not in FSHD1 myotubes. Graph shows the results of 4 FSHD1 and 4 FSHD2 cell lines. Results of control cell lines are not shown, DUX4 transcript was not detectable. Error bars show SD, n indicates number of independent cell lines, and significance was tested by 2-way ANOVA followed by Bonferroni multiple comparison test. *= P
    Figure Legend Snippet: Treatment of FSHD2 myotube cultures with EZH2 inhibitor GSK126 increases DUX4 levels. ( A ) Western blot analysis of control, FSHD1, and FSHD2 myotube samples after treatment with 0, 1, 2, and 4 μM GSK126. Blots were probed with H3K27me3 antibody and H3 antibody as loading control. Shown are the reduced ratios of H3K27me3:H3 signal intensities in samples treated with GSK126 compared to the untreated sample as a result of EZH2 inhibition. ( B ) qRT-PCR analysis of DUX4 expression in FSHD1 and FSHD2 myotubes after GSK126 treatment shows that relative DUX4 transcript levels are significantly increased in FSHD2 samples treated with 2 μM GSK126 but not in FSHD1 myotubes. Graph shows the results of 4 FSHD1 and 4 FSHD2 cell lines. Results of control cell lines are not shown, DUX4 transcript was not detectable. Error bars show SD, n indicates number of independent cell lines, and significance was tested by 2-way ANOVA followed by Bonferroni multiple comparison test. *= P

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

    SMCHD1, but not SUV39H1 and Cohesin, regulates DUX4 expression. Western blot confirmation of ( A ) SMCHD1, ( B ) SUV39H1, ( D ) RAD21, and E ) SMC3 knockdown in control myotubes expressing the indicated lentiviral transduced shRNAs. Tubulin was used as a loading control. Representative blots of at least duplicate experiments are shown. ( C , F ) Standard gel electrophoresis analysis of DUX4 expression upon lentiviral knockdown of SMCHD1, SUV39H1, RAD21 and SMC3 in control myotubes. Only depletion of SMCHD1 resulted in reproducible activation of DUX4 transcription. Representative gel photos are shown of at least duplicate experiments. The PCR fragment visible in one RAD21 knockdown was sequenced and shown to be the product of an a-specific amplification. ( G ) Western blot analysis confirms a 2-3 fold increase of SMCHD1 expression upon its lentiviral transduction in 2 FSHD1 and 1 FSHD2 myotube cultures. GFP transduced myotubes served as a negative control. Numbers indicate normalized relative expression levels of SMCHD1 using Tubulin as a loading control followed by setting normalized SMCHD1 levels of GFP transduced samples (only expressing endogenous SMCHD1) to 1. ( H ) Expression levels of DUX4 were significantly reduced upon ectopic expression of SMCHD1 in 2 FSHD1 and 2 FSHD2 myotube cultures. Relative DUX4 expression was calculated for each sample by normalization to GUSB and GAPDH housekeeping genes. Bars show values of each samples adjusted to the expression value of GFP transduced sample as 1. ( I ) Expression levels of the DUX4 target genes RFPL2, ZSCAN4, and TRIM43 showed a significant reduction upon ectopic expression of SMCHD1 in 2 FSHD1 and 2 FSHD2 myotubes, concordant with decreased DUX4 protein expression. Expression levels were normalized as described for panel Figure 1A . For panel H, I: Error bars display SD and significance was calculated using a 2-tailed Student's t-test. All P
    Figure Legend Snippet: SMCHD1, but not SUV39H1 and Cohesin, regulates DUX4 expression. Western blot confirmation of ( A ) SMCHD1, ( B ) SUV39H1, ( D ) RAD21, and E ) SMC3 knockdown in control myotubes expressing the indicated lentiviral transduced shRNAs. Tubulin was used as a loading control. Representative blots of at least duplicate experiments are shown. ( C , F ) Standard gel electrophoresis analysis of DUX4 expression upon lentiviral knockdown of SMCHD1, SUV39H1, RAD21 and SMC3 in control myotubes. Only depletion of SMCHD1 resulted in reproducible activation of DUX4 transcription. Representative gel photos are shown of at least duplicate experiments. The PCR fragment visible in one RAD21 knockdown was sequenced and shown to be the product of an a-specific amplification. ( G ) Western blot analysis confirms a 2-3 fold increase of SMCHD1 expression upon its lentiviral transduction in 2 FSHD1 and 1 FSHD2 myotube cultures. GFP transduced myotubes served as a negative control. Numbers indicate normalized relative expression levels of SMCHD1 using Tubulin as a loading control followed by setting normalized SMCHD1 levels of GFP transduced samples (only expressing endogenous SMCHD1) to 1. ( H ) Expression levels of DUX4 were significantly reduced upon ectopic expression of SMCHD1 in 2 FSHD1 and 2 FSHD2 myotube cultures. Relative DUX4 expression was calculated for each sample by normalization to GUSB and GAPDH housekeeping genes. Bars show values of each samples adjusted to the expression value of GFP transduced sample as 1. ( I ) Expression levels of the DUX4 target genes RFPL2, ZSCAN4, and TRIM43 showed a significant reduction upon ectopic expression of SMCHD1 in 2 FSHD1 and 2 FSHD2 myotubes, concordant with decreased DUX4 protein expression. Expression levels were normalized as described for panel Figure 1A . For panel H, I: Error bars display SD and significance was calculated using a 2-tailed Student's t-test. All P

    Techniques Used: Polyacrylamide Gel Electrophoresis, Expressing, Western Blot, Nucleic Acid Electrophoresis, Activation Assay, Polymerase Chain Reaction, Amplification, Transduction, Negative Control

    7) Product Images from "Poly(I:C) and CpG-ODN combined aerosolization to treat lung metastases and counter the immunosuppressive microenvironment"

    Article Title: Poly(I:C) and CpG-ODN combined aerosolization to treat lung metastases and counter the immunosuppressive microenvironment

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2015.1040214

    Up-modulation of NKG2D ligand expression on dacarbazine-treated B16 melanoma cells. ( A ) MULT1 and RAE1 expression on the cell surface of B16 melanoma cells analyzed by flow cytometry after 24-h culture in complete medium alone (PBS) or supplemented with
    Figure Legend Snippet: Up-modulation of NKG2D ligand expression on dacarbazine-treated B16 melanoma cells. ( A ) MULT1 and RAE1 expression on the cell surface of B16 melanoma cells analyzed by flow cytometry after 24-h culture in complete medium alone (PBS) or supplemented with

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    Antitumor activity of aerosol CpG-ODN/Poly(I:C) combined with dacarbazine (DTIC) on B16 experimental lung metastases. Number of macroscopic B16 melanoma lung metastases in mice untreated (7 mice) or treated with aerosol CpG-ODN/Poly(I:C) (8 mice), DTIC
    Figure Legend Snippet: Antitumor activity of aerosol CpG-ODN/Poly(I:C) combined with dacarbazine (DTIC) on B16 experimental lung metastases. Number of macroscopic B16 melanoma lung metastases in mice untreated (7 mice) or treated with aerosol CpG-ODN/Poly(I:C) (8 mice), DTIC

    Techniques Used: Activity Assay, Mouse Assay

    8) Product Images from "Nexilin/NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP"

    Article Title: Nexilin/NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31328-2

    Nexilin/ NEXN expression is controlled by YAP and by myocardin family coactivators. Panel A shows phosphorylation of YAP (P-YAP) in human coronary artery SMCs (HCASMCs) prior to and at different times after addition of sphingosine-1-phosphate (S1P). Panel B shows compiled data on YAP phosphorylation (N = 9). In panel C, qRT-PCR for NEXN and CTGF at different times after S1P addition is shown (N = 9). Panel D shows NEXN mRNA expression following adenoviral transduction of YAP1 and S1P treatment, individually and in combination (N = 11). Panel E shows the effect of dual silencing of YAP( YAP1) and TAZ ( WWTR1 ) on NEXN mRNA expression under basal conditions and after S1P stimulation of human bladder SMCs (HBSMCs) (N = 9). Panel F shows the mRNA level of NEXN after overexpression of MRTF-A/ MKL1 and YAP1 , alone and in combination (N = 9). Panel G shows the mRNA level of NEXN in control conditions and following overexpression of MRTF-A/ MKL1 , MRTF-B/ MKL2 and myocardin/ MYOCD (HBSMCs, N = 6–8; HCASMCs, N = 6). Panel H shows western blots for Nexilin following overexpression of MRTFs in bladder (top) and coronary artery (bottom) SMCs, and panel I shows compiled protein data (N = 9). Panel J shows the mRNA level for SRF following silencing of SRF , YAP/TAZ, or both. Panel K shows the associated reduction of the NEXN mRNA (N = 6). Panel L shows the effect of pharmacological inhibition of MRTF/SRF signaling in bladder and coronary artery SMCs (HBSMCs, N = 12; HCASMCs, N = 9).
    Figure Legend Snippet: Nexilin/ NEXN expression is controlled by YAP and by myocardin family coactivators. Panel A shows phosphorylation of YAP (P-YAP) in human coronary artery SMCs (HCASMCs) prior to and at different times after addition of sphingosine-1-phosphate (S1P). Panel B shows compiled data on YAP phosphorylation (N = 9). In panel C, qRT-PCR for NEXN and CTGF at different times after S1P addition is shown (N = 9). Panel D shows NEXN mRNA expression following adenoviral transduction of YAP1 and S1P treatment, individually and in combination (N = 11). Panel E shows the effect of dual silencing of YAP( YAP1) and TAZ ( WWTR1 ) on NEXN mRNA expression under basal conditions and after S1P stimulation of human bladder SMCs (HBSMCs) (N = 9). Panel F shows the mRNA level of NEXN after overexpression of MRTF-A/ MKL1 and YAP1 , alone and in combination (N = 9). Panel G shows the mRNA level of NEXN in control conditions and following overexpression of MRTF-A/ MKL1 , MRTF-B/ MKL2 and myocardin/ MYOCD (HBSMCs, N = 6–8; HCASMCs, N = 6). Panel H shows western blots for Nexilin following overexpression of MRTFs in bladder (top) and coronary artery (bottom) SMCs, and panel I shows compiled protein data (N = 9). Panel J shows the mRNA level for SRF following silencing of SRF , YAP/TAZ, or both. Panel K shows the associated reduction of the NEXN mRNA (N = 6). Panel L shows the effect of pharmacological inhibition of MRTF/SRF signaling in bladder and coronary artery SMCs (HBSMCs, N = 12; HCASMCs, N = 9).

    Techniques Used: Expressing, Quantitative RT-PCR, Transduction, Over Expression, Western Blot, Inhibition

    Knockdown of Nexilin/ NEXN reduces actin polymerization and SMC marker expression. NEXN was silenced using a short hairpin adenoviral construct (sh-NEXN) and the contents of NEXN and a number of SMC differentiation markers were examined at the mRNA (panel A, N = 6–9) and protein levels (panels B and C, N = 12). Panel D shows that depolymerization of actin using LatB reduces SRF expression in both human bladder and coronary artery SMCs (HBSMCs, N = 8; HCASMCs, N = 9). Panel E shows phalloidin staining of actin filaments in control cell (Null) and after NEXN silencing (sh-NEXN). The scale bar represents 50 μm. Panel F shows a sedimentation assay to determine filamentous (F-) and globular (G-) actin in control cells (null) and after silencing of NEXN . Panel G shows the normalized F- to G-actin ratio in bladder and coronary artery SMCs (HBSMCs, N = 12; HCASMCs, N = 6). Panel H shows that polymerization of actin using jasplakinolide (Jasp) increases MYH11 in HBSMCs in control conditions and after NEXN silencing (N = 6). Panel I shows cell density at different times following the creation of a cell-free area in the culture dish (N = 9–10). Panel J shows the speed of cell movement within the cell-free area (N = 19 and 22 motile cells in Null and sh- NEXN , respectively). Panel K shows a cell viability assay comparing control and NEXN -silenced cells (N = 12 throughout). The effect of NEXN silencing on cell migration was confirmed using an independent siRNA in L (N = 9). The associated repression of the NEXN protein is shown in ( M ) (N = 6). The schematic illustration in panel N summarizes our findings regarding the transcriptional control of NEXN and its impact on actin polymerization and cell motility. Several aspects of this model, including the involvement of a ( G ) protein-coupled receptor in the S1P effect, were not directly tested here, and they are thus drawn in grey.
    Figure Legend Snippet: Knockdown of Nexilin/ NEXN reduces actin polymerization and SMC marker expression. NEXN was silenced using a short hairpin adenoviral construct (sh-NEXN) and the contents of NEXN and a number of SMC differentiation markers were examined at the mRNA (panel A, N = 6–9) and protein levels (panels B and C, N = 12). Panel D shows that depolymerization of actin using LatB reduces SRF expression in both human bladder and coronary artery SMCs (HBSMCs, N = 8; HCASMCs, N = 9). Panel E shows phalloidin staining of actin filaments in control cell (Null) and after NEXN silencing (sh-NEXN). The scale bar represents 50 μm. Panel F shows a sedimentation assay to determine filamentous (F-) and globular (G-) actin in control cells (null) and after silencing of NEXN . Panel G shows the normalized F- to G-actin ratio in bladder and coronary artery SMCs (HBSMCs, N = 12; HCASMCs, N = 6). Panel H shows that polymerization of actin using jasplakinolide (Jasp) increases MYH11 in HBSMCs in control conditions and after NEXN silencing (N = 6). Panel I shows cell density at different times following the creation of a cell-free area in the culture dish (N = 9–10). Panel J shows the speed of cell movement within the cell-free area (N = 19 and 22 motile cells in Null and sh- NEXN , respectively). Panel K shows a cell viability assay comparing control and NEXN -silenced cells (N = 12 throughout). The effect of NEXN silencing on cell migration was confirmed using an independent siRNA in L (N = 9). The associated repression of the NEXN protein is shown in ( M ) (N = 6). The schematic illustration in panel N summarizes our findings regarding the transcriptional control of NEXN and its impact on actin polymerization and cell motility. Several aspects of this model, including the involvement of a ( G ) protein-coupled receptor in the S1P effect, were not directly tested here, and they are thus drawn in grey.

    Techniques Used: Marker, Expressing, Construct, Staining, Sedimentation, Viability Assay, Migration

    NEXN correlates with gene products that control and respond to changes in actin polymerization and Nexilin is reduced by depolymerization of actin. Correlations of NEXN versus all other RNAs in the top-ten NEXN expressing tissues were examined (data from GTExPortal). The sum of correlation coefficients for individual RNAs across tissues was calculated (R sum ) and the positive extreme of this distribution was plotted ( A ). Actin controlling and responding gene products represented in the extreme are highlighted in blue colors. Examples of NEXN correlations in the human coronary artery (N = 133) are shown in panels B through ( D ). P-values and Spearman Rho values are given in the respective panels. Panels E and F show mRNA data for NEXN in cultured human bladder (HBSMCs, N = 8) and coronary artery (HCASMCs, N = 9) SMCs after treatment with Latrunculin B (LatB). Panels H and I show protein data for Nexilin/ NEXN in the presence and absence of LatB (HBSMCs, 300 nM, N = 12; HCASMCs, 100 nM, N = 10). The top micrographs in panel J shows confocal imaging of YAP (red) on the left, and YAP (green) and CAV1 (red) on the right. The bottom row shows YAP (red) and Nexilin (green). The high magnification overlay at the bottom right shows partial colocalization of YAP and Nexilin at the cell membrane in yellow. All micrographs are from cross-sectioned HBSMCs and white scale bars represent 5 μm throughout.
    Figure Legend Snippet: NEXN correlates with gene products that control and respond to changes in actin polymerization and Nexilin is reduced by depolymerization of actin. Correlations of NEXN versus all other RNAs in the top-ten NEXN expressing tissues were examined (data from GTExPortal). The sum of correlation coefficients for individual RNAs across tissues was calculated (R sum ) and the positive extreme of this distribution was plotted ( A ). Actin controlling and responding gene products represented in the extreme are highlighted in blue colors. Examples of NEXN correlations in the human coronary artery (N = 133) are shown in panels B through ( D ). P-values and Spearman Rho values are given in the respective panels. Panels E and F show mRNA data for NEXN in cultured human bladder (HBSMCs, N = 8) and coronary artery (HCASMCs, N = 9) SMCs after treatment with Latrunculin B (LatB). Panels H and I show protein data for Nexilin/ NEXN in the presence and absence of LatB (HBSMCs, 300 nM, N = 12; HCASMCs, 100 nM, N = 10). The top micrographs in panel J shows confocal imaging of YAP (red) on the left, and YAP (green) and CAV1 (red) on the right. The bottom row shows YAP (red) and Nexilin (green). The high magnification overlay at the bottom right shows partial colocalization of YAP and Nexilin at the cell membrane in yellow. All micrographs are from cross-sectioned HBSMCs and white scale bars represent 5 μm throughout.

    Techniques Used: Expressing, Cell Culture, Imaging

    9) Product Images from "Dual Effect of Chrysanthemum indicum Extract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and Resorption In Vitro"

    Article Title: Dual Effect of Chrysanthemum indicum Extract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and Resorption In Vitro

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2014/176049

    CIE downregulates RANKL-induced early signals and marker genes during osteoclastogenesis. (a) BMMs were pretreated with DMSO (control) or CIE (50 μ g/mL) for 1 h in the presence of M-CSF (30 ng/mL) and were stimulated with RANKL (100 ng/mL) for the indicated times. Whole-cell lysates were used for western blot analysis with the specified antibodies. β -Actin served as the internal control. (b) BMMs were stimulated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of CIE (50 μ g/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent and the mRNA expression levels of OSCAR, TRAP, integrin α v, β 3, DC-STAMP, OC-STAMP, cathepsin K, and ICAM-1 were evaluated by real-time PCR. * P
    Figure Legend Snippet: CIE downregulates RANKL-induced early signals and marker genes during osteoclastogenesis. (a) BMMs were pretreated with DMSO (control) or CIE (50 μ g/mL) for 1 h in the presence of M-CSF (30 ng/mL) and were stimulated with RANKL (100 ng/mL) for the indicated times. Whole-cell lysates were used for western blot analysis with the specified antibodies. β -Actin served as the internal control. (b) BMMs were stimulated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of CIE (50 μ g/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent and the mRNA expression levels of OSCAR, TRAP, integrin α v, β 3, DC-STAMP, OC-STAMP, cathepsin K, and ICAM-1 were evaluated by real-time PCR. * P

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

    CIE promotes ascorbic acid and β -glycerol phosphate mediated osteoblast differentiation. (a) Primary osteoblasts were treated with various concentrations of CIE for 7 days in the presence of 50 μ g/mL ascorbic acid and 10 mM β -glycerol phosphate. ALP-positive cells were stained with ALP solution. (b) Primary osteoblasts were treated with various concentrations of CIE for 21 days. Calcium accumulation within the osteoblasts was stained with ARS solution. Stained calcium deposits were dissolved by 10% CPC buffer to measure the level of staining. (c) Primary osteoblasts were stimulated with ascorbic acid (50 μ g/mL) and β -glycerol phosphate in the presence of CIE (50 μ g/mL) or DMSO. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of Runx2, ALP, Col1 α , and OPN were evaluated by real-time PCR. * P
    Figure Legend Snippet: CIE promotes ascorbic acid and β -glycerol phosphate mediated osteoblast differentiation. (a) Primary osteoblasts were treated with various concentrations of CIE for 7 days in the presence of 50 μ g/mL ascorbic acid and 10 mM β -glycerol phosphate. ALP-positive cells were stained with ALP solution. (b) Primary osteoblasts were treated with various concentrations of CIE for 21 days. Calcium accumulation within the osteoblasts was stained with ARS solution. Stained calcium deposits were dissolved by 10% CPC buffer to measure the level of staining. (c) Primary osteoblasts were stimulated with ascorbic acid (50 μ g/mL) and β -glycerol phosphate in the presence of CIE (50 μ g/mL) or DMSO. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of Runx2, ALP, Col1 α , and OPN were evaluated by real-time PCR. * P

    Techniques Used: ALP Assay, Staining, Isolation, Expressing, Real-time Polymerase Chain Reaction

    CIE suppresses RANKL-induced c-Fos and NFATc1 expression. (a) Effects of CIE on levels of c-Fos and NFATc1 protein expression were evaluated using western blot analysis. β -Actin was used as the internal control. (b) BMMs were stimulated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of CIE (50 μ g/mL) for the specified times. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of c-Fos and NFATc1 were evaluated using real-time PCR. * P
    Figure Legend Snippet: CIE suppresses RANKL-induced c-Fos and NFATc1 expression. (a) Effects of CIE on levels of c-Fos and NFATc1 protein expression were evaluated using western blot analysis. β -Actin was used as the internal control. (b) BMMs were stimulated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of CIE (50 μ g/mL) for the specified times. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of c-Fos and NFATc1 were evaluated using real-time PCR. * P

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

    10) Product Images from "Efficient and Simple Production of Insulin-Producing Cells from Embryonal Carcinoma Stem Cells Using Mouse Neonate Pancreas Extract, As a Natural Inducer"

    Article Title: Efficient and Simple Production of Insulin-Producing Cells from Embryonal Carcinoma Stem Cells Using Mouse Neonate Pancreas Extract, As a Natural Inducer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0090885

    Quantitative analysis of genes involved in differentiation of pancreatic cells derived from P19 EC cells. The cells were cultured as EBs in different concentrations of MPE (mouse pancreas extract). (A) Relative gene expression of PDX-1, INS1, and INS2 in different concentration of MPE. (B) Comparison of expression of Oct3/4, Sox-2, Nanog (undifferentiated stem cell markers), and EP300, CREB1 (differentiated stem cell transcription factors) between P19 EC (embryonal carcinoma) cells and IPCs (insulin-producing cells). The data are expressed as relative gene expression to β-2M and are presented as mean±SD. The means with different letters are significantly different at P = 0.05.
    Figure Legend Snippet: Quantitative analysis of genes involved in differentiation of pancreatic cells derived from P19 EC cells. The cells were cultured as EBs in different concentrations of MPE (mouse pancreas extract). (A) Relative gene expression of PDX-1, INS1, and INS2 in different concentration of MPE. (B) Comparison of expression of Oct3/4, Sox-2, Nanog (undifferentiated stem cell markers), and EP300, CREB1 (differentiated stem cell transcription factors) between P19 EC (embryonal carcinoma) cells and IPCs (insulin-producing cells). The data are expressed as relative gene expression to β-2M and are presented as mean±SD. The means with different letters are significantly different at P = 0.05.

    Techniques Used: Derivative Assay, Cell Culture, Expressing, Concentration Assay

    Determination of secreted (A) and secreted versus intracellular (B) insulin in P19 undifferentiated EC cells (P19), spontaneous differentiated EBs (EB) and the MPE-treated cells. Significant insulin concentration was observed in MPE–treated IPCs. To normalize the amount of insulin secretion, the total protein of the cells in each well was measured by the Bradford method. The experiment was performed in triplicate. Each value represents mean ± SD.
    Figure Legend Snippet: Determination of secreted (A) and secreted versus intracellular (B) insulin in P19 undifferentiated EC cells (P19), spontaneous differentiated EBs (EB) and the MPE-treated cells. Significant insulin concentration was observed in MPE–treated IPCs. To normalize the amount of insulin secretion, the total protein of the cells in each well was measured by the Bradford method. The experiment was performed in triplicate. Each value represents mean ± SD.

    Techniques Used: Concentration Assay

    Fluorescence micrographs illustrating the expression of pancreatic β cell markers. Staining of IPCs with antibodies against proinsulin+insulin (A) and insulin receptor beta (C) showing that most of the P19 cells treated with MPE express pancreatic β cell markers. (E) Control for immunostaining, the primary antibody was omitted. B, D F are phase contrast images of the same field shown in A, C E respectively. Scale bars: 40 µm.
    Figure Legend Snippet: Fluorescence micrographs illustrating the expression of pancreatic β cell markers. Staining of IPCs with antibodies against proinsulin+insulin (A) and insulin receptor beta (C) showing that most of the P19 cells treated with MPE express pancreatic β cell markers. (E) Control for immunostaining, the primary antibody was omitted. B, D F are phase contrast images of the same field shown in A, C E respectively. Scale bars: 40 µm.

    Techniques Used: Fluorescence, Expressing, Staining, Immunostaining

    DTZ staining of differentiated IPCs, derived from P19 EC cells. IPCs formed pancreatic islet-like structures (A). A cell cluster distinctly stained crimson red by DTZ is apparent (B). Individual cells are DTZ-positive in spontaneous differentiated EBs (C). Untreated EC cells are not stained (D). Scale bars: A 100 µm, B, C D 200 µm.
    Figure Legend Snippet: DTZ staining of differentiated IPCs, derived from P19 EC cells. IPCs formed pancreatic islet-like structures (A). A cell cluster distinctly stained crimson red by DTZ is apparent (B). Individual cells are DTZ-positive in spontaneous differentiated EBs (C). Untreated EC cells are not stained (D). Scale bars: A 100 µm, B, C D 200 µm.

    Techniques Used: Staining, Derivative Assay

    11) Product Images from "Combining nitric oxide release with anti-inflammatory activity preserves nigrostriatal dopaminergic innervation and prevents motor impairment in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease"

    Article Title: Combining nitric oxide release with anti-inflammatory activity preserves nigrostriatal dopaminergic innervation and prevents motor impairment in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-7-83

    HCT1026 inhibits MPTP-induced loss of striatal TH and DAT mRNAs expression . Ageing (9-11 month-old) C57Bl/6 mice fed with a control (ct) or HCT1026 diets (30 mg kg -1 ) starting at -10 d, underwent an MPTP treatment according to the subchronic injection paradigm, as described. Age-matched mice fed with the different diets received physiologic saline and served as controls. Mice were sacrificed at different time-intervals after MPTP. Striatal tissue samples were processed for semi-quantitative RT-PCR analysis as described. Total RNA isolated and cDNA synthesized using Retroscript Kit (see Materials and Methods) following the manufacturer's directions. The 250 ng of cDNA were used for PCR, by using Super Taq DNA polymerase with specific primer pairs for TH (620 bp) and DAT (328 bp), and Classic S18 Standard (495 bp). Samples from PCR reactions were separated electrophoretically on 2% agarose gel containing 0,2 μg/ml of ethidium bromide (B-D, F-H). Fluorescent bands of amplified gene products were captured by using Gel Logic 200 Imaging System (Kodak), values normalized against S18 and ratios expressed as percent of control, within each experimental group (A, E). Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p
    Figure Legend Snippet: HCT1026 inhibits MPTP-induced loss of striatal TH and DAT mRNAs expression . Ageing (9-11 month-old) C57Bl/6 mice fed with a control (ct) or HCT1026 diets (30 mg kg -1 ) starting at -10 d, underwent an MPTP treatment according to the subchronic injection paradigm, as described. Age-matched mice fed with the different diets received physiologic saline and served as controls. Mice were sacrificed at different time-intervals after MPTP. Striatal tissue samples were processed for semi-quantitative RT-PCR analysis as described. Total RNA isolated and cDNA synthesized using Retroscript Kit (see Materials and Methods) following the manufacturer's directions. The 250 ng of cDNA were used for PCR, by using Super Taq DNA polymerase with specific primer pairs for TH (620 bp) and DAT (328 bp), and Classic S18 Standard (495 bp). Samples from PCR reactions were separated electrophoretically on 2% agarose gel containing 0,2 μg/ml of ethidium bromide (B-D, F-H). Fluorescent bands of amplified gene products were captured by using Gel Logic 200 Imaging System (Kodak), values normalized against S18 and ratios expressed as percent of control, within each experimental group (A, E). Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p

    Techniques Used: Expressing, Mouse Assay, Injection, Quantitative RT-PCR, Isolation, Synthesized, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Imaging

    12) Product Images from "Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines"

    Article Title: Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines

    Journal: BMC Research Notes

    doi: 10.1186/s13104-017-2959-0

    Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p
    Figure Legend Snippet: Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Synthesized, Expressing, Construct, Activated Clotting Time Assay

    Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method
    Figure Legend Snippet: Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method

    Techniques Used: Isolation, Purification

    13) Product Images from "SP-A2 contributes to miRNA-mediated sex differences in response to oxidative stress: pro-inflammatory, anti-apoptotic, and anti-oxidant pathways are involved"

    Article Title: SP-A2 contributes to miRNA-mediated sex differences in response to oxidative stress: pro-inflammatory, anti-apoptotic, and anti-oxidant pathways are involved

    Journal: Biology of Sex Differences

    doi: 10.1186/s13293-017-0158-2

    Effect of O 3 in males. a mRNA levels of GAPDH, SOD2, CAT, IL-6, STAT3, BCL2, FOXO1, FOXO3, BECN1, IKKβ, and NF-kB-p65 genes were measured in AM of male SP-A2 mice 4 h after O 3 exposure. mRNA levels were measured by qRT-PCR and normalized to GAPDH. STAT3 mRNA levels were significantly increased by 5-fold ( p
    Figure Legend Snippet: Effect of O 3 in males. a mRNA levels of GAPDH, SOD2, CAT, IL-6, STAT3, BCL2, FOXO1, FOXO3, BECN1, IKKβ, and NF-kB-p65 genes were measured in AM of male SP-A2 mice 4 h after O 3 exposure. mRNA levels were measured by qRT-PCR and normalized to GAPDH. STAT3 mRNA levels were significantly increased by 5-fold ( p

    Techniques Used: Mouse Assay, Quantitative RT-PCR

    14) Product Images from "Mbd2-CP2c loop drives adult-type globin gene expression and definitive erythropoiesis"

    Article Title: Mbd2-CP2c loop drives adult-type globin gene expression and definitive erythropoiesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky193

    p66α- and Mbd2-dependent degradation of CP2c and CP2b. ( A ) Immunoblot analysis of the CBP proteins expression in differentiating Mbd2 KD, Mbd3 KD and Mbd DKD MEL cells in vitro . ( B ) Quantification of the CBP proteins expression in uninduced cells in (A). n = 2. ( C ) RT-PCR analysis of the expression of CBP mRNAs in the uninduced Mbd2 KD and DKD cells. Values of uninduced cells shown in Supplementary Figure S5C are highlighted. n = 2. ( D ) Immunoblot analysis of CP2c protein expression in 293T cells transiently transfected of the Mbd2, Mbd3, shMbd2 and shMbd3 expression vectors, in combination, in the presence or absence of proteasome inhibitor MG132. ( E ) Immunoblot analysis of the CP2c expression in undifferentiated p66α OE and KD MEL cells in the presence or absence of MG132. (F and G) Immunoblot ( F ) and RT-qPCR ( G ) analyses of the CP2c, CP2b, and Pias1 expression in uninduced p66α OE and KD MEL cells. Values in uninduced cells shown in Supplementary Figures S5F and S5H are highlighted. n = 2.
    Figure Legend Snippet: p66α- and Mbd2-dependent degradation of CP2c and CP2b. ( A ) Immunoblot analysis of the CBP proteins expression in differentiating Mbd2 KD, Mbd3 KD and Mbd DKD MEL cells in vitro . ( B ) Quantification of the CBP proteins expression in uninduced cells in (A). n = 2. ( C ) RT-PCR analysis of the expression of CBP mRNAs in the uninduced Mbd2 KD and DKD cells. Values of uninduced cells shown in Supplementary Figure S5C are highlighted. n = 2. ( D ) Immunoblot analysis of CP2c protein expression in 293T cells transiently transfected of the Mbd2, Mbd3, shMbd2 and shMbd3 expression vectors, in combination, in the presence or absence of proteasome inhibitor MG132. ( E ) Immunoblot analysis of the CP2c expression in undifferentiated p66α OE and KD MEL cells in the presence or absence of MG132. (F and G) Immunoblot ( F ) and RT-qPCR ( G ) analyses of the CP2c, CP2b, and Pias1 expression in uninduced p66α OE and KD MEL cells. Values in uninduced cells shown in Supplementary Figures S5F and S5H are highlighted. n = 2.

    Techniques Used: Expressing, In Vitro, Reverse Transcription Polymerase Chain Reaction, Transfection, Quantitative RT-PCR

    Disruption of the Mbd2-p66α interplay in Mbd2-NuRD shows phenotypes analogous to Mbd2 downregulation. Quantification of the α- and β-globin expression in p66α Δ1 OE MEL cells by Immunoblot ( A ) and RT-PCR ( B ) analyses. n = 2. ( C ) Functional hemoglobin synthesis analysis by benzidine staining. n = 2. Quantification of the CP2c and CP2b expression in the p66α Δ1 OE MEL cells by Immunoblot ( D ) and RT-qPCR ( E ) analyses. D and E are values of the uninduced (d0) and induced (d3) states shown in Supplementary Figure S8B and C , respectively. n = 2. ( F ) Functional hemoglobin synthesis analysis by benzidine staining in undifferentiated Mbd2 KD and p66α Δ1 OE MEL cells, in the presence or absence of the peptides inhibiting the CP2c's DNA binding activity (5C and 5-2C). n = 2. The corresponding immunoblots of CBP proteins are shown in Supplementary Figure S8D .
    Figure Legend Snippet: Disruption of the Mbd2-p66α interplay in Mbd2-NuRD shows phenotypes analogous to Mbd2 downregulation. Quantification of the α- and β-globin expression in p66α Δ1 OE MEL cells by Immunoblot ( A ) and RT-PCR ( B ) analyses. n = 2. ( C ) Functional hemoglobin synthesis analysis by benzidine staining. n = 2. Quantification of the CP2c and CP2b expression in the p66α Δ1 OE MEL cells by Immunoblot ( D ) and RT-qPCR ( E ) analyses. D and E are values of the uninduced (d0) and induced (d3) states shown in Supplementary Figure S8B and C , respectively. n = 2. ( F ) Functional hemoglobin synthesis analysis by benzidine staining in undifferentiated Mbd2 KD and p66α Δ1 OE MEL cells, in the presence or absence of the peptides inhibiting the CP2c's DNA binding activity (5C and 5-2C). n = 2. The corresponding immunoblots of CBP proteins are shown in Supplementary Figure S8D .

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Functional Assay, Staining, Quantitative RT-PCR, Binding Assay, Activity Assay, Western Blot

    15) Product Images from "HER2 signaling regulates the tumor immune microenvironment and trastuzumab efficacy"

    Article Title: HER2 signaling regulates the tumor immune microenvironment and trastuzumab efficacy

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2018.1512942

    Inhibition of the production of CCL2 in HER2+ tumor cells by ER activity. A) Left panel: WB analysis of pHER2, HER2, and ER; right panel: qRT-PCR quantification of PGR mRNA in HER2 + BC cell lines maintained in standard medium conditions. All performed independent experiments are shown. B) NF-kB activity in HER2+ BC cell lines as determined by luciferase assay. All performed independent experiments are shown. C) CCL2 mRNA (left panel) and protein (right panel) quantification by qRT-PCR and ELISA, respectively, in HER2+ BC cell lines. All performed independent experiments are shown. D) WB analysis of pHER2, HER2, and ER in BT474 and ZR75.30 treated with or without 100 nM fulvestrant for 24 h. E-H) PGR mRNA quantification by qRT-PCR ( E ), NF-kB activity by luciferase assay ( F ), CCL2 mRNA quantification by qRT-PCR ( G ), and CCL2 protein quantification by ELISA ( H ) in BT474 and ZR75.30 cells treated with or without fulvestrant as in D. Data are representative of two experiments. *p
    Figure Legend Snippet: Inhibition of the production of CCL2 in HER2+ tumor cells by ER activity. A) Left panel: WB analysis of pHER2, HER2, and ER; right panel: qRT-PCR quantification of PGR mRNA in HER2 + BC cell lines maintained in standard medium conditions. All performed independent experiments are shown. B) NF-kB activity in HER2+ BC cell lines as determined by luciferase assay. All performed independent experiments are shown. C) CCL2 mRNA (left panel) and protein (right panel) quantification by qRT-PCR and ELISA, respectively, in HER2+ BC cell lines. All performed independent experiments are shown. D) WB analysis of pHER2, HER2, and ER in BT474 and ZR75.30 treated with or without 100 nM fulvestrant for 24 h. E-H) PGR mRNA quantification by qRT-PCR ( E ), NF-kB activity by luciferase assay ( F ), CCL2 mRNA quantification by qRT-PCR ( G ), and CCL2 protein quantification by ELISA ( H ) in BT474 and ZR75.30 cells treated with or without fulvestrant as in D. Data are representative of two experiments. *p

    Techniques Used: Inhibition, Activity Assay, Western Blot, Quantitative RT-PCR, Luciferase, Enzyme-linked Immunosorbent Assay

    Changes of chemokine expression levels upon stimulation or blockage of HER2 receptor signaling. A-C) Chemokine mRNA expression in cells as evaluated by qRT-PCR after 6-h treatment with EGF ( A ), HRG ( B ), and trastuzumab ( C ). Data are expressed as fold to untreated cells (dotted lines) and refer to independent experiments in BT474 (●), ZR75.30 (▲), and SKBr3 (■). D) CCL2 release upon stimulation with 20 ng/ml EGF and HRG for 24 h in BT474 (●) and SKBr3 (■) as evaluated by ELISA. Data are expressed as fold to untreated cells (dotted line) and refer to independent experiments. E) CCL2 gene expression in 50 HER2+ BC biopsies belonging to the GSE70360 dataset obtained before (pre) and after (post) treatment with one cycle of trastuzumab alone. p-value by paired t-test. F) Representative expression of HER2 and CCL2 in tumors derived from d16HER2 transgenic mice as evaluated by immunofluorescence. Scale bars: 10 μm. Right panel shows quantification of CCL2 positivity in HER2-positive and HER2-negative areas. Data are the means ± SD (n = 12). p-value by unpaired t-test.
    Figure Legend Snippet: Changes of chemokine expression levels upon stimulation or blockage of HER2 receptor signaling. A-C) Chemokine mRNA expression in cells as evaluated by qRT-PCR after 6-h treatment with EGF ( A ), HRG ( B ), and trastuzumab ( C ). Data are expressed as fold to untreated cells (dotted lines) and refer to independent experiments in BT474 (●), ZR75.30 (▲), and SKBr3 (■). D) CCL2 release upon stimulation with 20 ng/ml EGF and HRG for 24 h in BT474 (●) and SKBr3 (■) as evaluated by ELISA. Data are expressed as fold to untreated cells (dotted line) and refer to independent experiments. E) CCL2 gene expression in 50 HER2+ BC biopsies belonging to the GSE70360 dataset obtained before (pre) and after (post) treatment with one cycle of trastuzumab alone. p-value by paired t-test. F) Representative expression of HER2 and CCL2 in tumors derived from d16HER2 transgenic mice as evaluated by immunofluorescence. Scale bars: 10 μm. Right panel shows quantification of CCL2 positivity in HER2-positive and HER2-negative areas. Data are the means ± SD (n = 12). p-value by unpaired t-test.

    Techniques Used: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay, Transgenic Assay, Mouse Assay, Immunofluorescence

    CCL2 expression is regulated by HER2 signaling in SKBr3 and BT474 cells. A-B) CCL2 mRNA ( A ) and protein ( B ) quantification by qRT-PCR and ELISA, respectively, in SKBr3 cells treated with EGF or HRG and/or with PI3Ki (LY294002), MEKi (UO126), or NF-kBi (BAY 11–7082) inhibitors for 6 h. C ) CCL2 mRNA expression in BT474 cells treated as in A. Data are expressed as fold to EGF/HRG treated cells (dotted lines) and are representative of at least two experiments. *p
    Figure Legend Snippet: CCL2 expression is regulated by HER2 signaling in SKBr3 and BT474 cells. A-B) CCL2 mRNA ( A ) and protein ( B ) quantification by qRT-PCR and ELISA, respectively, in SKBr3 cells treated with EGF or HRG and/or with PI3Ki (LY294002), MEKi (UO126), or NF-kBi (BAY 11–7082) inhibitors for 6 h. C ) CCL2 mRNA expression in BT474 cells treated as in A. Data are expressed as fold to EGF/HRG treated cells (dotted lines) and are representative of at least two experiments. *p

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

    16) Product Images from "Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance"

    Article Title: Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.150342

    Viral deletion of Shh in gustatory neurons has little effect on taste bud number, size or innervation density. (A) Sagittal schematic of the brain depicting stereotaxic injection of the NST (red) in the brainstem (blue). (B) Experimental design for AAV5-Cre injection of Shh CreERT2/flox ; R26R tdTomato (AAV-ShhcKO) mice. (C) AAV5-Cre deletes Shh in gustatory neurons that project to NST and innervate FFP (indicated by the cross). (D-H) AAV5-Cre injection into the NST activates tdTomato + expression in gVII neurons of control (D) and mutant (E) mice, reduces Shh expression in gVII in mutants (F), and reveals tdTomato + nerve fibers (arrows) innervating the anterior tongue of controls (G) and mutants (H). (I-N) FFP taste buds (K8 + , green) in both control (I-K) and AAV-ShhcKO (L-N) mice are innervated by AAV5-tdTomato + neurites (arrows). (O,R) Typical FFP number is not affected by AAV5-ShhcKO (O), whereas atypical FFP numbers increase but not significantly (R). (P-T) Taste bud size (P) and density of tdTomato + neurites (Q) of typical FFP are similar in controls and AAV-ShhcKO mice, whereas taste buds of atypical FFP (S) are larger in mutants, with increased density of tdTomato + neurites (T). Nuclei are counterstained with Draq5 (blue); white dashed lines delimit basement membrane. (D,E,G,H) Images of whole ganglia and tongues. Scale bars: 150 μm in D,E; 1 mm in G,H. n =3 mice per condition. Data are mean±s.d., except Q, which is the median with interquartile range. Results were analyzed using Student's t -test (F,O,P,R-T) or Mann–Whitney U-test (Q). * P ≤0.05, **P
    Figure Legend Snippet: Viral deletion of Shh in gustatory neurons has little effect on taste bud number, size or innervation density. (A) Sagittal schematic of the brain depicting stereotaxic injection of the NST (red) in the brainstem (blue). (B) Experimental design for AAV5-Cre injection of Shh CreERT2/flox ; R26R tdTomato (AAV-ShhcKO) mice. (C) AAV5-Cre deletes Shh in gustatory neurons that project to NST and innervate FFP (indicated by the cross). (D-H) AAV5-Cre injection into the NST activates tdTomato + expression in gVII neurons of control (D) and mutant (E) mice, reduces Shh expression in gVII in mutants (F), and reveals tdTomato + nerve fibers (arrows) innervating the anterior tongue of controls (G) and mutants (H). (I-N) FFP taste buds (K8 + , green) in both control (I-K) and AAV-ShhcKO (L-N) mice are innervated by AAV5-tdTomato + neurites (arrows). (O,R) Typical FFP number is not affected by AAV5-ShhcKO (O), whereas atypical FFP numbers increase but not significantly (R). (P-T) Taste bud size (P) and density of tdTomato + neurites (Q) of typical FFP are similar in controls and AAV-ShhcKO mice, whereas taste buds of atypical FFP (S) are larger in mutants, with increased density of tdTomato + neurites (T). Nuclei are counterstained with Draq5 (blue); white dashed lines delimit basement membrane. (D,E,G,H) Images of whole ganglia and tongues. Scale bars: 150 μm in D,E; 1 mm in G,H. n =3 mice per condition. Data are mean±s.d., except Q, which is the median with interquartile range. Results were analyzed using Student's t -test (F,O,P,R-T) or Mann–Whitney U-test (Q). * P ≤0.05, **P

    Techniques Used: Injection, Mouse Assay, Expressing, Mutagenesis, MANN-WHITNEY

    Genetic deletion of Shh in Shh + cells, including gustatory neurons, minimally affects taste buds. (A) Shh CreERT2/flox ; R26R tdTomato (Shh-ShhcKO) and genetic control mice were given tamoxifen for 4 days and harvested at 35 days. (B) In tamoxifen-treated Shh-ShhcKO mice, Shh is deleted permanently from ganglion cells and nerves (indicated by the cross), but transiently from taste buds (see text). (C,D) tdTomato reports Shh deletion in tongue (C) and gVII (D) of mutant mice. (E) Quantitative PCR reveals that neither Shh nor Gli1 expression is significantly reduced in mutant tongue epithelium. (F) Expression of Shh , but not Gli1, is significantly reduced in mutant gVII. (G,H) Typical FFP number does not differ between mutants and controls (G), although atypical FFP increase in mutants (H). (I,J) The size of taste buds in typical (I) and atypical (J) FFP in mutants does not differ from controls. (K) Shh-tdTomato + neurites (red) innervate a taste bud (K8 + , green) in an atypical FFP in a Shh-ShhcKO mouse (Shh-descendent taste cell, arrow). (L) The proportion of FFP innervated by P2X2 + fibers is not affected by Shh-ShhcKO. (M,N) P2X2 + innervation density of taste buds in typical (M) and atypical (N) FFP does not differ between controls and Shh-ShhcKO mice. Nuclei counterstained with Draq5 (blue); white dashed lines delimit basement membrane; white solid lines delimit epithelial surface. C and D are images of whole tongue and gVII. K is a compressed confocal z -stack. Scale bars: 1 mm in C; 150 μm in D; 10 μm in K. n =3-5 mice per condition. Data are mean±s.d., except E and F, which are mean±s.e.m; I and N represent the median with interquartile range. Results were analyzed using Student's t -test (E-H,J,L,M) or Mann–Whitney U-test (I,N). *** P
    Figure Legend Snippet: Genetic deletion of Shh in Shh + cells, including gustatory neurons, minimally affects taste buds. (A) Shh CreERT2/flox ; R26R tdTomato (Shh-ShhcKO) and genetic control mice were given tamoxifen for 4 days and harvested at 35 days. (B) In tamoxifen-treated Shh-ShhcKO mice, Shh is deleted permanently from ganglion cells and nerves (indicated by the cross), but transiently from taste buds (see text). (C,D) tdTomato reports Shh deletion in tongue (C) and gVII (D) of mutant mice. (E) Quantitative PCR reveals that neither Shh nor Gli1 expression is significantly reduced in mutant tongue epithelium. (F) Expression of Shh , but not Gli1, is significantly reduced in mutant gVII. (G,H) Typical FFP number does not differ between mutants and controls (G), although atypical FFP increase in mutants (H). (I,J) The size of taste buds in typical (I) and atypical (J) FFP in mutants does not differ from controls. (K) Shh-tdTomato + neurites (red) innervate a taste bud (K8 + , green) in an atypical FFP in a Shh-ShhcKO mouse (Shh-descendent taste cell, arrow). (L) The proportion of FFP innervated by P2X2 + fibers is not affected by Shh-ShhcKO. (M,N) P2X2 + innervation density of taste buds in typical (M) and atypical (N) FFP does not differ between controls and Shh-ShhcKO mice. Nuclei counterstained with Draq5 (blue); white dashed lines delimit basement membrane; white solid lines delimit epithelial surface. C and D are images of whole tongue and gVII. K is a compressed confocal z -stack. Scale bars: 1 mm in C; 150 μm in D; 10 μm in K. n =3-5 mice per condition. Data are mean±s.d., except E and F, which are mean±s.e.m; I and N represent the median with interquartile range. Results were analyzed using Student's t -test (E-H,J,L,M) or Mann–Whitney U-test (I,N). *** P

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

    17) Product Images from "Nexilin/NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP"

    Article Title: Nexilin/NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31328-2

    Knockdown of Nexilin/ NEXN reduces actin polymerization and SMC marker expression. NEXN was silenced using a short hairpin adenoviral construct (sh-NEXN) and the contents of NEXN and a number of SMC differentiation markers were examined at the mRNA (panel A, N = 6–9) and protein levels (panels B and C, N = 12). Panel D shows that depolymerization of actin using LatB reduces SRF expression in both human bladder and coronary artery SMCs (HBSMCs, N = 8; HCASMCs, N = 9). Panel E shows phalloidin staining of actin filaments in control cell (Null) and after NEXN silencing (sh-NEXN). The scale bar represents 50 μm. Panel F shows a sedimentation assay to determine filamentous (F-) and globular (G-) actin in control cells (null) and after silencing of NEXN . Panel G shows the normalized F- to G-actin ratio in bladder and coronary artery SMCs (HBSMCs, N = 12; HCASMCs, N = 6). Panel H shows that polymerization of actin using jasplakinolide (Jasp) increases MYH11 in HBSMCs in control conditions and after NEXN silencing (N = 6). Panel I shows cell density at different times following the creation of a cell-free area in the culture dish (N = 9–10). Panel J shows the speed of cell movement within the cell-free area (N = 19 and 22 motile cells in Null and sh- NEXN , respectively). Panel K shows a cell viability assay comparing control and NEXN -silenced cells (N = 12 throughout). The effect of NEXN silencing on cell migration was confirmed using an independent siRNA in L (N = 9). The associated repression of the NEXN protein is shown in ( M ) (N = 6). The schematic illustration in panel N summarizes our findings regarding the transcriptional control of NEXN and its impact on actin polymerization and cell motility. Several aspects of this model, including the involvement of a ( G ) protein-coupled receptor in the S1P effect, were not directly tested here, and they are thus drawn in grey.
    Figure Legend Snippet: Knockdown of Nexilin/ NEXN reduces actin polymerization and SMC marker expression. NEXN was silenced using a short hairpin adenoviral construct (sh-NEXN) and the contents of NEXN and a number of SMC differentiation markers were examined at the mRNA (panel A, N = 6–9) and protein levels (panels B and C, N = 12). Panel D shows that depolymerization of actin using LatB reduces SRF expression in both human bladder and coronary artery SMCs (HBSMCs, N = 8; HCASMCs, N = 9). Panel E shows phalloidin staining of actin filaments in control cell (Null) and after NEXN silencing (sh-NEXN). The scale bar represents 50 μm. Panel F shows a sedimentation assay to determine filamentous (F-) and globular (G-) actin in control cells (null) and after silencing of NEXN . Panel G shows the normalized F- to G-actin ratio in bladder and coronary artery SMCs (HBSMCs, N = 12; HCASMCs, N = 6). Panel H shows that polymerization of actin using jasplakinolide (Jasp) increases MYH11 in HBSMCs in control conditions and after NEXN silencing (N = 6). Panel I shows cell density at different times following the creation of a cell-free area in the culture dish (N = 9–10). Panel J shows the speed of cell movement within the cell-free area (N = 19 and 22 motile cells in Null and sh- NEXN , respectively). Panel K shows a cell viability assay comparing control and NEXN -silenced cells (N = 12 throughout). The effect of NEXN silencing on cell migration was confirmed using an independent siRNA in L (N = 9). The associated repression of the NEXN protein is shown in ( M ) (N = 6). The schematic illustration in panel N summarizes our findings regarding the transcriptional control of NEXN and its impact on actin polymerization and cell motility. Several aspects of this model, including the involvement of a ( G ) protein-coupled receptor in the S1P effect, were not directly tested here, and they are thus drawn in grey.

    Techniques Used: Marker, Expressing, Construct, Staining, Sedimentation, Viability Assay, Migration

    NEXN correlates with gene products that control and respond to changes in actin polymerization and Nexilin is reduced by depolymerization of actin. Correlations of NEXN versus all other RNAs in the top-ten NEXN expressing tissues were examined (data from GTExPortal). The sum of correlation coefficients for individual RNAs across tissues was calculated (R sum ) and the positive extreme of this distribution was plotted ( A ). Actin controlling and responding gene products represented in the extreme are highlighted in blue colors. Examples of NEXN correlations in the human coronary artery (N = 133) are shown in panels B through ( D ). P-values and Spearman Rho values are given in the respective panels. Panels E and F show mRNA data for NEXN in cultured human bladder (HBSMCs, N = 8) and coronary artery (HCASMCs, N = 9) SMCs after treatment with Latrunculin B (LatB). Panels H and I show protein data for Nexilin/ NEXN in the presence and absence of LatB (HBSMCs, 300 nM, N = 12; HCASMCs, 100 nM, N = 10). The top micrographs in panel J shows confocal imaging of YAP (red) on the left, and YAP (green) and CAV1 (red) on the right. The bottom row shows YAP (red) and Nexilin (green). The high magnification overlay at the bottom right shows partial colocalization of YAP and Nexilin at the cell membrane in yellow. All micrographs are from cross-sectioned HBSMCs and white scale bars represent 5 μm throughout.
    Figure Legend Snippet: NEXN correlates with gene products that control and respond to changes in actin polymerization and Nexilin is reduced by depolymerization of actin. Correlations of NEXN versus all other RNAs in the top-ten NEXN expressing tissues were examined (data from GTExPortal). The sum of correlation coefficients for individual RNAs across tissues was calculated (R sum ) and the positive extreme of this distribution was plotted ( A ). Actin controlling and responding gene products represented in the extreme are highlighted in blue colors. Examples of NEXN correlations in the human coronary artery (N = 133) are shown in panels B through ( D ). P-values and Spearman Rho values are given in the respective panels. Panels E and F show mRNA data for NEXN in cultured human bladder (HBSMCs, N = 8) and coronary artery (HCASMCs, N = 9) SMCs after treatment with Latrunculin B (LatB). Panels H and I show protein data for Nexilin/ NEXN in the presence and absence of LatB (HBSMCs, 300 nM, N = 12; HCASMCs, 100 nM, N = 10). The top micrographs in panel J shows confocal imaging of YAP (red) on the left, and YAP (green) and CAV1 (red) on the right. The bottom row shows YAP (red) and Nexilin (green). The high magnification overlay at the bottom right shows partial colocalization of YAP and Nexilin at the cell membrane in yellow. All micrographs are from cross-sectioned HBSMCs and white scale bars represent 5 μm throughout.

    Techniques Used: Expressing, Cell Culture, Imaging

    18) Product Images from "Mice harboring the human SLC30A8 R138X loss-of-function mutation have increased insulin secretory capacity"

    Article Title: Mice harboring the human SLC30A8 R138X loss-of-function mutation have increased insulin secretory capacity

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1721418115

    Analysis of Slc30a8 RNA and protein in islets from male R138X mice on chow diet. ( A ) Slc30a8 RNA in situ hybridization of pancreatic islets isolated from wild-type, knockout, and R138X mice. KO islets were used as negative control. Red, glucagon RNA; green, insulin RNA; white, Slc30a8 RNA. ( B ) Quantification of islet Slc30a8 RNA levels using qPCR analysis. n.d., not detected. ( C ) Western blot of islets isolated from chow-fed WT, KO, and R138X mice. KO islets were used as negative control. The arrow indicates SLC30A8 protein; asterisks denote unspecific bands. ( D ) Dithizone staining of pancreatic islets isolated from WT, KO, and R138X mice.
    Figure Legend Snippet: Analysis of Slc30a8 RNA and protein in islets from male R138X mice on chow diet. ( A ) Slc30a8 RNA in situ hybridization of pancreatic islets isolated from wild-type, knockout, and R138X mice. KO islets were used as negative control. Red, glucagon RNA; green, insulin RNA; white, Slc30a8 RNA. ( B ) Quantification of islet Slc30a8 RNA levels using qPCR analysis. n.d., not detected. ( C ) Western blot of islets isolated from chow-fed WT, KO, and R138X mice. KO islets were used as negative control. The arrow indicates SLC30A8 protein; asterisks denote unspecific bands. ( D ) Dithizone staining of pancreatic islets isolated from WT, KO, and R138X mice.

    Techniques Used: Mouse Assay, RNA In Situ Hybridization, Isolation, Knock-Out, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Staining

    19) Product Images from "Aconitum pseudo-laeve var. erectum Inhibits Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclastogenesis via the c-Fos/nuclear Factor of Activated T-Cells, Cytoplasmic 1 Signaling Pathway and Prevents Lipopolysaccharide-Induced Bone Loss in Mice"

    Article Title: Aconitum pseudo-laeve var. erectum Inhibits Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclastogenesis via the c-Fos/nuclear Factor of Activated T-Cells, Cytoplasmic 1 Signaling Pathway and Prevents Lipopolysaccharide-Induced Bone Loss in Mice

    Journal: Molecules

    doi: 10.3390/molecules190811628

    Aconitum pseudo-laeve var. erectum (APE) suppresses receptor activator of nuclear factor kappa-B ligand (RANKL)-induced c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) expression without stimulating early signal pathways. ( A ) Bone marrow macrophages (BMMs) were pretreated with DMSO (control) or APE (200 µg/mL) for 1 h in the presence of macrophage colony-stimulating factor (M-CSF; 30 ng/mL) and were stimulated with RANKL (100 ng/mL) for the indicated times. Whole-cell lysates underwent western blot analysis with the various indicated antibodies. β-actin served as the internal control; ( B ) BMMs were stimulated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of APE (200 µg/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of c-Fos and NFATc1 were evaluated using real-time PCR; ( C ) Effects of APE on protein expression levels of c-Fos and NFATc1 were evaluated using western blot analysis. β-actin was used as the internal control; ( D ) BMMs were infected with retroviruses expressing pMX-IRES-EGFP (pMX), pMX-NFATc1-EGFP, and pMX-cFos-EGFP. Infected BMMs were cultured with or without APE (200 µg/mL) in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days. After culturing, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) (left). The TRAP-positive multinucleated osteoclasts were counted (right).
    Figure Legend Snippet: Aconitum pseudo-laeve var. erectum (APE) suppresses receptor activator of nuclear factor kappa-B ligand (RANKL)-induced c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) expression without stimulating early signal pathways. ( A ) Bone marrow macrophages (BMMs) were pretreated with DMSO (control) or APE (200 µg/mL) for 1 h in the presence of macrophage colony-stimulating factor (M-CSF; 30 ng/mL) and were stimulated with RANKL (100 ng/mL) for the indicated times. Whole-cell lysates underwent western blot analysis with the various indicated antibodies. β-actin served as the internal control; ( B ) BMMs were stimulated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of APE (200 µg/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of c-Fos and NFATc1 were evaluated using real-time PCR; ( C ) Effects of APE on protein expression levels of c-Fos and NFATc1 were evaluated using western blot analysis. β-actin was used as the internal control; ( D ) BMMs were infected with retroviruses expressing pMX-IRES-EGFP (pMX), pMX-NFATc1-EGFP, and pMX-cFos-EGFP. Infected BMMs were cultured with or without APE (200 µg/mL) in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days. After culturing, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) (left). The TRAP-positive multinucleated osteoclasts were counted (right).

    Techniques Used: Expressing, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Infection, Cell Culture, Staining

    Aconitum pseudo-laeve var. erectum (APE) down-regulates the expression of osteoclast-specific marker genes. Bone marrow macrophages (BMMs) were stimulated with receptor activator of nuclear factor kappa-B ligand (RANKL; 100 ng/mL) and macrophage colony-stimulating factor (M-CSF; 30 ng/mL) in the presence or absence of APE (200 µg/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of osteoclast-associated receptor (OSCAR), tartrate-resistant acid phosphatase (TRAP), Atp6v0d2, Cathepsin K, dendritic cell-specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), calcitonin receptor, and matrix metallopeptidase 9 (MMP-9) were evaluated by real-time PCR.
    Figure Legend Snippet: Aconitum pseudo-laeve var. erectum (APE) down-regulates the expression of osteoclast-specific marker genes. Bone marrow macrophages (BMMs) were stimulated with receptor activator of nuclear factor kappa-B ligand (RANKL; 100 ng/mL) and macrophage colony-stimulating factor (M-CSF; 30 ng/mL) in the presence or absence of APE (200 µg/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of osteoclast-associated receptor (OSCAR), tartrate-resistant acid phosphatase (TRAP), Atp6v0d2, Cathepsin K, dendritic cell-specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), calcitonin receptor, and matrix metallopeptidase 9 (MMP-9) were evaluated by real-time PCR.

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

    20) Product Images from "microRNA-155 deficiency impairs dendritic cell function in breast cancer"

    Article Title: microRNA-155 deficiency impairs dendritic cell function in breast cancer

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2016.1232223

    miR-155 affects DC migration by epigenetically regulating CCR7 expression. (A) In vivo migration of CFSE-labeled DCs toward draining lymph nodes was measured by flow cytometry. WT or miR-155 −/− BMDCs were pulsed with tumor cell lysate and ECM, labeled with CFSE, and implanted into the groins of tumor-bearing WT mice. Typical scatter plots (left) and percentages (right) of CFSE + population are shown (n = 4). (B) In vitro migration of WT or miR-155 −/− BMDCs pulsed with tumor cell lysate and ECM was determined by trans-well migration assay. Immature BMDCs maintained in DC medium were used as controls. Representative fluorescence images are shown (left). Cells were counted in 10 random fields per sample at 200 × magnification and quantified (right) (n = 3). (C) CCR7 mRNA level in DCs isolated from spleen, tumor, and lymph node of WT or miR-155 −/− mice carrying EO771 tumors was determined by RT-PCR (n = 3). (D) CCR7 mRNA level in BMDCs treated with tumor lysate and ECM in vitro was determined by RT-PCR; immature BMDCs served as controls (n = 3). (E) and (F) Cell surface CCR7 expression on DCs isolated from spleen (E) or lymph node (F) of tumor-bearing WT or miR-155 −/− mice was determined by flow cytometry. MFIs of CCR7 from three independent experiments are shown. (G) Quantified MFI of CCR7 on BMDCs matured by tumor cell lysate and ECM in vitro was shown. (H) Enrichment of H3K27me3 at Ccr7 promoter (left) and first intron (right) in WT and miR-155 −/− BMDCs treated with tumor lysate and ECM was determined by qPCR. (I and J) Jarid2 protein levels in DCs isolated from lymph nodes and spleens of WT and miR-155 −/− tumor-bearing mice (I), and in tumor-associated antigen-treated BMDCs (J) were detected by western blot; relative intensities of Jarid2 were labeled under the bands. (K) ChIP-qPCR was performed to detect the recruitment of Suz12 at the Ccr7 promoter (left) and first intron (right) in WT and miR-155 −/− BMDCs. (A), (C), (E) and (F), (H), and (K) Student's t test. (B), (D), and (G) two-way ANOVA followed by the Tukey multiple comparison test. # p
    Figure Legend Snippet: miR-155 affects DC migration by epigenetically regulating CCR7 expression. (A) In vivo migration of CFSE-labeled DCs toward draining lymph nodes was measured by flow cytometry. WT or miR-155 −/− BMDCs were pulsed with tumor cell lysate and ECM, labeled with CFSE, and implanted into the groins of tumor-bearing WT mice. Typical scatter plots (left) and percentages (right) of CFSE + population are shown (n = 4). (B) In vitro migration of WT or miR-155 −/− BMDCs pulsed with tumor cell lysate and ECM was determined by trans-well migration assay. Immature BMDCs maintained in DC medium were used as controls. Representative fluorescence images are shown (left). Cells were counted in 10 random fields per sample at 200 × magnification and quantified (right) (n = 3). (C) CCR7 mRNA level in DCs isolated from spleen, tumor, and lymph node of WT or miR-155 −/− mice carrying EO771 tumors was determined by RT-PCR (n = 3). (D) CCR7 mRNA level in BMDCs treated with tumor lysate and ECM in vitro was determined by RT-PCR; immature BMDCs served as controls (n = 3). (E) and (F) Cell surface CCR7 expression on DCs isolated from spleen (E) or lymph node (F) of tumor-bearing WT or miR-155 −/− mice was determined by flow cytometry. MFIs of CCR7 from three independent experiments are shown. (G) Quantified MFI of CCR7 on BMDCs matured by tumor cell lysate and ECM in vitro was shown. (H) Enrichment of H3K27me3 at Ccr7 promoter (left) and first intron (right) in WT and miR-155 −/− BMDCs treated with tumor lysate and ECM was determined by qPCR. (I and J) Jarid2 protein levels in DCs isolated from lymph nodes and spleens of WT and miR-155 −/− tumor-bearing mice (I), and in tumor-associated antigen-treated BMDCs (J) were detected by western blot; relative intensities of Jarid2 were labeled under the bands. (K) ChIP-qPCR was performed to detect the recruitment of Suz12 at the Ccr7 promoter (left) and first intron (right) in WT and miR-155 −/− BMDCs. (A), (C), (E) and (F), (H), and (K) Student's t test. (B), (D), and (G) two-way ANOVA followed by the Tukey multiple comparison test. # p

    Techniques Used: Migration, Expressing, In Vivo, Labeling, Flow Cytometry, Cytometry, Mouse Assay, In Vitro, Fluorescence, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation

    21) Product Images from "SMCHD1 regulates a limited set of gene clusters on autosomal chromosomes"

    Article Title: SMCHD1 regulates a limited set of gene clusters on autosomal chromosomes

    Journal: Skeletal Muscle

    doi: 10.1186/s13395-017-0129-7

    The effects of SMCHD1 KD in non-DUX4 permissive 4qB cells on PCDH β cluster expression. The expression level of SMCHD1 after SMCHD1 shRNA KD in comparison with control KD shRNAs targeting luciferase and GFP is shown by RT-qPCR analysis in primary ( a ) myoblast and ( b ) myotube cells. RNA expression analysis was then assessed for PCDH β cluster isoform members in primary ( c ) myoblast and ( d ) myotube cells. Results represent qRT-PCR analysis of the indicated gene after normalization to the internal control gene GUS1 . For each gene, the value of expression in the control shRNA samples was then arbitrarily set to 1. * P
    Figure Legend Snippet: The effects of SMCHD1 KD in non-DUX4 permissive 4qB cells on PCDH β cluster expression. The expression level of SMCHD1 after SMCHD1 shRNA KD in comparison with control KD shRNAs targeting luciferase and GFP is shown by RT-qPCR analysis in primary ( a ) myoblast and ( b ) myotube cells. RNA expression analysis was then assessed for PCDH β cluster isoform members in primary ( c ) myoblast and ( d ) myotube cells. Results represent qRT-PCR analysis of the indicated gene after normalization to the internal control gene GUS1 . For each gene, the value of expression in the control shRNA samples was then arbitrarily set to 1. * P

    Techniques Used: Expressing, shRNA, Luciferase, Quantitative RT-PCR, RNA Expression

    22) Product Images from "Mice harboring the human SLC30A8 R138X loss-of-function mutation have increased insulin secretory capacity"

    Article Title: Mice harboring the human SLC30A8 R138X loss-of-function mutation have increased insulin secretory capacity

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1721418115

    Analysis of Slc30a8 RNA and protein in islets from male R138X mice on chow diet. ( A ) Slc30a8 RNA in situ hybridization of pancreatic islets isolated from wild-type, knockout, and R138X mice. KO islets were used as negative control. Red, glucagon RNA; green, insulin RNA; white, Slc30a8 RNA. ( B ) Quantification of islet Slc30a8 RNA levels using qPCR analysis. n.d., not detected. ( C ) Western blot of islets isolated from chow-fed WT, KO, and R138X mice. KO islets were used as negative control. The arrow indicates SLC30A8 protein; asterisks denote unspecific bands. ( D ) Dithizone staining of pancreatic islets isolated from WT, KO, and R138X mice.
    Figure Legend Snippet: Analysis of Slc30a8 RNA and protein in islets from male R138X mice on chow diet. ( A ) Slc30a8 RNA in situ hybridization of pancreatic islets isolated from wild-type, knockout, and R138X mice. KO islets were used as negative control. Red, glucagon RNA; green, insulin RNA; white, Slc30a8 RNA. ( B ) Quantification of islet Slc30a8 RNA levels using qPCR analysis. n.d., not detected. ( C ) Western blot of islets isolated from chow-fed WT, KO, and R138X mice. KO islets were used as negative control. The arrow indicates SLC30A8 protein; asterisks denote unspecific bands. ( D ) Dithizone staining of pancreatic islets isolated from WT, KO, and R138X mice.

    Techniques Used: Mouse Assay, RNA In Situ Hybridization, Isolation, Knock-Out, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Staining

    23) Product Images from "Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47"

    Article Title: Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47

    Journal: European Journal of Human Genetics

    doi: 10.1038/ejhg.2011.150

    ( a ) Expression levels of CT47 mRNA in different SCLC cell lines relative to the expression in human testis. Commercially available human total testis RNA and RNA isolated from SCLC lines were used for cDNA synthesis under identical conditions. CT47 expression levels are normalized to the expression levels measured in the testis. Different SCLC lines have different, but low levels of CT47 expression compared to the testis. Relative abundance of histone modifications and EZH2 at the CT47 promoter ( b ), exon 3 ( c ) and the distal region in SCLC cell lines ( d ). SCLC cell lines show individual variation in the abundance of different histone modifications. Generally, a loss of the repressive chromatin mark H3K9me3 can be observed in SCLCs. H3K27me3 levels are higher in SCLCs at the promoter and exon 3 region than in LCLs, but lower at the distal region. The relative abundance of the PRC2 component EZH2 responsible for generating H2K27me3 is dramatically reduced in SCLCs compared to LCLs at all region studied. ( e ) DNA methylation levels at CpGs located in the CT47 promoter region in LCL and SCLC samples. The methylation level of seven different CpGs, next to the transcriptional start site of CT47 , was determined by bisulfite sequencing and quantified by ESME program. There is a significant difference between the methylation level of LCLs and SCLCs at every CpG tested ( P
    Figure Legend Snippet: ( a ) Expression levels of CT47 mRNA in different SCLC cell lines relative to the expression in human testis. Commercially available human total testis RNA and RNA isolated from SCLC lines were used for cDNA synthesis under identical conditions. CT47 expression levels are normalized to the expression levels measured in the testis. Different SCLC lines have different, but low levels of CT47 expression compared to the testis. Relative abundance of histone modifications and EZH2 at the CT47 promoter ( b ), exon 3 ( c ) and the distal region in SCLC cell lines ( d ). SCLC cell lines show individual variation in the abundance of different histone modifications. Generally, a loss of the repressive chromatin mark H3K9me3 can be observed in SCLCs. H3K27me3 levels are higher in SCLCs at the promoter and exon 3 region than in LCLs, but lower at the distal region. The relative abundance of the PRC2 component EZH2 responsible for generating H2K27me3 is dramatically reduced in SCLCs compared to LCLs at all region studied. ( e ) DNA methylation levels at CpGs located in the CT47 promoter region in LCL and SCLC samples. The methylation level of seven different CpGs, next to the transcriptional start site of CT47 , was determined by bisulfite sequencing and quantified by ESME program. There is a significant difference between the methylation level of LCLs and SCLCs at every CpG tested ( P

    Techniques Used: Expressing, Isolation, DNA Methylation Assay, Methylation, Methylation Sequencing

    24) Product Images from "Efficient and Simple Production of Insulin-Producing Cells from Embryonal Carcinoma Stem Cells Using Mouse Neonate Pancreas Extract, As a Natural Inducer"

    Article Title: Efficient and Simple Production of Insulin-Producing Cells from Embryonal Carcinoma Stem Cells Using Mouse Neonate Pancreas Extract, As a Natural Inducer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0090885

    Quantitative analysis of genes involved in differentiation of pancreatic cells derived from P19 EC cells. The cells were cultured as EBs in different concentrations of MPE (mouse pancreas extract). (A) Relative gene expression of PDX-1, INS1, and INS2 in different concentration of MPE. (B) Comparison of expression of Oct3/4, Sox-2, Nanog (undifferentiated stem cell markers), and EP300, CREB1 (differentiated stem cell transcription factors) between P19 EC (embryonal carcinoma) cells and IPCs (insulin-producing cells). The data are expressed as relative gene expression to β-2M and are presented as mean±SD. The means with different letters are significantly different at P = 0.05.
    Figure Legend Snippet: Quantitative analysis of genes involved in differentiation of pancreatic cells derived from P19 EC cells. The cells were cultured as EBs in different concentrations of MPE (mouse pancreas extract). (A) Relative gene expression of PDX-1, INS1, and INS2 in different concentration of MPE. (B) Comparison of expression of Oct3/4, Sox-2, Nanog (undifferentiated stem cell markers), and EP300, CREB1 (differentiated stem cell transcription factors) between P19 EC (embryonal carcinoma) cells and IPCs (insulin-producing cells). The data are expressed as relative gene expression to β-2M and are presented as mean±SD. The means with different letters are significantly different at P = 0.05.

    Techniques Used: Derivative Assay, Cell Culture, Expressing, Concentration Assay

    Representative micrographs showing morphology of exponentially growing P19 cells at low (A) and high (B) magnification. Undifferentiated cells are tightly packed polygonal cells, with large nucleoli and high nucleus–cytoplasm ratio. An embroyid body replated on 0.1% gelatin coated petri dish (C) differentiated spontaneously or induced by MPE. The cells spread out from the attached EB. Scale bars: A C 20 µm, B 10 µm.
    Figure Legend Snippet: Representative micrographs showing morphology of exponentially growing P19 cells at low (A) and high (B) magnification. Undifferentiated cells are tightly packed polygonal cells, with large nucleoli and high nucleus–cytoplasm ratio. An embroyid body replated on 0.1% gelatin coated petri dish (C) differentiated spontaneously or induced by MPE. The cells spread out from the attached EB. Scale bars: A C 20 µm, B 10 µm.

    Techniques Used:

    Determination of secreted (A) and secreted versus intracellular (B) insulin in P19 undifferentiated EC cells (P19), spontaneous differentiated EBs (EB) and the MPE-treated cells. Significant insulin concentration was observed in MPE–treated IPCs. To normalize the amount of insulin secretion, the total protein of the cells in each well was measured by the Bradford method. The experiment was performed in triplicate. Each value represents mean ± SD.
    Figure Legend Snippet: Determination of secreted (A) and secreted versus intracellular (B) insulin in P19 undifferentiated EC cells (P19), spontaneous differentiated EBs (EB) and the MPE-treated cells. Significant insulin concentration was observed in MPE–treated IPCs. To normalize the amount of insulin secretion, the total protein of the cells in each well was measured by the Bradford method. The experiment was performed in triplicate. Each value represents mean ± SD.

    Techniques Used: Concentration Assay

    Fluorescence micrographs illustrating the expression of pancreatic β cell markers. Staining of IPCs with antibodies against proinsulin+insulin (A) and insulin receptor beta (C) showing that most of the P19 cells treated with MPE express pancreatic β cell markers. (E) Control for immunostaining, the primary antibody was omitted. B, D F are phase contrast images of the same field shown in A, C E respectively. Scale bars: 40 µm.
    Figure Legend Snippet: Fluorescence micrographs illustrating the expression of pancreatic β cell markers. Staining of IPCs with antibodies against proinsulin+insulin (A) and insulin receptor beta (C) showing that most of the P19 cells treated with MPE express pancreatic β cell markers. (E) Control for immunostaining, the primary antibody was omitted. B, D F are phase contrast images of the same field shown in A, C E respectively. Scale bars: 40 µm.

    Techniques Used: Fluorescence, Expressing, Staining, Immunostaining

    DTZ staining of differentiated IPCs, derived from P19 EC cells. IPCs formed pancreatic islet-like structures (A). A cell cluster distinctly stained crimson red by DTZ is apparent (B). Individual cells are DTZ-positive in spontaneous differentiated EBs (C). Untreated EC cells are not stained (D). Scale bars: A 100 µm, B, C D 200 µm.
    Figure Legend Snippet: DTZ staining of differentiated IPCs, derived from P19 EC cells. IPCs formed pancreatic islet-like structures (A). A cell cluster distinctly stained crimson red by DTZ is apparent (B). Individual cells are DTZ-positive in spontaneous differentiated EBs (C). Untreated EC cells are not stained (D). Scale bars: A 100 µm, B, C D 200 µm.

    Techniques Used: Staining, Derivative Assay

    25) Product Images from "Combining nitric oxide release with anti-inflammatory activity preserves nigrostriatal dopaminergic innervation and prevents motor impairment in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease"

    Article Title: Combining nitric oxide release with anti-inflammatory activity preserves nigrostriatal dopaminergic innervation and prevents motor impairment in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-7-83

    HCT1026 inhibits MPTP-induced loss of striatal TH and DAT mRNAs expression . Ageing (9-11 month-old) C57Bl/6 mice fed with a control (ct) or HCT1026 diets (30 mg kg -1 ) starting at -10 d, underwent an MPTP treatment according to the subchronic injection paradigm, as described. Age-matched mice fed with the different diets received physiologic saline and served as controls. Mice were sacrificed at different time-intervals after MPTP. Striatal tissue samples were processed for semi-quantitative RT-PCR analysis as described. Total RNA isolated and cDNA synthesized using Retroscript Kit (see Materials and Methods) following the manufacturer's directions. The 250 ng of cDNA were used for PCR, by using Super Taq DNA polymerase with specific primer pairs for TH (620 bp) and DAT (328 bp), and Classic S18 Standard (495 bp). Samples from PCR reactions were separated electrophoretically on 2% agarose gel containing 0,2 μg/ml of ethidium bromide (B-D, F-H). Fluorescent bands of amplified gene products were captured by using Gel Logic 200 Imaging System (Kodak), values normalized against S18 and ratios expressed as percent of control, within each experimental group (A, E). Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p
    Figure Legend Snippet: HCT1026 inhibits MPTP-induced loss of striatal TH and DAT mRNAs expression . Ageing (9-11 month-old) C57Bl/6 mice fed with a control (ct) or HCT1026 diets (30 mg kg -1 ) starting at -10 d, underwent an MPTP treatment according to the subchronic injection paradigm, as described. Age-matched mice fed with the different diets received physiologic saline and served as controls. Mice were sacrificed at different time-intervals after MPTP. Striatal tissue samples were processed for semi-quantitative RT-PCR analysis as described. Total RNA isolated and cDNA synthesized using Retroscript Kit (see Materials and Methods) following the manufacturer's directions. The 250 ng of cDNA were used for PCR, by using Super Taq DNA polymerase with specific primer pairs for TH (620 bp) and DAT (328 bp), and Classic S18 Standard (495 bp). Samples from PCR reactions were separated electrophoretically on 2% agarose gel containing 0,2 μg/ml of ethidium bromide (B-D, F-H). Fluorescent bands of amplified gene products were captured by using Gel Logic 200 Imaging System (Kodak), values normalized against S18 and ratios expressed as percent of control, within each experimental group (A, E). Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p

    Techniques Used: Expressing, Mouse Assay, Injection, Quantitative RT-PCR, Isolation, Synthesized, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Imaging

    26) Product Images from "Characterization of stress response in human retinal epithelial cells"

    Article Title: Characterization of stress response in human retinal epithelial cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2012.01652.x

    Analysis of LEI/L-DNase II in caspase-independent apoptosis. ( A ) Indirect immunofluorescence experiments were performed on ARPE-19 cells untreated or treated with HMA for 24 hrs at the indicated concentrations or with 250 μM etoposide for the indicated times. DAPI (blue) or anti-LEI/L-DNase II antibody (red) was used. Scale bar: 25 μm. ( B ) ARPE-19 cells were treated with 40 μM HMA for 24 hrs and analysed by Western blot using anti-LEI/L-DNase II antibody. β actin was used as loading control. Right panel shows the quantification of the LEI/L-DNase II western. Black bars represent LEI/actin ratio, white bar represents L-DNase II/actin ratio. There is an increase of LEI expression and of L-DNase II in treated cells. Means between treated and untreated cells are statistically different as compared by using t -test ( P
    Figure Legend Snippet: Analysis of LEI/L-DNase II in caspase-independent apoptosis. ( A ) Indirect immunofluorescence experiments were performed on ARPE-19 cells untreated or treated with HMA for 24 hrs at the indicated concentrations or with 250 μM etoposide for the indicated times. DAPI (blue) or anti-LEI/L-DNase II antibody (red) was used. Scale bar: 25 μm. ( B ) ARPE-19 cells were treated with 40 μM HMA for 24 hrs and analysed by Western blot using anti-LEI/L-DNase II antibody. β actin was used as loading control. Right panel shows the quantification of the LEI/L-DNase II western. Black bars represent LEI/actin ratio, white bar represents L-DNase II/actin ratio. There is an increase of LEI expression and of L-DNase II in treated cells. Means between treated and untreated cells are statistically different as compared by using t -test ( P

    Techniques Used: Immunofluorescence, Western Blot, Expressing

    HMA induces autophagy. ( A ) ARPE 19 cells were untreated or treated with rapamycin for 2 hrs, with HMA or etoposide for 24 hrs and then probed with anti-LC3 antibody. Scale bar 25 μm. ( B ) ARPE-19 cells were treated as before and analysed by Western blot using anti-beclin 1, ATG-7, AGT5-12 and LC3 antibodies. γ tubulin was used as a loading control (left panel). On middle and right panels, cells were treated as before and analysed using anti-ERK and phospho-ERK (*) antibodies (middle panel) or with anti JNK1 or phosphorylated JNK1 (*) antibodies (right panel). Under the western images the quantification of the bands is reported showing a significant increase of Beclin 1 (Means are different from each other as calculated from a one-way anova test ( P
    Figure Legend Snippet: HMA induces autophagy. ( A ) ARPE 19 cells were untreated or treated with rapamycin for 2 hrs, with HMA or etoposide for 24 hrs and then probed with anti-LC3 antibody. Scale bar 25 μm. ( B ) ARPE-19 cells were treated as before and analysed by Western blot using anti-beclin 1, ATG-7, AGT5-12 and LC3 antibodies. γ tubulin was used as a loading control (left panel). On middle and right panels, cells were treated as before and analysed using anti-ERK and phospho-ERK (*) antibodies (middle panel) or with anti JNK1 or phosphorylated JNK1 (*) antibodies (right panel). Under the western images the quantification of the bands is reported showing a significant increase of Beclin 1 (Means are different from each other as calculated from a one-way anova test ( P

    Techniques Used: Western Blot

    Morphological changes induced by HMA. ( A ) ARPE-19 cells were treated with the indicated HMA concentrations for 24 hrs, stained with DAPI and observed at the optical microscope. Fields in squares were magnified (lower panel). Scale bar 25 μm. ( B ) Electron microscope analysis was performed on ARPE-19 cells untreated (upper left panel) or treated with HMA (middle and right upper panels). In HMA-treated cells squares show vacuoles (middle upper panel). The left upper panel shows a high magnification image of an autophagosome (see the double membrane indicated by two red arrows) containing a mitochondrion. Blue arrow shows the double membrane of the mitochondrion. The green arrow indicates a mithocondrial creast. Bottom panel: Higher magnifications show the nucleus (N), endoplasmic reticulum (ER), autophagosomes (A). In (N), white arrow shows chromatin condensation, black arrow indicates the nuclear pore and arrow head the dilatation of the nuclear membrane. Scale bar: 0.5 μm.
    Figure Legend Snippet: Morphological changes induced by HMA. ( A ) ARPE-19 cells were treated with the indicated HMA concentrations for 24 hrs, stained with DAPI and observed at the optical microscope. Fields in squares were magnified (lower panel). Scale bar 25 μm. ( B ) Electron microscope analysis was performed on ARPE-19 cells untreated (upper left panel) or treated with HMA (middle and right upper panels). In HMA-treated cells squares show vacuoles (middle upper panel). The left upper panel shows a high magnification image of an autophagosome (see the double membrane indicated by two red arrows) containing a mitochondrion. Blue arrow shows the double membrane of the mitochondrion. The green arrow indicates a mithocondrial creast. Bottom panel: Higher magnifications show the nucleus (N), endoplasmic reticulum (ER), autophagosomes (A). In (N), white arrow shows chromatin condensation, black arrow indicates the nuclear pore and arrow head the dilatation of the nuclear membrane. Scale bar: 0.5 μm.

    Techniques Used: Staining, Microscopy

    Role of autophagy in HMA response. ( A ) ARPE-19 cells were treated with HMA in the absence or presence of 1 μM rapamycin or 5 mM 3-MA. After 2 or 4 hrs of treatment, cells were trypsinized and counted. A total of 1000 cells were seeded into 6-well plates. Seven days after seeding, cells were stained with cresyl violet. Controls using rapamycin or 3-MA alone have show no difference with the control (not shown) ( B ) Using image analysis, the surface covered by the cells was measured and plotted. For 2 hrs of treatment all means were significantly different ( P
    Figure Legend Snippet: Role of autophagy in HMA response. ( A ) ARPE-19 cells were treated with HMA in the absence or presence of 1 μM rapamycin or 5 mM 3-MA. After 2 or 4 hrs of treatment, cells were trypsinized and counted. A total of 1000 cells were seeded into 6-well plates. Seven days after seeding, cells were stained with cresyl violet. Controls using rapamycin or 3-MA alone have show no difference with the control (not shown) ( B ) Using image analysis, the surface covered by the cells was measured and plotted. For 2 hrs of treatment all means were significantly different ( P

    Techniques Used: Staining

    Indirect Immunofluorescence analysis of PARP-1 activity and AIF-dependent parthanatos. ( A ) ARPE-19 cells treated with either HMA or etoposide were probed with anti-PAR antibody to evaluate PAR synthesis. ( B ) Evaluation of AIF localization in HMA-treated ARPE-19 cells. Experiments were carried out with anti-mtHSP70 (to label mitochondria, red fluorescence) and anti-AIF (green fluorescence) antibodies. Scale bar 25 μm.
    Figure Legend Snippet: Indirect Immunofluorescence analysis of PARP-1 activity and AIF-dependent parthanatos. ( A ) ARPE-19 cells treated with either HMA or etoposide were probed with anti-PAR antibody to evaluate PAR synthesis. ( B ) Evaluation of AIF localization in HMA-treated ARPE-19 cells. Experiments were carried out with anti-mtHSP70 (to label mitochondria, red fluorescence) and anti-AIF (green fluorescence) antibodies. Scale bar 25 μm.

    Techniques Used: Immunofluorescence, Activity Assay, Fluorescence

    HMA effect on ARPE-19 cell viability and proliferation. ARPE-19 cells were treated with increasing concentrations of HMA (20–120 μM) for up to 72 hrs. Cell incubation with 250 μM etoposide for up to 72 hrs was used as an internal standard for caspase-dependent apoptosis inducers. ( A ) Cell viability was evaluated by the MTT assay after 24, 48 and 72 hrs of treatment. Results are expressed as the mean ± SD of three independent experiments. All measurements were significantly different as calculated using a one way anova test P
    Figure Legend Snippet: HMA effect on ARPE-19 cell viability and proliferation. ARPE-19 cells were treated with increasing concentrations of HMA (20–120 μM) for up to 72 hrs. Cell incubation with 250 μM etoposide for up to 72 hrs was used as an internal standard for caspase-dependent apoptosis inducers. ( A ) Cell viability was evaluated by the MTT assay after 24, 48 and 72 hrs of treatment. Results are expressed as the mean ± SD of three independent experiments. All measurements were significantly different as calculated using a one way anova test P

    Techniques Used: Incubation, MTT Assay

    Analysis of caspase-dependent apoptosis in ARPE-19 cells treated with HMA. ( A ) ARPE-19 cells were treated with different HMA concentrations (40–120 μM) for 24 hrs and then analysed using Western blot. Etoposide (250 μM), administered for 24 hrs, was used as an internal standard. As a positive control for apoptosis, HeLa cells were treated with 100 μM etoposide for 3 hrs followed by 24 hrs of recovery in drug-free medium. The activation of caspases 3, 8 and 9 was investigated. Only caspase 3 was slightly activated in 120 μM HMA treated cells. ( B ) ARPE-19 cells were treated for 24 hrs with HMA (40–120 μM); 250 μM etoposide administered for 72 hrs was used as a pro-apoptotic drug. Long-term cultured (LTC) HeLa cells were used as a positive control for apoptosis. Nuclear DNA was extracted and loaded on a 1.8% agarose gel stained with ethidium bromide. No DNA degradation was visible in untreated cells or in cells treated with HMA 40 or 80 μM. A smear was observed in cells treated with 120 μM HMA and a faint ladder is seen in etoposide-treated ARPE-19 cells. ( C ) Upper panel. Western blot analysis of PARP-1 proteolysis was performed on untreated, HMA- or etoposide-treated ARPE-19 cells. HeLa cells treated with etoposide were used as a positive control. γ-Tubulin was used as a loading control. 113 kD: full length PARP-1; 89 kD: cleaved PARP-1. Lower panel shows a quantification of the expression of full length PARP-1 compared to γ-tubulin. At the used loading of proteins on the gel PARP-1 is not detectable in untreated or etoposide-treated cells, but its expression increases with HMA concentration. All the represented means are different from each other as calculated from a one way anova test ( P
    Figure Legend Snippet: Analysis of caspase-dependent apoptosis in ARPE-19 cells treated with HMA. ( A ) ARPE-19 cells were treated with different HMA concentrations (40–120 μM) for 24 hrs and then analysed using Western blot. Etoposide (250 μM), administered for 24 hrs, was used as an internal standard. As a positive control for apoptosis, HeLa cells were treated with 100 μM etoposide for 3 hrs followed by 24 hrs of recovery in drug-free medium. The activation of caspases 3, 8 and 9 was investigated. Only caspase 3 was slightly activated in 120 μM HMA treated cells. ( B ) ARPE-19 cells were treated for 24 hrs with HMA (40–120 μM); 250 μM etoposide administered for 72 hrs was used as a pro-apoptotic drug. Long-term cultured (LTC) HeLa cells were used as a positive control for apoptosis. Nuclear DNA was extracted and loaded on a 1.8% agarose gel stained with ethidium bromide. No DNA degradation was visible in untreated cells or in cells treated with HMA 40 or 80 μM. A smear was observed in cells treated with 120 μM HMA and a faint ladder is seen in etoposide-treated ARPE-19 cells. ( C ) Upper panel. Western blot analysis of PARP-1 proteolysis was performed on untreated, HMA- or etoposide-treated ARPE-19 cells. HeLa cells treated with etoposide were used as a positive control. γ-Tubulin was used as a loading control. 113 kD: full length PARP-1; 89 kD: cleaved PARP-1. Lower panel shows a quantification of the expression of full length PARP-1 compared to γ-tubulin. At the used loading of proteins on the gel PARP-1 is not detectable in untreated or etoposide-treated cells, but its expression increases with HMA concentration. All the represented means are different from each other as calculated from a one way anova test ( P

    Techniques Used: Western Blot, Positive Control, Activation Assay, Cell Culture, Agarose Gel Electrophoresis, Staining, Expressing, Concentration Assay

    27) Product Images from "Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein"

    Article Title: Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006758

    Generation and characterization of LCMV strains expressing a tagged L protein. (A) Viral titer of N- and C-terminal L protein-tagged Cl13 LCMV and WT Cl13 LCMV measured by focus forming assay at 72 hours post infection after reverse genetic rescue on BHK21 cells. ( B ) HEK293T cells were infected at a MOI of 0.01 with either Cl13 L-HA or with untagged Cl13. Supernatant was harvested and viral loads were measured at the indicated time points by focus forming assay. (C and D) C57BL/6J mice were infected with 2x10 6 FFU of the indicated viruses. Viral titers were determined in (C) blood at indicated time points and in (D) organs 20 days post infection. (E) C57BL/6J mice were infected with 2x10 6 FFU of the indicated viruses and the percentage of GP33-specific-tetramer + CD8 + T cells was quantified in the spleen at 8 days post infection. Each symbol and bar represents the mean ± SEM of three to five mice. Statistical significance was calculated by Two-way ANOVA (B- C ) or unpaired t-test ( D-E ). Significant p values were indicated as follows: ns—non significant, * p≤0.05,: ** p≤0.01.
    Figure Legend Snippet: Generation and characterization of LCMV strains expressing a tagged L protein. (A) Viral titer of N- and C-terminal L protein-tagged Cl13 LCMV and WT Cl13 LCMV measured by focus forming assay at 72 hours post infection after reverse genetic rescue on BHK21 cells. ( B ) HEK293T cells were infected at a MOI of 0.01 with either Cl13 L-HA or with untagged Cl13. Supernatant was harvested and viral loads were measured at the indicated time points by focus forming assay. (C and D) C57BL/6J mice were infected with 2x10 6 FFU of the indicated viruses. Viral titers were determined in (C) blood at indicated time points and in (D) organs 20 days post infection. (E) C57BL/6J mice were infected with 2x10 6 FFU of the indicated viruses and the percentage of GP33-specific-tetramer + CD8 + T cells was quantified in the spleen at 8 days post infection. Each symbol and bar represents the mean ± SEM of three to five mice. Statistical significance was calculated by Two-way ANOVA (B- C ) or unpaired t-test ( D-E ). Significant p values were indicated as follows: ns—non significant, * p≤0.05,: ** p≤0.01.

    Techniques Used: Expressing, Focus Forming Assay, Infection, Mouse Assay

    Functional screening for L protein interactors involved in LCMV life cycle. ( A ) Two independently generated HeLa S3 CRISPR-Cas9 targeted cell pools per gene of interest for 5 genes were infected in triplicate wells with LCMV Cl13 WT at a MOI of 0.01 and viral loads were measured at 36 hours post infection by focus forming assay. The obtained data were normalized to the non-target control and log2 transformed. ( B ) Two HeLa S3 CRISPR-Cas9 TRIM21-targeted cell pools were reconstituted either with TRIM21-expressing plasmid or with non-target control and 36 hour post transfection were infected in triplicate wells with LCMV Cl13 WT at a MOI of 0.01. Viral loads were measured at 36 hours post infection by focus forming assay. The obtained data were normalized to the non-target control and log2 transformed. ( C - D ) C57BL/6 and Trim21 -/- mice were infected with 2x10 6 FFU of the indicated viruses. Viral titers were determined in ( C ) blood at indicated time points and in ( D ) organs at 21 days post infection. The data shown in ( C ) is representative of two similar experiments. Each symbol and bar represents the mean ± SEM of three to five mice. Statistical significance was calculated by unpaired t-test ( A, B, D ) or by Two-way ANOVA ( C ). Significant p values were indicated as follows: ns—non significant, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.
    Figure Legend Snippet: Functional screening for L protein interactors involved in LCMV life cycle. ( A ) Two independently generated HeLa S3 CRISPR-Cas9 targeted cell pools per gene of interest for 5 genes were infected in triplicate wells with LCMV Cl13 WT at a MOI of 0.01 and viral loads were measured at 36 hours post infection by focus forming assay. The obtained data were normalized to the non-target control and log2 transformed. ( B ) Two HeLa S3 CRISPR-Cas9 TRIM21-targeted cell pools were reconstituted either with TRIM21-expressing plasmid or with non-target control and 36 hour post transfection were infected in triplicate wells with LCMV Cl13 WT at a MOI of 0.01. Viral loads were measured at 36 hours post infection by focus forming assay. The obtained data were normalized to the non-target control and log2 transformed. ( C - D ) C57BL/6 and Trim21 -/- mice were infected with 2x10 6 FFU of the indicated viruses. Viral titers were determined in ( C ) blood at indicated time points and in ( D ) organs at 21 days post infection. The data shown in ( C ) is representative of two similar experiments. Each symbol and bar represents the mean ± SEM of three to five mice. Statistical significance was calculated by unpaired t-test ( A, B, D ) or by Two-way ANOVA ( C ). Significant p values were indicated as follows: ns—non significant, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.

    Techniques Used: Functional Assay, Generated, CRISPR, Infection, Focus Forming Assay, Transformation Assay, Expressing, Plasmid Preparation, Transfection, Mouse Assay

    28) Product Images from "Activated kinase screening identifies the IKBKE oncogene as a positive regulator of autophagy"

    Article Title: Activated kinase screening identifies the IKBKE oncogene as a positive regulator of autophagy

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1517855

    IKBKE is required for autophagy induced by the ERBB2 breast oncogene. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon pharmacological inhibition of IKBKE activity by CYT387 (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P
    Figure Legend Snippet: IKBKE is required for autophagy induced by the ERBB2 breast oncogene. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon pharmacological inhibition of IKBKE activity by CYT387 (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P

    Techniques Used: Confocal Microscopy, Multiple Displacement Amplification, Expressing, Immunofluorescence, Transfection, Negative Control, Staining, Software, Inhibition, Activity Assay

    A role for autophagy in IKBKE-dependent normal breast epithelial cell transformation and TNBC proliferation. (a) Proliferation assay of MDA-MB-231 cells upon downregulation of endogenous IKBKE, ATG5, ULK1 protein levels by transfection with the indicated siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679, #680 and siRNA for ATG5 and ULK1 ). Cell viability was evaluated 72 h post-transfection by counting cells in triplicate with a Z2 Coulter Counter. Data were processed in Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Same as in (a), but using MDA-MB-468 TNBC cells. (c) Cell count of 1-7HB2 cell stably expressing IKBKE (WT), evaluating cell proliferation, by counting cells in triplicate with a Z2 Coulter Counter, upon downregulation (72 h post-transfection) of endogenous ATG5 and ULK1 with specific siRNA. A scrambled siRNA was used as a negative control. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Proliferation assay of 1-7HB2 cell stably expressing IKBKE (WT) upon pharmacological inhibition of autophagic activity by SAR-405 (10 μM) and Spautin-1 (100 μM). Cell proliferation was evaluated after 72-h treatment by counting cells in triplicate with a Z2 Coulter Counter. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.
    Figure Legend Snippet: A role for autophagy in IKBKE-dependent normal breast epithelial cell transformation and TNBC proliferation. (a) Proliferation assay of MDA-MB-231 cells upon downregulation of endogenous IKBKE, ATG5, ULK1 protein levels by transfection with the indicated siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679, #680 and siRNA for ATG5 and ULK1 ). Cell viability was evaluated 72 h post-transfection by counting cells in triplicate with a Z2 Coulter Counter. Data were processed in Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Same as in (a), but using MDA-MB-468 TNBC cells. (c) Cell count of 1-7HB2 cell stably expressing IKBKE (WT), evaluating cell proliferation, by counting cells in triplicate with a Z2 Coulter Counter, upon downregulation (72 h post-transfection) of endogenous ATG5 and ULK1 with specific siRNA. A scrambled siRNA was used as a negative control. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Proliferation assay of 1-7HB2 cell stably expressing IKBKE (WT) upon pharmacological inhibition of autophagic activity by SAR-405 (10 μM) and Spautin-1 (100 μM). Cell proliferation was evaluated after 72-h treatment by counting cells in triplicate with a Z2 Coulter Counter. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.

    Techniques Used: Transformation Assay, Proliferation Assay, Multiple Displacement Amplification, Transfection, Software, Cell Counting, Stable Transfection, Expressing, Negative Control, Inhibition, Activity Assay

    The IKBKE human oncogene induces autophagy in MDA-MB-231 breast cancer cells. (a) Endogenous IKBKE protein levels were tested in indicated cell lines, by WB analysis. (b) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and 2 unrelated specific siRNA against human IKBKE , #679 and #680). Where indicated, samples were treated with 400 nM BAF or with starvation medium (STV) for 5 h. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels, is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (c) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells by confocal microscopy analysis of MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 24 h. In these representative images, SQSTM1 is visualized in green and DAPI-stained nuclei in blue. SQSTM1-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Autophagic flux was evaluated in in MDA-MB-231 cells transfected with Scr siRNA and siRNA specific for IKBKE , Followed by rescue with IKBKE (WT) overexpression. Where indicated, 4 h treatment with 400 nM BAF was performed. In these representative images, LC3B is visualized in green, IKBKE (WT) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.
    Figure Legend Snippet: The IKBKE human oncogene induces autophagy in MDA-MB-231 breast cancer cells. (a) Endogenous IKBKE protein levels were tested in indicated cell lines, by WB analysis. (b) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and 2 unrelated specific siRNA against human IKBKE , #679 and #680). Where indicated, samples were treated with 400 nM BAF or with starvation medium (STV) for 5 h. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels, is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (c) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells by confocal microscopy analysis of MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 24 h. In these representative images, SQSTM1 is visualized in green and DAPI-stained nuclei in blue. SQSTM1-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Autophagic flux was evaluated in in MDA-MB-231 cells transfected with Scr siRNA and siRNA specific for IKBKE , Followed by rescue with IKBKE (WT) overexpression. Where indicated, 4 h treatment with 400 nM BAF was performed. In these representative images, LC3B is visualized in green, IKBKE (WT) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.

    Techniques Used: Multiple Displacement Amplification, Western Blot, Transfection, Confocal Microscopy, Staining, Software, Over Expression

    IKBKE is required for autophagy induced by the AKT transforming pathway. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon pharmacological inhibition of IKBKE activity by the CYT387 drug (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars, 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P
    Figure Legend Snippet: IKBKE is required for autophagy induced by the AKT transforming pathway. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon pharmacological inhibition of IKBKE activity by the CYT387 drug (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars, 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P

    Techniques Used: Confocal Microscopy, Multiple Displacement Amplification, Expressing, Immunofluorescence, Transfection, Negative Control, Staining, Software, Inhibition, Activity Assay

    29) Product Images from "C/EBPβ deletion in oncogenic Ras skin tumors is a synthetic lethal event"

    Article Title: C/EBPβ deletion in oncogenic Ras skin tumors is a synthetic lethal event

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1103-y

    Oncogenic Ras tumor regression following deletion of C/EBPβ is dependent on p53. a Schematic of the K14-CreER tam ;C/EBPβ flox/flox ;p53 flox/flox (DIKO) transgenic mouse model system. b Western blot analysis of C/EBPβ and p53 in epidermal lysates of tamoxifen-treated Cre, IKOβ, IKOp53, and DIKO mice. c IHC staining for C/EBPβ and p53 in normal DIKO mouse skin following vehicle and tamoxifen treatment. D dermis, E epidermis. Scale bar: 10 μm. d H E and IHC staining for C/EBPβ and p53 in DMBA/TPA-induced mouse squamous papillomas following tamoxifen dosing. e Tumor multiplicity in DMBA/TPA-induced skin squamous papilloma in Cre and DIKO mice dosed with tamoxifen at 19 weeks of skin tumor promotion (Cre N = 10 mice, DIKO N = 11 mice). f Tumor incidence in Cre and DIKO mice. g Tumor volume in Cre and DIKO mice. h Tumor multiplicity in Cre, IKOβ, IKOp53, and DIKO mice before and after tamoxifen collected 2 weeks after initial tamoxifen dose (Cre N = 5 mice, IKOβ N = 3 mice, IKOp53 N = 5 mice, DIKO N = 5 mice). # indicates IKOβ is significantly different from Cre p
    Figure Legend Snippet: Oncogenic Ras tumor regression following deletion of C/EBPβ is dependent on p53. a Schematic of the K14-CreER tam ;C/EBPβ flox/flox ;p53 flox/flox (DIKO) transgenic mouse model system. b Western blot analysis of C/EBPβ and p53 in epidermal lysates of tamoxifen-treated Cre, IKOβ, IKOp53, and DIKO mice. c IHC staining for C/EBPβ and p53 in normal DIKO mouse skin following vehicle and tamoxifen treatment. D dermis, E epidermis. Scale bar: 10 μm. d H E and IHC staining for C/EBPβ and p53 in DMBA/TPA-induced mouse squamous papillomas following tamoxifen dosing. e Tumor multiplicity in DMBA/TPA-induced skin squamous papilloma in Cre and DIKO mice dosed with tamoxifen at 19 weeks of skin tumor promotion (Cre N = 10 mice, DIKO N = 11 mice). f Tumor incidence in Cre and DIKO mice. g Tumor volume in Cre and DIKO mice. h Tumor multiplicity in Cre, IKOβ, IKOp53, and DIKO mice before and after tamoxifen collected 2 weeks after initial tamoxifen dose (Cre N = 5 mice, IKOβ N = 3 mice, IKOp53 N = 5 mice, DIKO N = 5 mice). # indicates IKOβ is significantly different from Cre p

    Techniques Used: Transgenic Assay, Western Blot, Mouse Assay, Immunohistochemistry, Staining

    Spatial and temporal regulation of C/EBPβ in epidermis and in pre-existing oncogenic Ras skin tumors. a Immunohistochemical (IHC) staining for C/EBPβ in a DMBA/TPA-induced mouse squamous papilloma. b Schematic of the K14-CreER tam ;C/EBPβ flox/flox (IKOβ) transgenic mouse model system. c IHC staining for C/EBPβ in IKOβ mouse skin following vehicle or tamoxifen dosing. D dermis, E epidermis. d Western blot analysis for C/EBPβ in epidermal lysates from tamoxifen-treated Cre and IKOβ mice. e H E and C/EBPβ IHC staining in DMBA/TPA-induced mouse squamous papilloma following vehicle and tamoxifen dosing. f DNA sequence of the 61st codon of Ha-Ras in mouse tail and DMBA/TPA-induced squamous papillomas
    Figure Legend Snippet: Spatial and temporal regulation of C/EBPβ in epidermis and in pre-existing oncogenic Ras skin tumors. a Immunohistochemical (IHC) staining for C/EBPβ in a DMBA/TPA-induced mouse squamous papilloma. b Schematic of the K14-CreER tam ;C/EBPβ flox/flox (IKOβ) transgenic mouse model system. c IHC staining for C/EBPβ in IKOβ mouse skin following vehicle or tamoxifen dosing. D dermis, E epidermis. d Western blot analysis for C/EBPβ in epidermal lysates from tamoxifen-treated Cre and IKOβ mice. e H E and C/EBPβ IHC staining in DMBA/TPA-induced mouse squamous papilloma following vehicle and tamoxifen dosing. f DNA sequence of the 61st codon of Ha-Ras in mouse tail and DMBA/TPA-induced squamous papillomas

    Techniques Used: Immunohistochemistry, Staining, Transgenic Assay, Western Blot, Mouse Assay, Sequencing

    Oncogenic Ras skin tumors depend on C/EBPβ for survival. a Tumor multiplicity in Cre and IKOβ before and after mice were dosed with tamoxifen starting at 19 weeks, b tumor incidence, and c tumor volume remaining (Cre mice n = 10, IKOβ mice n = 11). d Representative photographs of skin tumors before and after tamoxifen on the same mouse. e Tumor multiplicity in Cre and IKOβ mice before and after mice were dosed with tamoxifen starting at 34 weeks, and f tumor incidence (Cre mice n = 15, IKOβ mice n = 18). Data are expressed as means. *indicates significantly different from IKOβ controls p
    Figure Legend Snippet: Oncogenic Ras skin tumors depend on C/EBPβ for survival. a Tumor multiplicity in Cre and IKOβ before and after mice were dosed with tamoxifen starting at 19 weeks, b tumor incidence, and c tumor volume remaining (Cre mice n = 10, IKOβ mice n = 11). d Representative photographs of skin tumors before and after tamoxifen on the same mouse. e Tumor multiplicity in Cre and IKOβ mice before and after mice were dosed with tamoxifen starting at 34 weeks, and f tumor incidence (Cre mice n = 15, IKOβ mice n = 18). Data are expressed as means. *indicates significantly different from IKOβ controls p

    Techniques Used: Mouse Assay

    Skin tumors exhibit DNA damage and regressing C/EBPβ-deficient tumors do not display differences in proliferation, senescence, differentiation or inflammation. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen and tumors were collected two weeks after the initial vehicle or tamoxifen dose. a Quantification of IHC staining for Phospho-Histone H2A.X (γH2AX) in adjacent normal epidermis (vehicle n = 4 mice, tamoxifen n = 4 mice). b Quantification of γH2AX in tumor (vehicle n = 13 tumors, tamoxifen n = 8 tumors). c Quantification of IHC staining for BrdU incorporation in tumors (vehicle n = 17 tumors, tamoxifen n = 9 tumors). d Quantification of IHC staining for Ki67 in tumors (vehicle n = 13 tumors, tamoxifen n = 9 tumors). e Photograph of IHC staining for Keratin 5 (left) and Keratin 10 (right). Quantification of f neutrophil infiltration, g mononuclear leukocyte infiltration, and h total inflammation (vehicle n = 18 tumors, tamoxifen n = 18 tumors) from H E-stained tumor sections. i Quantification of CD4 IHC staining in tumor parenchyma and stroma (vehicle n = 7 tumors, tamoxifen n = 7 tumors). j Quantification of CD8 IHC staining in tumor parenchyma and stroma (vehicle n = 10 tumors, tamoxifen n = 9 tumors). k Quantification of F4/80 IHC staining in tumor parenchyma and stroma (vehicle n = 11 tumors, tamoxifen n = 9 tumors). Data are expressed as mean ± SD or percent counts. *indicates significantly different from controls p
    Figure Legend Snippet: Skin tumors exhibit DNA damage and regressing C/EBPβ-deficient tumors do not display differences in proliferation, senescence, differentiation or inflammation. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen and tumors were collected two weeks after the initial vehicle or tamoxifen dose. a Quantification of IHC staining for Phospho-Histone H2A.X (γH2AX) in adjacent normal epidermis (vehicle n = 4 mice, tamoxifen n = 4 mice). b Quantification of γH2AX in tumor (vehicle n = 13 tumors, tamoxifen n = 8 tumors). c Quantification of IHC staining for BrdU incorporation in tumors (vehicle n = 17 tumors, tamoxifen n = 9 tumors). d Quantification of IHC staining for Ki67 in tumors (vehicle n = 13 tumors, tamoxifen n = 9 tumors). e Photograph of IHC staining for Keratin 5 (left) and Keratin 10 (right). Quantification of f neutrophil infiltration, g mononuclear leukocyte infiltration, and h total inflammation (vehicle n = 18 tumors, tamoxifen n = 18 tumors) from H E-stained tumor sections. i Quantification of CD4 IHC staining in tumor parenchyma and stroma (vehicle n = 7 tumors, tamoxifen n = 7 tumors). j Quantification of CD8 IHC staining in tumor parenchyma and stroma (vehicle n = 10 tumors, tamoxifen n = 9 tumors). k Quantification of F4/80 IHC staining in tumor parenchyma and stroma (vehicle n = 11 tumors, tamoxifen n = 9 tumors). Data are expressed as mean ± SD or percent counts. *indicates significantly different from controls p

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining, BrdU Incorporation Assay

    Regressing C/EBPβ-deficient tumors display tumor-specific elevations in apoptosis and p53 protein, whereas adjacent C/EBPβ-depleted skin is unaffected. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen at 19 weeks and tumors were collected 2 weeks later. a Deletion of C/EBPβ was confirmed by IHC staining. b Photograph displaying apoptotic cells (left) and quantification of apoptosis in H E-stained tumors (right) (vehicle n = 18 tumors, tamoxifen n = 10 tumors). c Photograph displaying p53 IHC staining (left) and quantification of IHC staining for p53 (right) (vehicle n = 15 tumors, tamoxifen n = 12 tumors). d Quantification of apoptosis in H E-stained adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). e Quantification of IHC staining for p53 in adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). f Quantification of p53 mRNA from tumors (vehicle n = 3 tumors, tamoxifen n = 3 tumor). g Quantification of IHC staining for p53 phosphorylated on serine 18 (vehicle n = 9 tumors, tamoxifen n = 11 tumors). Data are expressed as mean ± SD. *indicates significantly different from controls p
    Figure Legend Snippet: Regressing C/EBPβ-deficient tumors display tumor-specific elevations in apoptosis and p53 protein, whereas adjacent C/EBPβ-depleted skin is unaffected. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen at 19 weeks and tumors were collected 2 weeks later. a Deletion of C/EBPβ was confirmed by IHC staining. b Photograph displaying apoptotic cells (left) and quantification of apoptosis in H E-stained tumors (right) (vehicle n = 18 tumors, tamoxifen n = 10 tumors). c Photograph displaying p53 IHC staining (left) and quantification of IHC staining for p53 (right) (vehicle n = 15 tumors, tamoxifen n = 12 tumors). d Quantification of apoptosis in H E-stained adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). e Quantification of IHC staining for p53 in adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). f Quantification of p53 mRNA from tumors (vehicle n = 3 tumors, tamoxifen n = 3 tumor). g Quantification of IHC staining for p53 phosphorylated on serine 18 (vehicle n = 9 tumors, tamoxifen n = 11 tumors). Data are expressed as mean ± SD. *indicates significantly different from controls p

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining

    30) Product Images from "Activated kinase screening identifies the IKBKE oncogene as a positive regulator of autophagy"

    Article Title: Activated kinase screening identifies the IKBKE oncogene as a positive regulator of autophagy

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1517855

    Screening of a library of MYR kinases to identify regulators of the autophagic process. (a) Schematic representation of the experimental approach used to perform the screening of 170 constitutively active kinases for their ability to modulate the autophagic process, in HeLa cells. (b) Normalized LC3B-II levels, obtained by western blot (WB) analysis, of samples transfected with each activated kinase, in Full Medium (FM) conditions. The graph shows also the mean value of the screening and its standard deviation (σ). (c) Same as in (b), but after 1 h of 100 nM bafilomycin A 1 (BAF) treatment. (d) Integration of results shown in (b) and (c), to show potential positive regulators of the autophagic flux, identified as kinases inducing normalized LC3B-II values higher than + σ in both FM and BAF conditions (in colors). (e) WB analysis to evaluate autophagic flux of HeLa cells upon myrFLAG-PRKAA1 overexpression. Transfection with an empty vector was used as a negative control. Transfection with HA-MAPK15 was used as a positive control for activation of the autophagic flux. Where indicated, 1 h treatment with 100 nM BAF was performed. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels (used as loading control), is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.
    Figure Legend Snippet: Screening of a library of MYR kinases to identify regulators of the autophagic process. (a) Schematic representation of the experimental approach used to perform the screening of 170 constitutively active kinases for their ability to modulate the autophagic process, in HeLa cells. (b) Normalized LC3B-II levels, obtained by western blot (WB) analysis, of samples transfected with each activated kinase, in Full Medium (FM) conditions. The graph shows also the mean value of the screening and its standard deviation (σ). (c) Same as in (b), but after 1 h of 100 nM bafilomycin A 1 (BAF) treatment. (d) Integration of results shown in (b) and (c), to show potential positive regulators of the autophagic flux, identified as kinases inducing normalized LC3B-II values higher than + σ in both FM and BAF conditions (in colors). (e) WB analysis to evaluate autophagic flux of HeLa cells upon myrFLAG-PRKAA1 overexpression. Transfection with an empty vector was used as a negative control. Transfection with HA-MAPK15 was used as a positive control for activation of the autophagic flux. Where indicated, 1 h treatment with 100 nM BAF was performed. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels (used as loading control), is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.

    Techniques Used: Western Blot, Transfection, Standard Deviation, Over Expression, Plasmid Preparation, Negative Control, Positive Control, Activation Assay

    31) Product Images from "Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47"

    Article Title: Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47

    Journal: European Journal of Human Genetics

    doi: 10.1038/ejhg.2011.150

    Relative abundance of different histone modifications at CT47 in human male (WA14) and female (WA9) pluripotent hESCs and in differentiated hEBs. The relative abundance of H3K4me2, associated with transcriptionally permissive chromatin, is increasing during differentiation in male and female samples. The repressive chromatin mark H3K9me3 is detected at high levels at pluripotent state and slightly decreases after differentiation. H3K27me3, associated with transcriptional repression, is detected in pluripotent state and increasing during differentiation.
    Figure Legend Snippet: Relative abundance of different histone modifications at CT47 in human male (WA14) and female (WA9) pluripotent hESCs and in differentiated hEBs. The relative abundance of H3K4me2, associated with transcriptionally permissive chromatin, is increasing during differentiation in male and female samples. The repressive chromatin mark H3K9me3 is detected at high levels at pluripotent state and slightly decreases after differentiation. H3K27me3, associated with transcriptional repression, is detected in pluripotent state and increasing during differentiation.

    Techniques Used:

    32) Product Images from "Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47"

    Article Title: Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47

    Journal: European Journal of Human Genetics

    doi: 10.1038/ejhg.2011.150

    ( a ) Expression levels of CT47 mRNA in different SCLC cell lines relative to the expression in human testis. Commercially available human total testis RNA and RNA isolated from SCLC lines were used for cDNA synthesis under identical conditions. CT47 expression levels are normalized to the expression levels measured in the testis. Different SCLC lines have different, but low levels of CT47 expression compared to the testis. Relative abundance of histone modifications and EZH2 at the CT47 promoter ( b ), exon 3 ( c ) and the distal region in SCLC cell lines ( d ). SCLC cell lines show individual variation in the abundance of different histone modifications. Generally, a loss of the repressive chromatin mark H3K9me3 can be observed in SCLCs. H3K27me3 levels are higher in SCLCs at the promoter and exon 3 region than in LCLs, but lower at the distal region. The relative abundance of the PRC2 component EZH2 responsible for generating H2K27me3 is dramatically reduced in SCLCs compared to LCLs at all region studied. ( e ) DNA methylation levels at CpGs located in the CT47 promoter region in LCL and SCLC samples. The methylation level of seven different CpGs, next to the transcriptional start site of CT47 , was determined by bisulfite sequencing and quantified by ESME program. There is a significant difference between the methylation level of LCLs and SCLCs at every CpG tested ( P
    Figure Legend Snippet: ( a ) Expression levels of CT47 mRNA in different SCLC cell lines relative to the expression in human testis. Commercially available human total testis RNA and RNA isolated from SCLC lines were used for cDNA synthesis under identical conditions. CT47 expression levels are normalized to the expression levels measured in the testis. Different SCLC lines have different, but low levels of CT47 expression compared to the testis. Relative abundance of histone modifications and EZH2 at the CT47 promoter ( b ), exon 3 ( c ) and the distal region in SCLC cell lines ( d ). SCLC cell lines show individual variation in the abundance of different histone modifications. Generally, a loss of the repressive chromatin mark H3K9me3 can be observed in SCLCs. H3K27me3 levels are higher in SCLCs at the promoter and exon 3 region than in LCLs, but lower at the distal region. The relative abundance of the PRC2 component EZH2 responsible for generating H2K27me3 is dramatically reduced in SCLCs compared to LCLs at all region studied. ( e ) DNA methylation levels at CpGs located in the CT47 promoter region in LCL and SCLC samples. The methylation level of seven different CpGs, next to the transcriptional start site of CT47 , was determined by bisulfite sequencing and quantified by ESME program. There is a significant difference between the methylation level of LCLs and SCLCs at every CpG tested ( P

    Techniques Used: Expressing, Isolation, DNA Methylation Assay, Methylation, Methylation Sequencing

    33) Product Images from "Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix"

    Article Title: Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-16960-8

    Comparison of different storage temperature and trehalose for FTA Elute card stored for 2 days. ( A ) Untreated or 50 mg/ml trehalose-treated 6 mm FTA Elute card disc punch-out spotted with 20 µl serum were dried and stored at RT, 4 °C or −20 °C for 2 days before RNA was extracted using QIAzol lysis reagent. 10 miRNAs were quantified by RT-qPCR and their copy numbers were presented. Neat refers to copy number of each individual miRNA obtained from 20 µl serum extracted directly using QIAzol lysis reagent. The individual % miRNA recovery was compared between untreated and trehalose-treated FTA Elute card disc punch-out. ( B ) Average % miRNA recovery of all 10 miRNAs was calculated and plotted to compare the effect of pre-treatment of FTA Elute card disc punch-out with 50mg/ml trehalose. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data between selected pairs. Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; **P ≤ 0.01; * P ≤ 0.05, n = 3).
    Figure Legend Snippet: Comparison of different storage temperature and trehalose for FTA Elute card stored for 2 days. ( A ) Untreated or 50 mg/ml trehalose-treated 6 mm FTA Elute card disc punch-out spotted with 20 µl serum were dried and stored at RT, 4 °C or −20 °C for 2 days before RNA was extracted using QIAzol lysis reagent. 10 miRNAs were quantified by RT-qPCR and their copy numbers were presented. Neat refers to copy number of each individual miRNA obtained from 20 µl serum extracted directly using QIAzol lysis reagent. The individual % miRNA recovery was compared between untreated and trehalose-treated FTA Elute card disc punch-out. ( B ) Average % miRNA recovery of all 10 miRNAs was calculated and plotted to compare the effect of pre-treatment of FTA Elute card disc punch-out with 50mg/ml trehalose. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data between selected pairs. Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; **P ≤ 0.01; * P ≤ 0.05, n = 3).

    Techniques Used: Lysis, Quantitative RT-PCR

    miRNA expression level in GC patient and paired controls. ( A ) 20 µl of each of the 10 GC patients and 10 paired control serum were spotted on individual 6 mm FTA Elute card disc punch-out. 8 miRNAs were quantified and miRNA recovery was compared with the sample in which RNA was isolated directly from 20 µl serum using QIAzol lysis reagent (neat). ( B ) Correlation of miRNA representation from neat serum or FTA Elute card discs. Each experimental condition consisted of 10 biological replicates and data were presented as mean ± SEM. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data for selected pairs (*** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns: not significant).
    Figure Legend Snippet: miRNA expression level in GC patient and paired controls. ( A ) 20 µl of each of the 10 GC patients and 10 paired control serum were spotted on individual 6 mm FTA Elute card disc punch-out. 8 miRNAs were quantified and miRNA recovery was compared with the sample in which RNA was isolated directly from 20 µl serum using QIAzol lysis reagent (neat). ( B ) Correlation of miRNA representation from neat serum or FTA Elute card discs. Each experimental condition consisted of 10 biological replicates and data were presented as mean ± SEM. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data for selected pairs (*** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns: not significant).

    Techniques Used: Expressing, Isolation, Lysis

    Effect on miRNA yield during long-term FTA Elute card storage. ( A ) 20 µl serum spotted on trehalose-treated 6 mm FTA Elute card disc punch-out was subjected to accelerated aging testing of 1, 6 and 12 months before RNA was extracted using QIAzol lysis reagent. 10 miRNAs were quantified. The copy number of each miRNA was compared against the copy number obtained after storage at RT for 2 days. ( B ) Average % miRNA recovery of all 10 miRNAs for accelerated aging of 1, 6, 12 months was determined and compared with 2 days. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data. Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; **P ≤ 0.01; * P ≤ 0.05; ns: not significant, n = 3).
    Figure Legend Snippet: Effect on miRNA yield during long-term FTA Elute card storage. ( A ) 20 µl serum spotted on trehalose-treated 6 mm FTA Elute card disc punch-out was subjected to accelerated aging testing of 1, 6 and 12 months before RNA was extracted using QIAzol lysis reagent. 10 miRNAs were quantified. The copy number of each miRNA was compared against the copy number obtained after storage at RT for 2 days. ( B ) Average % miRNA recovery of all 10 miRNAs for accelerated aging of 1, 6, 12 months was determined and compared with 2 days. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data. Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; **P ≤ 0.01; * P ≤ 0.05; ns: not significant, n = 3).

    Techniques Used: Lysis

    miRNA recovery from serum-spotted FTA Elute cards using water and heat was inefficient. ( A ) RNA was extracted directly from 20 µl serum (black) or 20 µl plasma collected in EDTA-containing tube (plasma-EDTA) (white) using QIAzol lysis reagent. 10 miRNAs were quantified by RT-qPCR based on their copy numbers. RNA was extracted from a 6 mm FTA Elute card disc punch-out spotted with 20 µl serum (black) or 20 µl plasma-EDTA (white), using water and heating at 95 °C for 30 min. 10 miRNAs were quantified by RT-qPCR and their copy numbers ( B ) and % miRNA recovery ( C ) were presented. (% recovery = (Copy number of miRNA extracted from FTA Elute card disc punch-out/Copy number of miRNA extracted directly from serum) × 100%). Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns: not significant, Student’s t -test, n = 3).
    Figure Legend Snippet: miRNA recovery from serum-spotted FTA Elute cards using water and heat was inefficient. ( A ) RNA was extracted directly from 20 µl serum (black) or 20 µl plasma collected in EDTA-containing tube (plasma-EDTA) (white) using QIAzol lysis reagent. 10 miRNAs were quantified by RT-qPCR based on their copy numbers. RNA was extracted from a 6 mm FTA Elute card disc punch-out spotted with 20 µl serum (black) or 20 µl plasma-EDTA (white), using water and heating at 95 °C for 30 min. 10 miRNAs were quantified by RT-qPCR and their copy numbers ( B ) and % miRNA recovery ( C ) were presented. (% recovery = (Copy number of miRNA extracted from FTA Elute card disc punch-out/Copy number of miRNA extracted directly from serum) × 100%). Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns: not significant, Student’s t -test, n = 3).

    Techniques Used: Lysis, Quantitative RT-PCR

    34) Product Images from "DUX4 promotes transcription of FRG2 by directly activating its promoter in facioscapulohumeral muscular dystrophy"

    Article Title: DUX4 promotes transcription of FRG2 by directly activating its promoter in facioscapulohumeral muscular dystrophy

    Journal: Skeletal Muscle

    doi: 10.1186/2044-5040-4-19

    FRG2 activation in FSHD derived differentiating myoblasts. (A) Schematic representation of the FSHD locus on chromosome 4q35. Rectangles indicate the different genes, arrows their transcriptional direction. Triangles represent D4Z4 repeat units and the single inverted repeat unit upstream of FRG2 . Each unit contains the full DUX4 ORF, only the last repeat unit produces a stable transcript in FSHD patients. The dashed line indicates the duplicated region present on chromosome 10q26. (B) qRT-PCR analysis of mean FRG2 expression levels in control (C), FSHD1 (F1) and FSHD2 (F2) derived proliferating myoblasts (MB) and differentiating myotubes (MT) shows the significant activation of FRG2 during differentiation only in F1 and F2 derived cells. Relative expression was determined using GAPDH and GUSB as reference genes. Sample numbers are indicated and error bars represent the standard error of the mean. Asterisks indicate significant differences based on a one-way ANOVA ( P = 0.0014), followed by pairwise comparison using Bonferroni correction, NS = non-significant. (C) Genomic snapshot (location indicated at the bottom) of RNA sequencing data of two control, two FSHD1 and two FSHD2 derived proliferating myoblasts (MB) and differentiating myotubes (MT) confirms the full length expression of FRG2 in differentiating myotubes originating from FSHD individuals.
    Figure Legend Snippet: FRG2 activation in FSHD derived differentiating myoblasts. (A) Schematic representation of the FSHD locus on chromosome 4q35. Rectangles indicate the different genes, arrows their transcriptional direction. Triangles represent D4Z4 repeat units and the single inverted repeat unit upstream of FRG2 . Each unit contains the full DUX4 ORF, only the last repeat unit produces a stable transcript in FSHD patients. The dashed line indicates the duplicated region present on chromosome 10q26. (B) qRT-PCR analysis of mean FRG2 expression levels in control (C), FSHD1 (F1) and FSHD2 (F2) derived proliferating myoblasts (MB) and differentiating myotubes (MT) shows the significant activation of FRG2 during differentiation only in F1 and F2 derived cells. Relative expression was determined using GAPDH and GUSB as reference genes. Sample numbers are indicated and error bars represent the standard error of the mean. Asterisks indicate significant differences based on a one-way ANOVA ( P = 0.0014), followed by pairwise comparison using Bonferroni correction, NS = non-significant. (C) Genomic snapshot (location indicated at the bottom) of RNA sequencing data of two control, two FSHD1 and two FSHD2 derived proliferating myoblasts (MB) and differentiating myotubes (MT) confirms the full length expression of FRG2 in differentiating myotubes originating from FSHD individuals.

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

    35) Product Images from "DUX4 promotes transcription of FRG2 by directly activating its promoter in facioscapulohumeral muscular dystrophy"

    Article Title: DUX4 promotes transcription of FRG2 by directly activating its promoter in facioscapulohumeral muscular dystrophy

    Journal: Skeletal Muscle

    doi: 10.1186/2044-5040-4-19

    FRG2 activation in FSHD derived differentiating myoblasts. (A) Schematic representation of the FSHD locus on chromosome 4q35. Rectangles indicate the different genes, arrows their transcriptional direction. Triangles represent D4Z4 repeat units and the single inverted repeat unit upstream of FRG2 . Each unit contains the full DUX4 ORF, only the last repeat unit produces a stable transcript in FSHD patients. The dashed line indicates the duplicated region present on chromosome 10q26. (B) qRT-PCR analysis of mean FRG2 expression levels in control (C), FSHD1 (F1) and FSHD2 (F2) derived proliferating myoblasts (MB) and differentiating myotubes (MT) shows the significant activation of FRG2 during differentiation only in F1 and F2 derived cells. Relative expression was determined using GAPDH and GUSB as reference genes. Sample numbers are indicated and error bars represent the standard error of the mean. Asterisks indicate significant differences based on a one-way ANOVA ( P = 0.0014), followed by pairwise comparison using Bonferroni correction, NS = non-significant. (C) Genomic snapshot (location indicated at the bottom) of RNA sequencing data of two control, two FSHD1 and two FSHD2 derived proliferating myoblasts (MB) and differentiating myotubes (MT) confirms the full length expression of FRG2 in differentiating myotubes originating from FSHD individuals.
    Figure Legend Snippet: FRG2 activation in FSHD derived differentiating myoblasts. (A) Schematic representation of the FSHD locus on chromosome 4q35. Rectangles indicate the different genes, arrows their transcriptional direction. Triangles represent D4Z4 repeat units and the single inverted repeat unit upstream of FRG2 . Each unit contains the full DUX4 ORF, only the last repeat unit produces a stable transcript in FSHD patients. The dashed line indicates the duplicated region present on chromosome 10q26. (B) qRT-PCR analysis of mean FRG2 expression levels in control (C), FSHD1 (F1) and FSHD2 (F2) derived proliferating myoblasts (MB) and differentiating myotubes (MT) shows the significant activation of FRG2 during differentiation only in F1 and F2 derived cells. Relative expression was determined using GAPDH and GUSB as reference genes. Sample numbers are indicated and error bars represent the standard error of the mean. Asterisks indicate significant differences based on a one-way ANOVA ( P = 0.0014), followed by pairwise comparison using Bonferroni correction, NS = non-significant. (C) Genomic snapshot (location indicated at the bottom) of RNA sequencing data of two control, two FSHD1 and two FSHD2 derived proliferating myoblasts (MB) and differentiating myotubes (MT) confirms the full length expression of FRG2 in differentiating myotubes originating from FSHD individuals.

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

    36) Product Images from "Tendinosis-like changes in denervated rat Achilles tendon"

    Article Title: Tendinosis-like changes in denervated rat Achilles tendon

    Journal: BMC Musculoskeletal Disorders

    doi: 10.1186/s12891-018-2353-7

    Denervated tendons express molecules associated with tendinosis. ( a ) Expression of genes related to tendinosis using qRT-PCR: substance P ( Tac1 ) and its preferred receptor NK-1R ( Tacr1 ). ( b ) Immunohistochemistry showing positive staining for NK-1R in a denervated tendon. Bar = 5 μm. ** indicates p
    Figure Legend Snippet: Denervated tendons express molecules associated with tendinosis. ( a ) Expression of genes related to tendinosis using qRT-PCR: substance P ( Tac1 ) and its preferred receptor NK-1R ( Tacr1 ). ( b ) Immunohistochemistry showing positive staining for NK-1R in a denervated tendon. Bar = 5 μm. ** indicates p

    Techniques Used: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining

    37) Product Images from "SMCHD1 regulates a limited set of gene clusters on autosomal chromosomes"

    Article Title: SMCHD1 regulates a limited set of gene clusters on autosomal chromosomes

    Journal: Skeletal Muscle

    doi: 10.1186/s13395-017-0129-7

    The effects of SMCHD1 KD in non-DUX4 permissive 4qB cells on PCDH β cluster expression. The expression level of SMCHD1 after SMCHD1 shRNA KD in comparison with control KD shRNAs targeting luciferase and GFP is shown by RT-qPCR analysis in primary ( a ) myoblast and ( b ) myotube cells. RNA expression analysis was then assessed for PCDH β cluster isoform members in primary ( c ) myoblast and ( d ) myotube cells. Results represent qRT-PCR analysis of the indicated gene after normalization to the internal control gene GUS1 . For each gene, the value of expression in the control shRNA samples was then arbitrarily set to 1. * P
    Figure Legend Snippet: The effects of SMCHD1 KD in non-DUX4 permissive 4qB cells on PCDH β cluster expression. The expression level of SMCHD1 after SMCHD1 shRNA KD in comparison with control KD shRNAs targeting luciferase and GFP is shown by RT-qPCR analysis in primary ( a ) myoblast and ( b ) myotube cells. RNA expression analysis was then assessed for PCDH β cluster isoform members in primary ( c ) myoblast and ( d ) myotube cells. Results represent qRT-PCR analysis of the indicated gene after normalization to the internal control gene GUS1 . For each gene, the value of expression in the control shRNA samples was then arbitrarily set to 1. * P

    Techniques Used: Expressing, shRNA, Luciferase, Quantitative RT-PCR, RNA Expression

    38) Product Images from "Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47"

    Article Title: Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47

    Journal: European Journal of Human Genetics

    doi: 10.1038/ejhg.2011.150

    ( a ) Expression levels of CT47 mRNA in different SCLC cell lines relative to the expression in human testis. Commercially available human total testis RNA and RNA isolated from SCLC lines were used for cDNA synthesis under identical conditions. CT47 expression levels are normalized to the expression levels measured in the testis. Different SCLC lines have different, but low levels of CT47 expression compared to the testis. Relative abundance of histone modifications and EZH2 at the CT47 promoter ( b ), exon 3 ( c ) and the distal region in SCLC cell lines ( d ). SCLC cell lines show individual variation in the abundance of different histone modifications. Generally, a loss of the repressive chromatin mark H3K9me3 can be observed in SCLCs. H3K27me3 levels are higher in SCLCs at the promoter and exon 3 region than in LCLs, but lower at the distal region. The relative abundance of the PRC2 component EZH2 responsible for generating H2K27me3 is dramatically reduced in SCLCs compared to LCLs at all region studied. ( e ) DNA methylation levels at CpGs located in the CT47 promoter region in LCL and SCLC samples. The methylation level of seven different CpGs, next to the transcriptional start site of CT47 , was determined by bisulfite sequencing and quantified by ESME program. There is a significant difference between the methylation level of LCLs and SCLCs at every CpG tested ( P
    Figure Legend Snippet: ( a ) Expression levels of CT47 mRNA in different SCLC cell lines relative to the expression in human testis. Commercially available human total testis RNA and RNA isolated from SCLC lines were used for cDNA synthesis under identical conditions. CT47 expression levels are normalized to the expression levels measured in the testis. Different SCLC lines have different, but low levels of CT47 expression compared to the testis. Relative abundance of histone modifications and EZH2 at the CT47 promoter ( b ), exon 3 ( c ) and the distal region in SCLC cell lines ( d ). SCLC cell lines show individual variation in the abundance of different histone modifications. Generally, a loss of the repressive chromatin mark H3K9me3 can be observed in SCLCs. H3K27me3 levels are higher in SCLCs at the promoter and exon 3 region than in LCLs, but lower at the distal region. The relative abundance of the PRC2 component EZH2 responsible for generating H2K27me3 is dramatically reduced in SCLCs compared to LCLs at all region studied. ( e ) DNA methylation levels at CpGs located in the CT47 promoter region in LCL and SCLC samples. The methylation level of seven different CpGs, next to the transcriptional start site of CT47 , was determined by bisulfite sequencing and quantified by ESME program. There is a significant difference between the methylation level of LCLs and SCLCs at every CpG tested ( P

    Techniques Used: Expressing, Isolation, DNA Methylation Assay, Methylation, Methylation Sequencing

    39) Product Images from "Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47"

    Article Title: Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47

    Journal: European Journal of Human Genetics

    doi: 10.1038/ejhg.2011.150

    Relative abundance of different histone modifications at CT47 in human male (WA14) and female (WA9) pluripotent hESCs and in differentiated hEBs. The relative abundance of H3K4me2, associated with transcriptionally permissive chromatin, is increasing during differentiation in male and female samples. The repressive chromatin mark H3K9me3 is detected at high levels at pluripotent state and slightly decreases after differentiation. H3K27me3, associated with transcriptional repression, is detected in pluripotent state and increasing during differentiation.
    Figure Legend Snippet: Relative abundance of different histone modifications at CT47 in human male (WA14) and female (WA9) pluripotent hESCs and in differentiated hEBs. The relative abundance of H3K4me2, associated with transcriptionally permissive chromatin, is increasing during differentiation in male and female samples. The repressive chromatin mark H3K9me3 is detected at high levels at pluripotent state and slightly decreases after differentiation. H3K27me3, associated with transcriptional repression, is detected in pluripotent state and increasing during differentiation.

    Techniques Used:

    40) Product Images from "Activated kinase screening identifies the IKBKE oncogene as a positive regulator of autophagy"

    Article Title: Activated kinase screening identifies the IKBKE oncogene as a positive regulator of autophagy

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1517855

    IKBKE is required for autophagy induced by the ERBB2 breast oncogene. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon pharmacological inhibition of IKBKE activity by CYT387 (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P
    Figure Legend Snippet: IKBKE is required for autophagy induced by the ERBB2 breast oncogene. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon pharmacological inhibition of IKBKE activity by CYT387 (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P

    Techniques Used: Confocal Microscopy, Multiple Displacement Amplification, Expressing, Immunofluorescence, Transfection, Negative Control, Staining, Software, Inhibition, Activity Assay

    A role for autophagy in IKBKE-dependent normal breast epithelial cell transformation and TNBC proliferation. (a) Proliferation assay of MDA-MB-231 cells upon downregulation of endogenous IKBKE, ATG5, ULK1 protein levels by transfection with the indicated siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679, #680 and siRNA for ATG5 and ULK1 ). Cell viability was evaluated 72 h post-transfection by counting cells in triplicate with a Z2 Coulter Counter. Data were processed in Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Same as in (a), but using MDA-MB-468 TNBC cells. (c) Cell count of 1-7HB2 cell stably expressing IKBKE (WT), evaluating cell proliferation, by counting cells in triplicate with a Z2 Coulter Counter, upon downregulation (72 h post-transfection) of endogenous ATG5 and ULK1 with specific siRNA. A scrambled siRNA was used as a negative control. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Proliferation assay of 1-7HB2 cell stably expressing IKBKE (WT) upon pharmacological inhibition of autophagic activity by SAR-405 (10 μM) and Spautin-1 (100 μM). Cell proliferation was evaluated after 72-h treatment by counting cells in triplicate with a Z2 Coulter Counter. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.
    Figure Legend Snippet: A role for autophagy in IKBKE-dependent normal breast epithelial cell transformation and TNBC proliferation. (a) Proliferation assay of MDA-MB-231 cells upon downregulation of endogenous IKBKE, ATG5, ULK1 protein levels by transfection with the indicated siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679, #680 and siRNA for ATG5 and ULK1 ). Cell viability was evaluated 72 h post-transfection by counting cells in triplicate with a Z2 Coulter Counter. Data were processed in Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Same as in (a), but using MDA-MB-468 TNBC cells. (c) Cell count of 1-7HB2 cell stably expressing IKBKE (WT), evaluating cell proliferation, by counting cells in triplicate with a Z2 Coulter Counter, upon downregulation (72 h post-transfection) of endogenous ATG5 and ULK1 with specific siRNA. A scrambled siRNA was used as a negative control. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Proliferation assay of 1-7HB2 cell stably expressing IKBKE (WT) upon pharmacological inhibition of autophagic activity by SAR-405 (10 μM) and Spautin-1 (100 μM). Cell proliferation was evaluated after 72-h treatment by counting cells in triplicate with a Z2 Coulter Counter. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.

    Techniques Used: Transformation Assay, Proliferation Assay, Multiple Displacement Amplification, Transfection, Software, Cell Counting, Stable Transfection, Expressing, Negative Control, Inhibition, Activity Assay

    The IKBKE human oncogene induces autophagy in MDA-MB-231 breast cancer cells. (a) Endogenous IKBKE protein levels were tested in indicated cell lines, by WB analysis. (b) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and 2 unrelated specific siRNA against human IKBKE , #679 and #680). Where indicated, samples were treated with 400 nM BAF or with starvation medium (STV) for 5 h. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels, is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (c) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells by confocal microscopy analysis of MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 24 h. In these representative images, SQSTM1 is visualized in green and DAPI-stained nuclei in blue. SQSTM1-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Autophagic flux was evaluated in in MDA-MB-231 cells transfected with Scr siRNA and siRNA specific for IKBKE , Followed by rescue with IKBKE (WT) overexpression. Where indicated, 4 h treatment with 400 nM BAF was performed. In these representative images, LC3B is visualized in green, IKBKE (WT) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.
    Figure Legend Snippet: The IKBKE human oncogene induces autophagy in MDA-MB-231 breast cancer cells. (a) Endogenous IKBKE protein levels were tested in indicated cell lines, by WB analysis. (b) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and 2 unrelated specific siRNA against human IKBKE , #679 and #680). Where indicated, samples were treated with 400 nM BAF or with starvation medium (STV) for 5 h. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels, is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (c) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells by confocal microscopy analysis of MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 24 h. In these representative images, SQSTM1 is visualized in green and DAPI-stained nuclei in blue. SQSTM1-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Autophagic flux was evaluated in in MDA-MB-231 cells transfected with Scr siRNA and siRNA specific for IKBKE , Followed by rescue with IKBKE (WT) overexpression. Where indicated, 4 h treatment with 400 nM BAF was performed. In these representative images, LC3B is visualized in green, IKBKE (WT) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.

    Techniques Used: Multiple Displacement Amplification, Western Blot, Transfection, Confocal Microscopy, Staining, Software, Over Expression

    IKBKE is required for autophagy induced by the AKT transforming pathway. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon pharmacological inhibition of IKBKE activity by the CYT387 drug (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars, 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P
    Figure Legend Snippet: IKBKE is required for autophagy induced by the AKT transforming pathway. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon pharmacological inhibition of IKBKE activity by the CYT387 drug (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars, 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P

    Techniques Used: Confocal Microscopy, Multiple Displacement Amplification, Expressing, Immunofluorescence, Transfection, Negative Control, Staining, Software, Inhibition, Activity Assay

    41) Product Images from "CHRAC/ACF contribute to the repressive ground state of chromatin"

    Article Title: CHRAC/ACF contribute to the repressive ground state of chromatin

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800024

    (A) ChIP-qPCR to verify the recruitment of ISWI to the UAS sites. “UAS” and “5′ LacZ” denote the regions amplified by qPCR. Bars denote average % input enrichment (n = 3 biological replicates) ± SEM. “neg ctrl” represents a negative control locus (encompassing the Spt4 gene). (B) RT–qPCR assesses expression changes of the LacZ gene in early embryos (2–8 h) upon ACF1 tethering. Bars denote average fold-change over the control (GAL4DBD alone) (n = 3 biological replicates) ± SEM. (C) Screenshot of the genome browser representing the LacZ/mini-white reporter locus. Each track shows nucleosome dyad densities for each replicate of the corresponding genotype (as described in Fig 1A ). (D) Same as (C) but depicting the genomic region surrounding the integration site of the LacZ reporter transgene (dashed line).
    Figure Legend Snippet: (A) ChIP-qPCR to verify the recruitment of ISWI to the UAS sites. “UAS” and “5′ LacZ” denote the regions amplified by qPCR. Bars denote average % input enrichment (n = 3 biological replicates) ± SEM. “neg ctrl” represents a negative control locus (encompassing the Spt4 gene). (B) RT–qPCR assesses expression changes of the LacZ gene in early embryos (2–8 h) upon ACF1 tethering. Bars denote average fold-change over the control (GAL4DBD alone) (n = 3 biological replicates) ± SEM. (C) Screenshot of the genome browser representing the LacZ/mini-white reporter locus. Each track shows nucleosome dyad densities for each replicate of the corresponding genotype (as described in Fig 1A ). (D) Same as (C) but depicting the genomic region surrounding the integration site of the LacZ reporter transgene (dashed line).

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, Negative Control, Quantitative RT-PCR, Expressing

    ACF can be tethered to a reporter locus through GAL4-DBD. (A) Schematic illustration of the transgenes used for testing the effects of ACF1 recruitment. ACF1 is fused to the GAL4 DNA-binding domain (GAL4DBD) at either the N-terminus or the C-terminus. A transgene containing the GAL4DBD alone (GAL4) is used as a negative control. The reporter transgene contains five UAS Gal4 (UAS) 5′ of LacZ and mini-white genes. (B) Western blot detection of ACF1 in an embryo nuclear extract (0–16 h AEL). Endogenous and fusion proteins were detected with a specific ACF1 antibody (blue channel); the ACF1-GAL4 fusions are FLAG-tagged and detected with an anti-FLAG antibody (magenta channel). Asterisks indicate the expressed transgenic ACF1-GAL4 fusions. Embryos containing a transgene coding for the GAL4DBD alone (GAL4) are included as a negative control. Lamin serves as a loading control. (C) ChIP-qPCR monitors the recruitment of ACF1 to UAS in 0- to 12-h embryos. The immunoprecipitation was conducted using ACF1 and FLAG antibodies. “UAS” and “5′ LacZ” denote the regions amplified by qPCR. Bars denote average % Input enrichment (n = 3 biological replicates) ± SEM. “neg ctrl” represents a negative control locus (encompassing the Spt4 gene).
    Figure Legend Snippet: ACF can be tethered to a reporter locus through GAL4-DBD. (A) Schematic illustration of the transgenes used for testing the effects of ACF1 recruitment. ACF1 is fused to the GAL4 DNA-binding domain (GAL4DBD) at either the N-terminus or the C-terminus. A transgene containing the GAL4DBD alone (GAL4) is used as a negative control. The reporter transgene contains five UAS Gal4 (UAS) 5′ of LacZ and mini-white genes. (B) Western blot detection of ACF1 in an embryo nuclear extract (0–16 h AEL). Endogenous and fusion proteins were detected with a specific ACF1 antibody (blue channel); the ACF1-GAL4 fusions are FLAG-tagged and detected with an anti-FLAG antibody (magenta channel). Asterisks indicate the expressed transgenic ACF1-GAL4 fusions. Embryos containing a transgene coding for the GAL4DBD alone (GAL4) are included as a negative control. Lamin serves as a loading control. (C) ChIP-qPCR monitors the recruitment of ACF1 to UAS in 0- to 12-h embryos. The immunoprecipitation was conducted using ACF1 and FLAG antibodies. “UAS” and “5′ LacZ” denote the regions amplified by qPCR. Bars denote average % Input enrichment (n = 3 biological replicates) ± SEM. “neg ctrl” represents a negative control locus (encompassing the Spt4 gene).

    Techniques Used: Binding Assay, Negative Control, Western Blot, Transgenic Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Amplification

    42) Product Images from "Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells That Contribute to the Fibrous CapCharacterization of TCF21 downstream target regions identifies a transcriptional network linking multiple independent coronary artery disease loci"

    Article Title: Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells That Contribute to the Fibrous CapCharacterization of TCF21 downstream target regions identifies a transcriptional network linking multiple independent coronary artery disease loci

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005155

    Differential TCF21 gene expression in fibrous cap of stable vs. ruptured atherosclerotic plaque. A) Microdissection laser capture was employed to harvest tissue from endarterectomy samples from vessels with stable plaque or evidence of plaque rupture. An example of a stable plaque and a ruptured plaque with necrotic core (NC) and related luminal thrombus (T) are shown. These representative sections show the types of plaque regions that were harvested for RNA isolation. B) qPCR quantitation of TCF21 mRNA levels in harvested tissue. TCF21 expression was significantly lower in fibrous cap tissue from the ruptured plaque.
    Figure Legend Snippet: Differential TCF21 gene expression in fibrous cap of stable vs. ruptured atherosclerotic plaque. A) Microdissection laser capture was employed to harvest tissue from endarterectomy samples from vessels with stable plaque or evidence of plaque rupture. An example of a stable plaque and a ruptured plaque with necrotic core (NC) and related luminal thrombus (T) are shown. These representative sections show the types of plaque regions that were harvested for RNA isolation. B) qPCR quantitation of TCF21 mRNA levels in harvested tissue. TCF21 expression was significantly lower in fibrous cap tissue from the ruptured plaque.

    Techniques Used: Expressing, Laser Capture Microdissection, Isolation, Real-time Polymerase Chain Reaction, Quantitation Assay

    43) Product Images from "Increased DUX4 expression during muscle differentiation correlates with decreased SMCHD1 protein levels at D4Z4 "

    Article Title: Increased DUX4 expression during muscle differentiation correlates with decreased SMCHD1 protein levels at D4Z4

    Journal: Epigenetics

    doi: 10.1080/15592294.2015.1113798

    Treatment of FSHD2 myotube cultures with EZH2 inhibitor GSK126 increases DUX4 levels. ( A ) Western blot analysis of control, FSHD1, and FSHD2 myotube samples after treatment with 0, 1, 2, and 4 μM GSK126. Blots were probed with H3K27me3 antibody and H3 antibody as loading control. Shown are the reduced ratios of H3K27me3:H3 signal intensities in samples treated with GSK126 compared to the untreated sample as a result of EZH2 inhibition. ( B ) qRT-PCR analysis of DUX4 expression in FSHD1 and FSHD2 myotubes after GSK126 treatment shows that relative DUX4 transcript levels are significantly increased in FSHD2 samples treated with 2 μM GSK126 but not in FSHD1 myotubes. Graph shows the results of 4 FSHD1 and 4 FSHD2 cell lines. Results of control cell lines are not shown, DUX4 transcript was not detectable. Error bars show SD, n indicates number of independent cell lines, and significance was tested by 2-way ANOVA followed by Bonferroni multiple comparison test. *= P
    Figure Legend Snippet: Treatment of FSHD2 myotube cultures with EZH2 inhibitor GSK126 increases DUX4 levels. ( A ) Western blot analysis of control, FSHD1, and FSHD2 myotube samples after treatment with 0, 1, 2, and 4 μM GSK126. Blots were probed with H3K27me3 antibody and H3 antibody as loading control. Shown are the reduced ratios of H3K27me3:H3 signal intensities in samples treated with GSK126 compared to the untreated sample as a result of EZH2 inhibition. ( B ) qRT-PCR analysis of DUX4 expression in FSHD1 and FSHD2 myotubes after GSK126 treatment shows that relative DUX4 transcript levels are significantly increased in FSHD2 samples treated with 2 μM GSK126 but not in FSHD1 myotubes. Graph shows the results of 4 FSHD1 and 4 FSHD2 cell lines. Results of control cell lines are not shown, DUX4 transcript was not detectable. Error bars show SD, n indicates number of independent cell lines, and significance was tested by 2-way ANOVA followed by Bonferroni multiple comparison test. *= P

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

    44) Product Images from "MiR-106b exhibits an anti-angiogenic function by inhibiting STAT3 expression in endothelial cells"

    Article Title: MiR-106b exhibits an anti-angiogenic function by inhibiting STAT3 expression in endothelial cells

    Journal: Lipids in Health and Disease

    doi: 10.1186/s12944-016-0216-5

    a - l QRT-PCR results of different candidate genes and western-blot results of STAT3 protein expression among groups under different treatments. a - h VEGFA, JAK1 and MAPK14 expression levels are not significantly affected by miR-106b up or down regulation in HUVECs ( P > 0.05, n = 6). Meanwhile, over-expressed miR-106b represses STAT3 mRNA expression in HUVECs, while miR-106b inhibition lead to an increase in STAT3 expression. Results are shown as mean ± SEM representative of at least three independent experiments. ** P
    Figure Legend Snippet: a - l QRT-PCR results of different candidate genes and western-blot results of STAT3 protein expression among groups under different treatments. a - h VEGFA, JAK1 and MAPK14 expression levels are not significantly affected by miR-106b up or down regulation in HUVECs ( P > 0.05, n = 6). Meanwhile, over-expressed miR-106b represses STAT3 mRNA expression in HUVECs, while miR-106b inhibition lead to an increase in STAT3 expression. Results are shown as mean ± SEM representative of at least three independent experiments. ** P

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

    45) Product Images from "Direct Regulation of TLR5 Expression by Caveolin-1"

    Article Title: Direct Regulation of TLR5 Expression by Caveolin-1

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2015.0213

    TLR5 expression decreases in caveolin-1 knockout mice. RNA and protein were isolated from small intestine (S.I.), skin, and spleen of wild type (WT) and caveolin-1 knockout (Cav1 −/− ) mice. (A) Caveolin-1, TLR4, and TLR5 mRNA levels were analyzed by RT-PCR with specific primers. (B) Caveolin-1, TLR4, and TLR5 protein expression was detected by Western blotting with specific antibodies. The β-actin was used as the loading control for both RT-PCR and Western blotting. Relative density of protein expression and mRNA levels were normalized to β-actin and represented by quantitative graphs. Data are presented as mean ± SD from five independent experiments (n = 3 mice/group); * p
    Figure Legend Snippet: TLR5 expression decreases in caveolin-1 knockout mice. RNA and protein were isolated from small intestine (S.I.), skin, and spleen of wild type (WT) and caveolin-1 knockout (Cav1 −/− ) mice. (A) Caveolin-1, TLR4, and TLR5 mRNA levels were analyzed by RT-PCR with specific primers. (B) Caveolin-1, TLR4, and TLR5 protein expression was detected by Western blotting with specific antibodies. The β-actin was used as the loading control for both RT-PCR and Western blotting. Relative density of protein expression and mRNA levels were normalized to β-actin and represented by quantitative graphs. Data are presented as mean ± SD from five independent experiments (n = 3 mice/group); * p

    Techniques Used: Expressing, Knock-Out, Mouse Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

    TLR5 is preserved in aged mice tissues. RNA and protein were isolated from small intestine (S.I.), skin, and spleen of young (Y) and aged (O) mice. (A) Caveolin-1, TLR4, and TLR5 mRNA levels were quantified by RT-PCR with specific primers. (B) Caveolin-1, TLR4, and TLR5 protein expression was detected by Western blotting with specific antibodies. The β-actin was used as the loading control for both RTPCR and Western blotting. The relative density of protein expression and mRNA levels were normalized to β-actin and represented by quantitative graphs. Data are presented as mean ± SD from five independent experiments (n = 3 mice/group); * p
    Figure Legend Snippet: TLR5 is preserved in aged mice tissues. RNA and protein were isolated from small intestine (S.I.), skin, and spleen of young (Y) and aged (O) mice. (A) Caveolin-1, TLR4, and TLR5 mRNA levels were quantified by RT-PCR with specific primers. (B) Caveolin-1, TLR4, and TLR5 protein expression was detected by Western blotting with specific antibodies. The β-actin was used as the loading control for both RTPCR and Western blotting. The relative density of protein expression and mRNA levels were normalized to β-actin and represented by quantitative graphs. Data are presented as mean ± SD from five independent experiments (n = 3 mice/group); * p

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

    TLR5 is conserved in senescent non-immune cells. RNA and protein were isolated from NS-HDF and S-HDF cells. (A) Caveolin-1, TLR4, and TLR5 protein expression was detected by Western blot with specific antibodies. (B) Caveolin-1, TLR4, and TLR5 mRNA levels were analyzed by RTPCR with specific primers. The β-actin was used as the loading control for both RT-PCR and Western blotting. The relative density of protein expression and mRNA levels were normalized to β-actin and represented by quantitative graphs. Data are presented as mean ± SD from five independent experiments; * p
    Figure Legend Snippet: TLR5 is conserved in senescent non-immune cells. RNA and protein were isolated from NS-HDF and S-HDF cells. (A) Caveolin-1, TLR4, and TLR5 protein expression was detected by Western blot with specific antibodies. (B) Caveolin-1, TLR4, and TLR5 mRNA levels were analyzed by RTPCR with specific primers. The β-actin was used as the loading control for both RT-PCR and Western blotting. The relative density of protein expression and mRNA levels were normalized to β-actin and represented by quantitative graphs. Data are presented as mean ± SD from five independent experiments; * p

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

    46) Product Images from "SP-A2 contributes to miRNA-mediated sex differences in response to oxidative stress: pro-inflammatory, anti-apoptotic, and anti-oxidant pathways are involved"

    Article Title: SP-A2 contributes to miRNA-mediated sex differences in response to oxidative stress: pro-inflammatory, anti-apoptotic, and anti-oxidant pathways are involved

    Journal: Biology of Sex Differences

    doi: 10.1186/s13293-017-0158-2

    Schematic representation for the role of miRNAs in mediating regulation of pathways involved in anti-apoptotic, inflammatory, and reactive oxygen species on AM of SP-A2 mice compared to KO mice in response to O 3 . The miRNAs and genes studied in the present study are highlighted with yellow. Up (↑) and down (↓) arrows indicate increase or decrease
    Figure Legend Snippet: Schematic representation for the role of miRNAs in mediating regulation of pathways involved in anti-apoptotic, inflammatory, and reactive oxygen species on AM of SP-A2 mice compared to KO mice in response to O 3 . The miRNAs and genes studied in the present study are highlighted with yellow. Up (↑) and down (↓) arrows indicate increase or decrease

    Techniques Used: Mouse Assay

    Volcano plots (as described in Fig. 1 ) indicating the statistical significance of SP-A2 AM miRNome expression levels compared to SP-A KO under FA or O 3 exposure for males and females. a Male SP-A2 mice compared to KO exposed to FA. b Female SP-A2 mice compared to KO after FA exposure. c Male SP-A2 mice compared to KO after O 3 exposure. d Female SP-A2 mice compared to KO after O 3 exposure. The shaded gray areas with a broken outline compare the significantly changed miRNAs in SP-A2 male O 3 exposed mice and SP-A1 male O 3 exposed mice (Fig. 1 )
    Figure Legend Snippet: Volcano plots (as described in Fig. 1 ) indicating the statistical significance of SP-A2 AM miRNome expression levels compared to SP-A KO under FA or O 3 exposure for males and females. a Male SP-A2 mice compared to KO exposed to FA. b Female SP-A2 mice compared to KO after FA exposure. c Male SP-A2 mice compared to KO after O 3 exposure. d Female SP-A2 mice compared to KO after O 3 exposure. The shaded gray areas with a broken outline compare the significantly changed miRNAs in SP-A2 male O 3 exposed mice and SP-A1 male O 3 exposed mice (Fig. 1 )

    Techniques Used: Expressing, Mouse Assay

    The effect of gonadectomy and OxS on AM miRNA expression profiles of SP-A2 mice. The differentially expressed miRNAs in SP-A2 non-gonadectomized (NGx) and gonadectomized (Gx) mice were identified after normalizing to corresponding NGx and Gx KO. NGx shows the miRNAs ( n = 37) that changed significantly (≥ 2-fold) in OxS when males were compared to females. Gx depicts the comparison of Gx values (male vs female) to NGx (male vs female). Out of the same 37 miRNAs (found to have their levels increased in NGx), 2 miRNAs (5.4%) showed a significant increase (≥ 2-fold) and 35 miRNAs (94.59%) showed a significant decrease (≤ 2-fold). Inset depicts the comparison, of the 37 differentially expressed miRNAs, between Gx male and female. Out of 37 miRNAs studied, 17 miRNAs (45.9%) are significantly increased in female (≥ 2-fold), and 7 miRNAs (18.9%) are significantly increased in male (≥ 2-fold)
    Figure Legend Snippet: The effect of gonadectomy and OxS on AM miRNA expression profiles of SP-A2 mice. The differentially expressed miRNAs in SP-A2 non-gonadectomized (NGx) and gonadectomized (Gx) mice were identified after normalizing to corresponding NGx and Gx KO. NGx shows the miRNAs ( n = 37) that changed significantly (≥ 2-fold) in OxS when males were compared to females. Gx depicts the comparison of Gx values (male vs female) to NGx (male vs female). Out of the same 37 miRNAs (found to have their levels increased in NGx), 2 miRNAs (5.4%) showed a significant increase (≥ 2-fold) and 35 miRNAs (94.59%) showed a significant decrease (≤ 2-fold). Inset depicts the comparison, of the 37 differentially expressed miRNAs, between Gx male and female. Out of 37 miRNAs studied, 17 miRNAs (45.9%) are significantly increased in female (≥ 2-fold), and 7 miRNAs (18.9%) are significantly increased in male (≥ 2-fold)

    Techniques Used: Expressing, Mouse Assay

    Venn diagrams showing the distribution of differentially expressed SP-A1 and SP-A2 AM miRNAs that changed ≥ 2-fold compared to SP-A KO AM, which are unique or common (overlapping areas) in FA and O 3 exposure. Also, the figure shows the effect of sex on the expression of miRNAs after FA and O 3 exposure. Venn diagrams are showing unique and commonly identified miRNAs among SP-A1 and SP-A2 male and female hTG mice. Out of the total 226 miRNAs identified in male and female SP-A1, 49 identified miRNAs are unique to male and 24 are unique to female; 153 miRNAs are identified in common after O 3 exposure. In the case of SP-A2, out of the 166 miRNAs identified, 48 miRNAs are unique to male and 100 are unique to female; 18 miRNAs were identified in common after O 3 exposure. However, under control conditions, more miRNAs ( n = 122) were in common in the SP-A1 male and female mice than in the SP-A2 ( n = 24)
    Figure Legend Snippet: Venn diagrams showing the distribution of differentially expressed SP-A1 and SP-A2 AM miRNAs that changed ≥ 2-fold compared to SP-A KO AM, which are unique or common (overlapping areas) in FA and O 3 exposure. Also, the figure shows the effect of sex on the expression of miRNAs after FA and O 3 exposure. Venn diagrams are showing unique and commonly identified miRNAs among SP-A1 and SP-A2 male and female hTG mice. Out of the total 226 miRNAs identified in male and female SP-A1, 49 identified miRNAs are unique to male and 24 are unique to female; 153 miRNAs are identified in common after O 3 exposure. In the case of SP-A2, out of the 166 miRNAs identified, 48 miRNAs are unique to male and 100 are unique to female; 18 miRNAs were identified in common after O 3 exposure. However, under control conditions, more miRNAs ( n = 122) were in common in the SP-A1 male and female mice than in the SP-A2 ( n = 24)

    Techniques Used: Expressing, Mouse Assay

    47) Product Images from "C/EBPβ deletion in oncogenic Ras skin tumors is a synthetic lethal event"

    Article Title: C/EBPβ deletion in oncogenic Ras skin tumors is a synthetic lethal event

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1103-y

    Oncogenic Ras tumor regression following deletion of C/EBPβ is dependent on p53. a Schematic of the K14-CreER tam ;C/EBPβ flox/flox ;p53 flox/flox (DIKO) transgenic mouse model system. b Western blot analysis of C/EBPβ and p53 in epidermal lysates of tamoxifen-treated Cre, IKOβ, IKOp53, and DIKO mice. c IHC staining for C/EBPβ and p53 in normal DIKO mouse skin following vehicle and tamoxifen treatment. D dermis, E epidermis. Scale bar: 10 μm. d H E and IHC staining for C/EBPβ and p53 in DMBA/TPA-induced mouse squamous papillomas following tamoxifen dosing. e Tumor multiplicity in DMBA/TPA-induced skin squamous papilloma in Cre and DIKO mice dosed with tamoxifen at 19 weeks of skin tumor promotion (Cre N = 10 mice, DIKO N = 11 mice). f Tumor incidence in Cre and DIKO mice. g Tumor volume in Cre and DIKO mice. h Tumor multiplicity in Cre, IKOβ, IKOp53, and DIKO mice before and after tamoxifen collected 2 weeks after initial tamoxifen dose (Cre N = 5 mice, IKOβ N = 3 mice, IKOp53 N = 5 mice, DIKO N = 5 mice). # indicates IKOβ is significantly different from Cre p
    Figure Legend Snippet: Oncogenic Ras tumor regression following deletion of C/EBPβ is dependent on p53. a Schematic of the K14-CreER tam ;C/EBPβ flox/flox ;p53 flox/flox (DIKO) transgenic mouse model system. b Western blot analysis of C/EBPβ and p53 in epidermal lysates of tamoxifen-treated Cre, IKOβ, IKOp53, and DIKO mice. c IHC staining for C/EBPβ and p53 in normal DIKO mouse skin following vehicle and tamoxifen treatment. D dermis, E epidermis. Scale bar: 10 μm. d H E and IHC staining for C/EBPβ and p53 in DMBA/TPA-induced mouse squamous papillomas following tamoxifen dosing. e Tumor multiplicity in DMBA/TPA-induced skin squamous papilloma in Cre and DIKO mice dosed with tamoxifen at 19 weeks of skin tumor promotion (Cre N = 10 mice, DIKO N = 11 mice). f Tumor incidence in Cre and DIKO mice. g Tumor volume in Cre and DIKO mice. h Tumor multiplicity in Cre, IKOβ, IKOp53, and DIKO mice before and after tamoxifen collected 2 weeks after initial tamoxifen dose (Cre N = 5 mice, IKOβ N = 3 mice, IKOp53 N = 5 mice, DIKO N = 5 mice). # indicates IKOβ is significantly different from Cre p

    Techniques Used: Transgenic Assay, Western Blot, Mouse Assay, Immunohistochemistry, Staining

    Regressing tumors display significant alterations in gene expression and enrichment of type I interferon response, p53, and TNF/death receptor signaling networks. Total RNA from three Cre and three IKOβ tumors from different mice were subjected to RNA sequencing. a Heatmap showing total gene expression. b Gene set enrichment analysis with top 40 statistically significant (FDR
    Figure Legend Snippet: Regressing tumors display significant alterations in gene expression and enrichment of type I interferon response, p53, and TNF/death receptor signaling networks. Total RNA from three Cre and three IKOβ tumors from different mice were subjected to RNA sequencing. a Heatmap showing total gene expression. b Gene set enrichment analysis with top 40 statistically significant (FDR

    Techniques Used: Expressing, Mouse Assay, RNA Sequencing Assay

    Spatial and temporal regulation of C/EBPβ in epidermis and in pre-existing oncogenic Ras skin tumors. a Immunohistochemical (IHC) staining for C/EBPβ in a DMBA/TPA-induced mouse squamous papilloma. b Schematic of the K14-CreER tam ;C/EBPβ flox/flox (IKOβ) transgenic mouse model system. c IHC staining for C/EBPβ in IKOβ mouse skin following vehicle or tamoxifen dosing. D dermis, E epidermis. d Western blot analysis for C/EBPβ in epidermal lysates from tamoxifen-treated Cre and IKOβ mice. e H E and C/EBPβ IHC staining in DMBA/TPA-induced mouse squamous papilloma following vehicle and tamoxifen dosing. f DNA sequence of the 61st codon of Ha-Ras in mouse tail and DMBA/TPA-induced squamous papillomas
    Figure Legend Snippet: Spatial and temporal regulation of C/EBPβ in epidermis and in pre-existing oncogenic Ras skin tumors. a Immunohistochemical (IHC) staining for C/EBPβ in a DMBA/TPA-induced mouse squamous papilloma. b Schematic of the K14-CreER tam ;C/EBPβ flox/flox (IKOβ) transgenic mouse model system. c IHC staining for C/EBPβ in IKOβ mouse skin following vehicle or tamoxifen dosing. D dermis, E epidermis. d Western blot analysis for C/EBPβ in epidermal lysates from tamoxifen-treated Cre and IKOβ mice. e H E and C/EBPβ IHC staining in DMBA/TPA-induced mouse squamous papilloma following vehicle and tamoxifen dosing. f DNA sequence of the 61st codon of Ha-Ras in mouse tail and DMBA/TPA-induced squamous papillomas

    Techniques Used: Immunohistochemistry, Staining, Transgenic Assay, Western Blot, Mouse Assay, Sequencing

    Oncogenic Ras skin tumors depend on C/EBPβ for survival. a Tumor multiplicity in Cre and IKOβ before and after mice were dosed with tamoxifen starting at 19 weeks, b tumor incidence, and c tumor volume remaining (Cre mice n = 10, IKOβ mice n = 11). d Representative photographs of skin tumors before and after tamoxifen on the same mouse. e Tumor multiplicity in Cre and IKOβ mice before and after mice were dosed with tamoxifen starting at 34 weeks, and f tumor incidence (Cre mice n = 15, IKOβ mice n = 18). Data are expressed as means. *indicates significantly different from IKOβ controls p
    Figure Legend Snippet: Oncogenic Ras skin tumors depend on C/EBPβ for survival. a Tumor multiplicity in Cre and IKOβ before and after mice were dosed with tamoxifen starting at 19 weeks, b tumor incidence, and c tumor volume remaining (Cre mice n = 10, IKOβ mice n = 11). d Representative photographs of skin tumors before and after tamoxifen on the same mouse. e Tumor multiplicity in Cre and IKOβ mice before and after mice were dosed with tamoxifen starting at 34 weeks, and f tumor incidence (Cre mice n = 15, IKOβ mice n = 18). Data are expressed as means. *indicates significantly different from IKOβ controls p

    Techniques Used: Mouse Assay

    Skin tumors exhibit DNA damage and regressing C/EBPβ-deficient tumors do not display differences in proliferation, senescence, differentiation or inflammation. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen and tumors were collected two weeks after the initial vehicle or tamoxifen dose. a Quantification of IHC staining for Phospho-Histone H2A.X (γH2AX) in adjacent normal epidermis (vehicle n = 4 mice, tamoxifen n = 4 mice). b Quantification of γH2AX in tumor (vehicle n = 13 tumors, tamoxifen n = 8 tumors). c Quantification of IHC staining for BrdU incorporation in tumors (vehicle n = 17 tumors, tamoxifen n = 9 tumors). d Quantification of IHC staining for Ki67 in tumors (vehicle n = 13 tumors, tamoxifen n = 9 tumors). e Photograph of IHC staining for Keratin 5 (left) and Keratin 10 (right). Quantification of f neutrophil infiltration, g mononuclear leukocyte infiltration, and h total inflammation (vehicle n = 18 tumors, tamoxifen n = 18 tumors) from H E-stained tumor sections. i Quantification of CD4 IHC staining in tumor parenchyma and stroma (vehicle n = 7 tumors, tamoxifen n = 7 tumors). j Quantification of CD8 IHC staining in tumor parenchyma and stroma (vehicle n = 10 tumors, tamoxifen n = 9 tumors). k Quantification of F4/80 IHC staining in tumor parenchyma and stroma (vehicle n = 11 tumors, tamoxifen n = 9 tumors). Data are expressed as mean ± SD or percent counts. *indicates significantly different from controls p
    Figure Legend Snippet: Skin tumors exhibit DNA damage and regressing C/EBPβ-deficient tumors do not display differences in proliferation, senescence, differentiation or inflammation. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen and tumors were collected two weeks after the initial vehicle or tamoxifen dose. a Quantification of IHC staining for Phospho-Histone H2A.X (γH2AX) in adjacent normal epidermis (vehicle n = 4 mice, tamoxifen n = 4 mice). b Quantification of γH2AX in tumor (vehicle n = 13 tumors, tamoxifen n = 8 tumors). c Quantification of IHC staining for BrdU incorporation in tumors (vehicle n = 17 tumors, tamoxifen n = 9 tumors). d Quantification of IHC staining for Ki67 in tumors (vehicle n = 13 tumors, tamoxifen n = 9 tumors). e Photograph of IHC staining for Keratin 5 (left) and Keratin 10 (right). Quantification of f neutrophil infiltration, g mononuclear leukocyte infiltration, and h total inflammation (vehicle n = 18 tumors, tamoxifen n = 18 tumors) from H E-stained tumor sections. i Quantification of CD4 IHC staining in tumor parenchyma and stroma (vehicle n = 7 tumors, tamoxifen n = 7 tumors). j Quantification of CD8 IHC staining in tumor parenchyma and stroma (vehicle n = 10 tumors, tamoxifen n = 9 tumors). k Quantification of F4/80 IHC staining in tumor parenchyma and stroma (vehicle n = 11 tumors, tamoxifen n = 9 tumors). Data are expressed as mean ± SD or percent counts. *indicates significantly different from controls p

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining, BrdU Incorporation Assay

    Regressing C/EBPβ-deficient tumors display tumor-specific elevations in apoptosis and p53 protein, whereas adjacent C/EBPβ-depleted skin is unaffected. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen at 19 weeks and tumors were collected 2 weeks later. a Deletion of C/EBPβ was confirmed by IHC staining. b Photograph displaying apoptotic cells (left) and quantification of apoptosis in H E-stained tumors (right) (vehicle n = 18 tumors, tamoxifen n = 10 tumors). c Photograph displaying p53 IHC staining (left) and quantification of IHC staining for p53 (right) (vehicle n = 15 tumors, tamoxifen n = 12 tumors). d Quantification of apoptosis in H E-stained adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). e Quantification of IHC staining for p53 in adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). f Quantification of p53 mRNA from tumors (vehicle n = 3 tumors, tamoxifen n = 3 tumor). g Quantification of IHC staining for p53 phosphorylated on serine 18 (vehicle n = 9 tumors, tamoxifen n = 11 tumors). Data are expressed as mean ± SD. *indicates significantly different from controls p
    Figure Legend Snippet: Regressing C/EBPβ-deficient tumors display tumor-specific elevations in apoptosis and p53 protein, whereas adjacent C/EBPβ-depleted skin is unaffected. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen at 19 weeks and tumors were collected 2 weeks later. a Deletion of C/EBPβ was confirmed by IHC staining. b Photograph displaying apoptotic cells (left) and quantification of apoptosis in H E-stained tumors (right) (vehicle n = 18 tumors, tamoxifen n = 10 tumors). c Photograph displaying p53 IHC staining (left) and quantification of IHC staining for p53 (right) (vehicle n = 15 tumors, tamoxifen n = 12 tumors). d Quantification of apoptosis in H E-stained adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). e Quantification of IHC staining for p53 in adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). f Quantification of p53 mRNA from tumors (vehicle n = 3 tumors, tamoxifen n = 3 tumor). g Quantification of IHC staining for p53 phosphorylated on serine 18 (vehicle n = 9 tumors, tamoxifen n = 11 tumors). Data are expressed as mean ± SD. *indicates significantly different from controls p

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining

    48) Product Images from "C/EBPβ deletion in oncogenic Ras skin tumors is a synthetic lethal event"

    Article Title: C/EBPβ deletion in oncogenic Ras skin tumors is a synthetic lethal event

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1103-y

    Oncogenic Ras tumor regression following deletion of C/EBPβ is dependent on p53. a Schematic of the K14-CreER tam ;C/EBPβ flox/flox ;p53 flox/flox (DIKO) transgenic mouse model system. b Western blot analysis of C/EBPβ and p53 in epidermal lysates of tamoxifen-treated Cre, IKOβ, IKOp53, and DIKO mice. c IHC staining for C/EBPβ and p53 in normal DIKO mouse skin following vehicle and tamoxifen treatment. D dermis, E epidermis. Scale bar: 10 μm. d H E and IHC staining for C/EBPβ and p53 in DMBA/TPA-induced mouse squamous papillomas following tamoxifen dosing. e Tumor multiplicity in DMBA/TPA-induced skin squamous papilloma in Cre and DIKO mice dosed with tamoxifen at 19 weeks of skin tumor promotion (Cre N = 10 mice, DIKO N = 11 mice). f Tumor incidence in Cre and DIKO mice. g Tumor volume in Cre and DIKO mice. h Tumor multiplicity in Cre, IKOβ, IKOp53, and DIKO mice before and after tamoxifen collected 2 weeks after initial tamoxifen dose (Cre N = 5 mice, IKOβ N = 3 mice, IKOp53 N = 5 mice, DIKO N = 5 mice). # indicates IKOβ is significantly different from Cre p
    Figure Legend Snippet: Oncogenic Ras tumor regression following deletion of C/EBPβ is dependent on p53. a Schematic of the K14-CreER tam ;C/EBPβ flox/flox ;p53 flox/flox (DIKO) transgenic mouse model system. b Western blot analysis of C/EBPβ and p53 in epidermal lysates of tamoxifen-treated Cre, IKOβ, IKOp53, and DIKO mice. c IHC staining for C/EBPβ and p53 in normal DIKO mouse skin following vehicle and tamoxifen treatment. D dermis, E epidermis. Scale bar: 10 μm. d H E and IHC staining for C/EBPβ and p53 in DMBA/TPA-induced mouse squamous papillomas following tamoxifen dosing. e Tumor multiplicity in DMBA/TPA-induced skin squamous papilloma in Cre and DIKO mice dosed with tamoxifen at 19 weeks of skin tumor promotion (Cre N = 10 mice, DIKO N = 11 mice). f Tumor incidence in Cre and DIKO mice. g Tumor volume in Cre and DIKO mice. h Tumor multiplicity in Cre, IKOβ, IKOp53, and DIKO mice before and after tamoxifen collected 2 weeks after initial tamoxifen dose (Cre N = 5 mice, IKOβ N = 3 mice, IKOp53 N = 5 mice, DIKO N = 5 mice). # indicates IKOβ is significantly different from Cre p

    Techniques Used: Transgenic Assay, Western Blot, Mouse Assay, Immunohistochemistry, Staining

    Regressing tumors display significant alterations in gene expression and enrichment of type I interferon response, p53, and TNF/death receptor signaling networks. Total RNA from three Cre and three IKOβ tumors from different mice were subjected to RNA sequencing. a Heatmap showing total gene expression. b Gene set enrichment analysis with top 40 statistically significant (FDR
    Figure Legend Snippet: Regressing tumors display significant alterations in gene expression and enrichment of type I interferon response, p53, and TNF/death receptor signaling networks. Total RNA from three Cre and three IKOβ tumors from different mice were subjected to RNA sequencing. a Heatmap showing total gene expression. b Gene set enrichment analysis with top 40 statistically significant (FDR

    Techniques Used: Expressing, Mouse Assay, RNA Sequencing Assay

    Spatial and temporal regulation of C/EBPβ in epidermis and in pre-existing oncogenic Ras skin tumors. a Immunohistochemical (IHC) staining for C/EBPβ in a DMBA/TPA-induced mouse squamous papilloma. b Schematic of the K14-CreER tam ;C/EBPβ flox/flox (IKOβ) transgenic mouse model system. c IHC staining for C/EBPβ in IKOβ mouse skin following vehicle or tamoxifen dosing. D dermis, E epidermis. d Western blot analysis for C/EBPβ in epidermal lysates from tamoxifen-treated Cre and IKOβ mice. e H E and C/EBPβ IHC staining in DMBA/TPA-induced mouse squamous papilloma following vehicle and tamoxifen dosing. f DNA sequence of the 61st codon of Ha-Ras in mouse tail and DMBA/TPA-induced squamous papillomas
    Figure Legend Snippet: Spatial and temporal regulation of C/EBPβ in epidermis and in pre-existing oncogenic Ras skin tumors. a Immunohistochemical (IHC) staining for C/EBPβ in a DMBA/TPA-induced mouse squamous papilloma. b Schematic of the K14-CreER tam ;C/EBPβ flox/flox (IKOβ) transgenic mouse model system. c IHC staining for C/EBPβ in IKOβ mouse skin following vehicle or tamoxifen dosing. D dermis, E epidermis. d Western blot analysis for C/EBPβ in epidermal lysates from tamoxifen-treated Cre and IKOβ mice. e H E and C/EBPβ IHC staining in DMBA/TPA-induced mouse squamous papilloma following vehicle and tamoxifen dosing. f DNA sequence of the 61st codon of Ha-Ras in mouse tail and DMBA/TPA-induced squamous papillomas

    Techniques Used: Immunohistochemistry, Staining, Transgenic Assay, Western Blot, Mouse Assay, Sequencing

    Oncogenic Ras skin tumors depend on C/EBPβ for survival. a Tumor multiplicity in Cre and IKOβ before and after mice were dosed with tamoxifen starting at 19 weeks, b tumor incidence, and c tumor volume remaining (Cre mice n = 10, IKOβ mice n = 11). d Representative photographs of skin tumors before and after tamoxifen on the same mouse. e Tumor multiplicity in Cre and IKOβ mice before and after mice were dosed with tamoxifen starting at 34 weeks, and f tumor incidence (Cre mice n = 15, IKOβ mice n = 18). Data are expressed as means. *indicates significantly different from IKOβ controls p
    Figure Legend Snippet: Oncogenic Ras skin tumors depend on C/EBPβ for survival. a Tumor multiplicity in Cre and IKOβ before and after mice were dosed with tamoxifen starting at 19 weeks, b tumor incidence, and c tumor volume remaining (Cre mice n = 10, IKOβ mice n = 11). d Representative photographs of skin tumors before and after tamoxifen on the same mouse. e Tumor multiplicity in Cre and IKOβ mice before and after mice were dosed with tamoxifen starting at 34 weeks, and f tumor incidence (Cre mice n = 15, IKOβ mice n = 18). Data are expressed as means. *indicates significantly different from IKOβ controls p

    Techniques Used: Mouse Assay

    Skin tumors exhibit DNA damage and regressing C/EBPβ-deficient tumors do not display differences in proliferation, senescence, differentiation or inflammation. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen and tumors were collected two weeks after the initial vehicle or tamoxifen dose. a Quantification of IHC staining for Phospho-Histone H2A.X (γH2AX) in adjacent normal epidermis (vehicle n = 4 mice, tamoxifen n = 4 mice). b Quantification of γH2AX in tumor (vehicle n = 13 tumors, tamoxifen n = 8 tumors). c Quantification of IHC staining for BrdU incorporation in tumors (vehicle n = 17 tumors, tamoxifen n = 9 tumors). d Quantification of IHC staining for Ki67 in tumors (vehicle n = 13 tumors, tamoxifen n = 9 tumors). e Photograph of IHC staining for Keratin 5 (left) and Keratin 10 (right). Quantification of f neutrophil infiltration, g mononuclear leukocyte infiltration, and h total inflammation (vehicle n = 18 tumors, tamoxifen n = 18 tumors) from H E-stained tumor sections. i Quantification of CD4 IHC staining in tumor parenchyma and stroma (vehicle n = 7 tumors, tamoxifen n = 7 tumors). j Quantification of CD8 IHC staining in tumor parenchyma and stroma (vehicle n = 10 tumors, tamoxifen n = 9 tumors). k Quantification of F4/80 IHC staining in tumor parenchyma and stroma (vehicle n = 11 tumors, tamoxifen n = 9 tumors). Data are expressed as mean ± SD or percent counts. *indicates significantly different from controls p
    Figure Legend Snippet: Skin tumors exhibit DNA damage and regressing C/EBPβ-deficient tumors do not display differences in proliferation, senescence, differentiation or inflammation. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen and tumors were collected two weeks after the initial vehicle or tamoxifen dose. a Quantification of IHC staining for Phospho-Histone H2A.X (γH2AX) in adjacent normal epidermis (vehicle n = 4 mice, tamoxifen n = 4 mice). b Quantification of γH2AX in tumor (vehicle n = 13 tumors, tamoxifen n = 8 tumors). c Quantification of IHC staining for BrdU incorporation in tumors (vehicle n = 17 tumors, tamoxifen n = 9 tumors). d Quantification of IHC staining for Ki67 in tumors (vehicle n = 13 tumors, tamoxifen n = 9 tumors). e Photograph of IHC staining for Keratin 5 (left) and Keratin 10 (right). Quantification of f neutrophil infiltration, g mononuclear leukocyte infiltration, and h total inflammation (vehicle n = 18 tumors, tamoxifen n = 18 tumors) from H E-stained tumor sections. i Quantification of CD4 IHC staining in tumor parenchyma and stroma (vehicle n = 7 tumors, tamoxifen n = 7 tumors). j Quantification of CD8 IHC staining in tumor parenchyma and stroma (vehicle n = 10 tumors, tamoxifen n = 9 tumors). k Quantification of F4/80 IHC staining in tumor parenchyma and stroma (vehicle n = 11 tumors, tamoxifen n = 9 tumors). Data are expressed as mean ± SD or percent counts. *indicates significantly different from controls p

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining, BrdU Incorporation Assay

    Regressing C/EBPβ-deficient tumors display tumor-specific elevations in apoptosis and p53 protein, whereas adjacent C/EBPβ-depleted skin is unaffected. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen at 19 weeks and tumors were collected 2 weeks later. a Deletion of C/EBPβ was confirmed by IHC staining. b Photograph displaying apoptotic cells (left) and quantification of apoptosis in H E-stained tumors (right) (vehicle n = 18 tumors, tamoxifen n = 10 tumors). c Photograph displaying p53 IHC staining (left) and quantification of IHC staining for p53 (right) (vehicle n = 15 tumors, tamoxifen n = 12 tumors). d Quantification of apoptosis in H E-stained adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). e Quantification of IHC staining for p53 in adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). f Quantification of p53 mRNA from tumors (vehicle n = 3 tumors, tamoxifen n = 3 tumor). g Quantification of IHC staining for p53 phosphorylated on serine 18 (vehicle n = 9 tumors, tamoxifen n = 11 tumors). Data are expressed as mean ± SD. *indicates significantly different from controls p
    Figure Legend Snippet: Regressing C/EBPβ-deficient tumors display tumor-specific elevations in apoptosis and p53 protein, whereas adjacent C/EBPβ-depleted skin is unaffected. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen at 19 weeks and tumors were collected 2 weeks later. a Deletion of C/EBPβ was confirmed by IHC staining. b Photograph displaying apoptotic cells (left) and quantification of apoptosis in H E-stained tumors (right) (vehicle n = 18 tumors, tamoxifen n = 10 tumors). c Photograph displaying p53 IHC staining (left) and quantification of IHC staining for p53 (right) (vehicle n = 15 tumors, tamoxifen n = 12 tumors). d Quantification of apoptosis in H E-stained adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). e Quantification of IHC staining for p53 in adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). f Quantification of p53 mRNA from tumors (vehicle n = 3 tumors, tamoxifen n = 3 tumor). g Quantification of IHC staining for p53 phosphorylated on serine 18 (vehicle n = 9 tumors, tamoxifen n = 11 tumors). Data are expressed as mean ± SD. *indicates significantly different from controls p

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining

    49) Product Images from "SMCHD1 regulates a limited set of gene clusters on autosomal chromosomes"

    Article Title: SMCHD1 regulates a limited set of gene clusters on autosomal chromosomes

    Journal: Skeletal Muscle

    doi: 10.1186/s13395-017-0129-7

    The effects of SMCHD1 KD in non-DUX4 permissive 4qB cells on PCDH β cluster expression. The expression level of SMCHD1 after SMCHD1 shRNA KD in comparison with control KD shRNAs targeting luciferase and GFP is shown by RT-qPCR analysis in primary ( a ) myoblast and ( b ) myotube cells. RNA expression analysis was then assessed for PCDH β cluster isoform members in primary ( c ) myoblast and ( d ) myotube cells. Results represent qRT-PCR analysis of the indicated gene after normalization to the internal control gene GUS1 . For each gene, the value of expression in the control shRNA samples was then arbitrarily set to 1. * P
    Figure Legend Snippet: The effects of SMCHD1 KD in non-DUX4 permissive 4qB cells on PCDH β cluster expression. The expression level of SMCHD1 after SMCHD1 shRNA KD in comparison with control KD shRNAs targeting luciferase and GFP is shown by RT-qPCR analysis in primary ( a ) myoblast and ( b ) myotube cells. RNA expression analysis was then assessed for PCDH β cluster isoform members in primary ( c ) myoblast and ( d ) myotube cells. Results represent qRT-PCR analysis of the indicated gene after normalization to the internal control gene GUS1 . For each gene, the value of expression in the control shRNA samples was then arbitrarily set to 1. * P

    Techniques Used: Expressing, shRNA, Luciferase, Quantitative RT-PCR, RNA Expression

    50) Product Images from "Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines"

    Article Title: Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines

    Journal: BMC Research Notes

    doi: 10.1186/s13104-017-2959-0

    Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p
    Figure Legend Snippet: Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Synthesized, Expressing, Construct, Activated Clotting Time Assay

    Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method
    Figure Legend Snippet: Schematic representation of the experimental setup. siRNAs direct site-specific cleavage of mRNAs, resulting in a 5′ and 3′ mRNA cleavage fragments. After RNA isolation total RNA samples consist of uncleaved mRNA transcripts and non-coding RNA, as well as undegraded 5′ and 3′ mRNA cleavage fragments. Purification of mRNA using poly-T beads excludes 5′ mRNA cleavage fragments and non-coding RNAs that are not polyadenylated. As indicated by the boxes, 5′ and 3′ primer sets could detect different species of RNA depending on the isolation method

    Techniques Used: Isolation, Purification

    51) Product Images from "Bleomycin Inhibits Proliferation via Schlafen-Mediated Cell Cycle Arrest in Mouse Alveolar Epithelial Cells"

    Article Title: Bleomycin Inhibits Proliferation via Schlafen-Mediated Cell Cycle Arrest in Mouse Alveolar Epithelial Cells

    Journal: Tuberculosis and Respiratory Diseases

    doi: 10.4046/trd.2017.0124

    The MLE-12 cells induced apoptosis by bleomycin (BLM). (A) MLE-12 cells were stained with Hoechst 33258 staining after BLM treatment for 24 hours. Scale bars=100 µm. Expression of cell apoptosis marker, BAX (B) and Bcl2 (C) was measured by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). BAX/Bcl2 ratio (D) was divided (B, C) that measured by qRT-PCR. * p
    Figure Legend Snippet: The MLE-12 cells induced apoptosis by bleomycin (BLM). (A) MLE-12 cells were stained with Hoechst 33258 staining after BLM treatment for 24 hours. Scale bars=100 µm. Expression of cell apoptosis marker, BAX (B) and Bcl2 (C) was measured by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). BAX/Bcl2 ratio (D) was divided (B, C) that measured by qRT-PCR. * p

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

    The evaluation of baicalein (Bai) affected MLE-12 cells. The MLE-12 cells were treated with Bai (0.01–100 µM) for 24 hours. (A) The cell viability was measured using MTT assay. Measured the p21 (B) and p53 (C) at the mRNA levels, which were associated with cell cycle. Bai exposed MLE-12 cells were measured expression of Slfn1 (D), Slfn2 (E), and Slfn4 (F) by quantitative real-time reverse transcription-polymerase chain reaction analysis. ** p
    Figure Legend Snippet: The evaluation of baicalein (Bai) affected MLE-12 cells. The MLE-12 cells were treated with Bai (0.01–100 µM) for 24 hours. (A) The cell viability was measured using MTT assay. Measured the p21 (B) and p53 (C) at the mRNA levels, which were associated with cell cycle. Bai exposed MLE-12 cells were measured expression of Slfn1 (D), Slfn2 (E), and Slfn4 (F) by quantitative real-time reverse transcription-polymerase chain reaction analysis. ** p

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

    In the MLE-12 cells was evaluated toxicity effects of by bleomycin (BLM). MLE-12 cells were treated BLM (1–10 µg/mL) for 24 hours. (A) MLE-12 cells were induced morphological change by BLM. Scale bars=100 µm. (B) MLE-12 cells were treated with BLM and determined cell viability using MTT assay. (C) Expression of alveolar epithelial cell marker, surfactant protein C (SPC) was measured by quantitative real-time reverse transcription-polymerase chain reaction. The graph was measured inflammatory cytokines of tumor necrosis factor α (TNF-α) (D), interleukin-6 (IL-6) (E), and transforming growth factor β1 (TGF-β1) (F) in cell culture supernatant in enzyme-linked immunosorbent assay. * p
    Figure Legend Snippet: In the MLE-12 cells was evaluated toxicity effects of by bleomycin (BLM). MLE-12 cells were treated BLM (1–10 µg/mL) for 24 hours. (A) MLE-12 cells were induced morphological change by BLM. Scale bars=100 µm. (B) MLE-12 cells were treated with BLM and determined cell viability using MTT assay. (C) Expression of alveolar epithelial cell marker, surfactant protein C (SPC) was measured by quantitative real-time reverse transcription-polymerase chain reaction. The graph was measured inflammatory cytokines of tumor necrosis factor α (TNF-α) (D), interleukin-6 (IL-6) (E), and transforming growth factor β1 (TGF-β1) (F) in cell culture supernatant in enzyme-linked immunosorbent assay. * p

    Techniques Used: MTT Assay, Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

    Evaluation of nuclear enlargement and cell cycle arrest induced in MLE-12 cells by bleomycin (BLM). (A) The image was obtained using DAPI staining in MLE-12 cells after BLM treatment. Scale bars=200 µm. (B) Propidium iodide (PI) staining was performed for cell cycle arrest in MLE-12 cells after BLM treatment. (C) Statistical analysis was each percentage of the field in the samples. * p
    Figure Legend Snippet: Evaluation of nuclear enlargement and cell cycle arrest induced in MLE-12 cells by bleomycin (BLM). (A) The image was obtained using DAPI staining in MLE-12 cells after BLM treatment. Scale bars=200 µm. (B) Propidium iodide (PI) staining was performed for cell cycle arrest in MLE-12 cells after BLM treatment. (C) Statistical analysis was each percentage of the field in the samples. * p

    Techniques Used: Staining

    Gene expression associated with cell cycle of bleomycin (BLM) treatment in MLE-12 cells. The MLE-12 cells were treated with BLM (1–10 µg/mL) for 24 hours. Measured the p21 (A), p53 (B), and proliferating cell nuclear antigen ( Pcna ) (C) at the mRNA levels, which were associated with cell cycle. BLM exposed MLE-12 cells were measured expression of Slfn1 (D), Slfn2 (E), and Slfn4 (F) by quantitative real-time reverse transcription-polymerase chain reaction analysis.
    Figure Legend Snippet: Gene expression associated with cell cycle of bleomycin (BLM) treatment in MLE-12 cells. The MLE-12 cells were treated with BLM (1–10 µg/mL) for 24 hours. Measured the p21 (A), p53 (B), and proliferating cell nuclear antigen ( Pcna ) (C) at the mRNA levels, which were associated with cell cycle. BLM exposed MLE-12 cells were measured expression of Slfn1 (D), Slfn2 (E), and Slfn4 (F) by quantitative real-time reverse transcription-polymerase chain reaction analysis.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    52) Product Images from "Nexilin/NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP"

    Article Title: Nexilin/NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31328-2

    Nexilin/ NEXN expression is controlled by YAP and by myocardin family coactivators. Panel A shows phosphorylation of YAP (P-YAP) in human coronary artery SMCs (HCASMCs) prior to and at different times after addition of sphingosine-1-phosphate (S1P). Panel B shows compiled data on YAP phosphorylation (N = 9). In panel C, qRT-PCR for NEXN and CTGF at different times after S1P addition is shown (N = 9). Panel D shows NEXN mRNA expression following adenoviral transduction of YAP1 and S1P treatment, individually and in combination (N = 11). Panel E shows the effect of dual silencing of YAP( YAP1) and TAZ ( WWTR1 ) on NEXN mRNA expression under basal conditions and after S1P stimulation of human bladder SMCs (HBSMCs) (N = 9). Panel F shows the mRNA level of NEXN after overexpression of MRTF-A/ MKL1 and YAP1 , alone and in combination (N = 9). Panel G shows the mRNA level of NEXN in control conditions and following overexpression of MRTF-A/ MKL1 , MRTF-B/ MKL2 and myocardin/ MYOCD (HBSMCs, N = 6–8; HCASMCs, N = 6). Panel H shows western blots for Nexilin following overexpression of MRTFs in bladder (top) and coronary artery (bottom) SMCs, and panel I shows compiled protein data (N = 9). Panel J shows the mRNA level for SRF following silencing of SRF , YAP/TAZ, or both. Panel K shows the associated reduction of the NEXN mRNA (N = 6). Panel L shows the effect of pharmacological inhibition of MRTF/SRF signaling in bladder and coronary artery SMCs (HBSMCs, N = 12; HCASMCs, N = 9).
    Figure Legend Snippet: Nexilin/ NEXN expression is controlled by YAP and by myocardin family coactivators. Panel A shows phosphorylation of YAP (P-YAP) in human coronary artery SMCs (HCASMCs) prior to and at different times after addition of sphingosine-1-phosphate (S1P). Panel B shows compiled data on YAP phosphorylation (N = 9). In panel C, qRT-PCR for NEXN and CTGF at different times after S1P addition is shown (N = 9). Panel D shows NEXN mRNA expression following adenoviral transduction of YAP1 and S1P treatment, individually and in combination (N = 11). Panel E shows the effect of dual silencing of YAP( YAP1) and TAZ ( WWTR1 ) on NEXN mRNA expression under basal conditions and after S1P stimulation of human bladder SMCs (HBSMCs) (N = 9). Panel F shows the mRNA level of NEXN after overexpression of MRTF-A/ MKL1 and YAP1 , alone and in combination (N = 9). Panel G shows the mRNA level of NEXN in control conditions and following overexpression of MRTF-A/ MKL1 , MRTF-B/ MKL2 and myocardin/ MYOCD (HBSMCs, N = 6–8; HCASMCs, N = 6). Panel H shows western blots for Nexilin following overexpression of MRTFs in bladder (top) and coronary artery (bottom) SMCs, and panel I shows compiled protein data (N = 9). Panel J shows the mRNA level for SRF following silencing of SRF , YAP/TAZ, or both. Panel K shows the associated reduction of the NEXN mRNA (N = 6). Panel L shows the effect of pharmacological inhibition of MRTF/SRF signaling in bladder and coronary artery SMCs (HBSMCs, N = 12; HCASMCs, N = 9).

    Techniques Used: Expressing, Quantitative RT-PCR, Transduction, Over Expression, Western Blot, Inhibition

    Knockdown of Nexilin/ NEXN reduces actin polymerization and SMC marker expression. NEXN was silenced using a short hairpin adenoviral construct (sh-NEXN) and the contents of NEXN and a number of SMC differentiation markers were examined at the mRNA (panel A, N = 6–9) and protein levels (panels B and C, N = 12). Panel D shows that depolymerization of actin using LatB reduces SRF expression in both human bladder and coronary artery SMCs (HBSMCs, N = 8; HCASMCs, N = 9). Panel E shows phalloidin staining of actin filaments in control cell (Null) and after NEXN silencing (sh-NEXN). The scale bar represents 50 μm. Panel F shows a sedimentation assay to determine filamentous (F-) and globular (G-) actin in control cells (null) and after silencing of NEXN . Panel G shows the normalized F- to G-actin ratio in bladder and coronary artery SMCs (HBSMCs, N = 12; HCASMCs, N = 6). Panel H shows that polymerization of actin using jasplakinolide (Jasp) increases MYH11 in HBSMCs in control conditions and after NEXN silencing (N = 6). Panel I shows cell density at different times following the creation of a cell-free area in the culture dish (N = 9–10). Panel J shows the speed of cell movement within the cell-free area (N = 19 and 22 motile cells in Null and sh- NEXN , respectively). Panel K shows a cell viability assay comparing control and NEXN -silenced cells (N = 12 throughout). The effect of NEXN silencing on cell migration was confirmed using an independent siRNA in L (N = 9). The associated repression of the NEXN protein is shown in ( M ) (N = 6). The schematic illustration in panel N summarizes our findings regarding the transcriptional control of NEXN and its impact on actin polymerization and cell motility. Several aspects of this model, including the involvement of a ( G ) protein-coupled receptor in the S1P effect, were not directly tested here, and they are thus drawn in grey.
    Figure Legend Snippet: Knockdown of Nexilin/ NEXN reduces actin polymerization and SMC marker expression. NEXN was silenced using a short hairpin adenoviral construct (sh-NEXN) and the contents of NEXN and a number of SMC differentiation markers were examined at the mRNA (panel A, N = 6–9) and protein levels (panels B and C, N = 12). Panel D shows that depolymerization of actin using LatB reduces SRF expression in both human bladder and coronary artery SMCs (HBSMCs, N = 8; HCASMCs, N = 9). Panel E shows phalloidin staining of actin filaments in control cell (Null) and after NEXN silencing (sh-NEXN). The scale bar represents 50 μm. Panel F shows a sedimentation assay to determine filamentous (F-) and globular (G-) actin in control cells (null) and after silencing of NEXN . Panel G shows the normalized F- to G-actin ratio in bladder and coronary artery SMCs (HBSMCs, N = 12; HCASMCs, N = 6). Panel H shows that polymerization of actin using jasplakinolide (Jasp) increases MYH11 in HBSMCs in control conditions and after NEXN silencing (N = 6). Panel I shows cell density at different times following the creation of a cell-free area in the culture dish (N = 9–10). Panel J shows the speed of cell movement within the cell-free area (N = 19 and 22 motile cells in Null and sh- NEXN , respectively). Panel K shows a cell viability assay comparing control and NEXN -silenced cells (N = 12 throughout). The effect of NEXN silencing on cell migration was confirmed using an independent siRNA in L (N = 9). The associated repression of the NEXN protein is shown in ( M ) (N = 6). The schematic illustration in panel N summarizes our findings regarding the transcriptional control of NEXN and its impact on actin polymerization and cell motility. Several aspects of this model, including the involvement of a ( G ) protein-coupled receptor in the S1P effect, were not directly tested here, and they are thus drawn in grey.

    Techniques Used: Marker, Expressing, Construct, Staining, Sedimentation, Viability Assay, Migration

    NEXN correlates with gene products that control and respond to changes in actin polymerization and Nexilin is reduced by depolymerization of actin. Correlations of NEXN versus all other RNAs in the top-ten NEXN expressing tissues were examined (data from GTExPortal). The sum of correlation coefficients for individual RNAs across tissues was calculated (R sum ) and the positive extreme of this distribution was plotted ( A ). Actin controlling and responding gene products represented in the extreme are highlighted in blue colors. Examples of NEXN correlations in the human coronary artery (N = 133) are shown in panels B through ( D ). P-values and Spearman Rho values are given in the respective panels. Panels E and F show mRNA data for NEXN in cultured human bladder (HBSMCs, N = 8) and coronary artery (HCASMCs, N = 9) SMCs after treatment with Latrunculin B (LatB). Panels H and I show protein data for Nexilin/ NEXN in the presence and absence of LatB (HBSMCs, 300 nM, N = 12; HCASMCs, 100 nM, N = 10). The top micrographs in panel J shows confocal imaging of YAP (red) on the left, and YAP (green) and CAV1 (red) on the right. The bottom row shows YAP (red) and Nexilin (green). The high magnification overlay at the bottom right shows partial colocalization of YAP and Nexilin at the cell membrane in yellow. All micrographs are from cross-sectioned HBSMCs and white scale bars represent 5 μm throughout.
    Figure Legend Snippet: NEXN correlates with gene products that control and respond to changes in actin polymerization and Nexilin is reduced by depolymerization of actin. Correlations of NEXN versus all other RNAs in the top-ten NEXN expressing tissues were examined (data from GTExPortal). The sum of correlation coefficients for individual RNAs across tissues was calculated (R sum ) and the positive extreme of this distribution was plotted ( A ). Actin controlling and responding gene products represented in the extreme are highlighted in blue colors. Examples of NEXN correlations in the human coronary artery (N = 133) are shown in panels B through ( D ). P-values and Spearman Rho values are given in the respective panels. Panels E and F show mRNA data for NEXN in cultured human bladder (HBSMCs, N = 8) and coronary artery (HCASMCs, N = 9) SMCs after treatment with Latrunculin B (LatB). Panels H and I show protein data for Nexilin/ NEXN in the presence and absence of LatB (HBSMCs, 300 nM, N = 12; HCASMCs, 100 nM, N = 10). The top micrographs in panel J shows confocal imaging of YAP (red) on the left, and YAP (green) and CAV1 (red) on the right. The bottom row shows YAP (red) and Nexilin (green). The high magnification overlay at the bottom right shows partial colocalization of YAP and Nexilin at the cell membrane in yellow. All micrographs are from cross-sectioned HBSMCs and white scale bars represent 5 μm throughout.

    Techniques Used: Expressing, Cell Culture, Imaging

    53) Product Images from "Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells That Contribute to the Fibrous CapCharacterization of TCF21 downstream target regions identifies a transcriptional network linking multiple independent coronary artery disease loci"

    Article Title: Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells That Contribute to the Fibrous CapCharacterization of TCF21 downstream target regions identifies a transcriptional network linking multiple independent coronary artery disease loci

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005155

    Differential TCF21 gene expression in fibrous cap of stable vs. ruptured atherosclerotic plaque. A) Microdissection laser capture was employed to harvest tissue from endarterectomy samples from vessels with stable plaque or evidence of plaque rupture. An example of a stable plaque and a ruptured plaque with necrotic core (NC) and related luminal thrombus (T) are shown. These representative sections show the types of plaque regions that were harvested for RNA isolation. B) qPCR quantitation of TCF21 mRNA levels in harvested tissue. TCF21 expression was significantly lower in fibrous cap tissue from the ruptured plaque.
    Figure Legend Snippet: Differential TCF21 gene expression in fibrous cap of stable vs. ruptured atherosclerotic plaque. A) Microdissection laser capture was employed to harvest tissue from endarterectomy samples from vessels with stable plaque or evidence of plaque rupture. An example of a stable plaque and a ruptured plaque with necrotic core (NC) and related luminal thrombus (T) are shown. These representative sections show the types of plaque regions that were harvested for RNA isolation. B) qPCR quantitation of TCF21 mRNA levels in harvested tissue. TCF21 expression was significantly lower in fibrous cap tissue from the ruptured plaque.

    Techniques Used: Expressing, Laser Capture Microdissection, Isolation, Real-time Polymerase Chain Reaction, Quantitation Assay

    54) Product Images from "SMCHD1 regulates a limited set of gene clusters on autosomal chromosomes"

    Article Title: SMCHD1 regulates a limited set of gene clusters on autosomal chromosomes

    Journal: Skeletal Muscle

    doi: 10.1186/s13395-017-0129-7

    The effects of SMCHD1 KD in non-DUX4 permissive 4qB cells on PCDH β cluster expression. The expression level of SMCHD1 after SMCHD1 shRNA KD in comparison with control KD shRNAs targeting luciferase and GFP is shown by RT-qPCR analysis in primary ( a ) myoblast and ( b ) myotube cells. RNA expression analysis was then assessed for PCDH β cluster isoform members in primary ( c ) myoblast and ( d ) myotube cells. Results represent qRT-PCR analysis of the indicated gene after normalization to the internal control gene GUS1 . For each gene, the value of expression in the control shRNA samples was then arbitrarily set to 1. * P
    Figure Legend Snippet: The effects of SMCHD1 KD in non-DUX4 permissive 4qB cells on PCDH β cluster expression. The expression level of SMCHD1 after SMCHD1 shRNA KD in comparison with control KD shRNAs targeting luciferase and GFP is shown by RT-qPCR analysis in primary ( a ) myoblast and ( b ) myotube cells. RNA expression analysis was then assessed for PCDH β cluster isoform members in primary ( c ) myoblast and ( d ) myotube cells. Results represent qRT-PCR analysis of the indicated gene after normalization to the internal control gene GUS1 . For each gene, the value of expression in the control shRNA samples was then arbitrarily set to 1. * P

    Techniques Used: Expressing, shRNA, Luciferase, Quantitative RT-PCR, RNA Expression

    55) Product Images from "Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines"

    Article Title: Optimal RNA isolation method and primer design to detect gene knockdown by qPCR when validating Drosophila transgenic RNAi lines

    Journal: BMC Research Notes

    doi: 10.1186/s13104-017-2959-0

    Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p
    Figure Legend Snippet: Primer location and RNA isolation method affect qPCR knockdown detection. qPCR was conducted on cDNA synthesized from total RNA samples and mRNA samples. Two primer sets—one amplifying 5′ of the siRNA cut site, the other amplifying 3′ of the siRNA cut site—were compared. Relative gene expression of a snr1 , b brm , c osa and d trr was measured by qPCR in third instar larvae after ubiquitous expression of UAS - RNAi constructs with Act - Gal4 . Expression levels were normalized to the reference genes, eIF2Bγ and βCOP . Shown here, are relative expression values compared to the UAS - mCherry - RNAi control (indicated by the dotted line). Asterisks directly above bars indicate a significant knockdown compared to the control, while asterisks above brackets indicate significant differences in gene expression between different conditions—total RNA vs. mRNA, 3′ vs. 5′ primer set (*p

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Synthesized, Expressing, Construct, Activated Clotting Time Assay

    56) Product Images from "C/EBPβ deletion in oncogenic Ras skin tumors is a synthetic lethal event"

    Article Title: C/EBPβ deletion in oncogenic Ras skin tumors is a synthetic lethal event

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1103-y

    Oncogenic Ras tumor regression following deletion of C/EBPβ is dependent on p53. a Schematic of the K14-CreER tam ;C/EBPβ flox/flox ;p53 flox/flox (DIKO) transgenic mouse model system. b Western blot analysis of C/EBPβ and p53 in epidermal lysates of tamoxifen-treated Cre, IKOβ, IKOp53, and DIKO mice. c IHC staining for C/EBPβ and p53 in normal DIKO mouse skin following vehicle and tamoxifen treatment. D dermis, E epidermis. Scale bar: 10 μm. d H E and IHC staining for C/EBPβ and p53 in DMBA/TPA-induced mouse squamous papillomas following tamoxifen dosing. e Tumor multiplicity in DMBA/TPA-induced skin squamous papilloma in Cre and DIKO mice dosed with tamoxifen at 19 weeks of skin tumor promotion (Cre N = 10 mice, DIKO N = 11 mice). f Tumor incidence in Cre and DIKO mice. g Tumor volume in Cre and DIKO mice. h Tumor multiplicity in Cre, IKOβ, IKOp53, and DIKO mice before and after tamoxifen collected 2 weeks after initial tamoxifen dose (Cre N = 5 mice, IKOβ N = 3 mice, IKOp53 N = 5 mice, DIKO N = 5 mice). # indicates IKOβ is significantly different from Cre p
    Figure Legend Snippet: Oncogenic Ras tumor regression following deletion of C/EBPβ is dependent on p53. a Schematic of the K14-CreER tam ;C/EBPβ flox/flox ;p53 flox/flox (DIKO) transgenic mouse model system. b Western blot analysis of C/EBPβ and p53 in epidermal lysates of tamoxifen-treated Cre, IKOβ, IKOp53, and DIKO mice. c IHC staining for C/EBPβ and p53 in normal DIKO mouse skin following vehicle and tamoxifen treatment. D dermis, E epidermis. Scale bar: 10 μm. d H E and IHC staining for C/EBPβ and p53 in DMBA/TPA-induced mouse squamous papillomas following tamoxifen dosing. e Tumor multiplicity in DMBA/TPA-induced skin squamous papilloma in Cre and DIKO mice dosed with tamoxifen at 19 weeks of skin tumor promotion (Cre N = 10 mice, DIKO N = 11 mice). f Tumor incidence in Cre and DIKO mice. g Tumor volume in Cre and DIKO mice. h Tumor multiplicity in Cre, IKOβ, IKOp53, and DIKO mice before and after tamoxifen collected 2 weeks after initial tamoxifen dose (Cre N = 5 mice, IKOβ N = 3 mice, IKOp53 N = 5 mice, DIKO N = 5 mice). # indicates IKOβ is significantly different from Cre p

    Techniques Used: Transgenic Assay, Western Blot, Mouse Assay, Immunohistochemistry, Staining

    Spatial and temporal regulation of C/EBPβ in epidermis and in pre-existing oncogenic Ras skin tumors. a Immunohistochemical (IHC) staining for C/EBPβ in a DMBA/TPA-induced mouse squamous papilloma. b Schematic of the K14-CreER tam ;C/EBPβ flox/flox (IKOβ) transgenic mouse model system. c IHC staining for C/EBPβ in IKOβ mouse skin following vehicle or tamoxifen dosing. D dermis, E epidermis. d Western blot analysis for C/EBPβ in epidermal lysates from tamoxifen-treated Cre and IKOβ mice. e H E and C/EBPβ IHC staining in DMBA/TPA-induced mouse squamous papilloma following vehicle and tamoxifen dosing. f DNA sequence of the 61st codon of Ha-Ras in mouse tail and DMBA/TPA-induced squamous papillomas
    Figure Legend Snippet: Spatial and temporal regulation of C/EBPβ in epidermis and in pre-existing oncogenic Ras skin tumors. a Immunohistochemical (IHC) staining for C/EBPβ in a DMBA/TPA-induced mouse squamous papilloma. b Schematic of the K14-CreER tam ;C/EBPβ flox/flox (IKOβ) transgenic mouse model system. c IHC staining for C/EBPβ in IKOβ mouse skin following vehicle or tamoxifen dosing. D dermis, E epidermis. d Western blot analysis for C/EBPβ in epidermal lysates from tamoxifen-treated Cre and IKOβ mice. e H E and C/EBPβ IHC staining in DMBA/TPA-induced mouse squamous papilloma following vehicle and tamoxifen dosing. f DNA sequence of the 61st codon of Ha-Ras in mouse tail and DMBA/TPA-induced squamous papillomas

    Techniques Used: Immunohistochemistry, Staining, Transgenic Assay, Western Blot, Mouse Assay, Sequencing

    Oncogenic Ras skin tumors depend on C/EBPβ for survival. a Tumor multiplicity in Cre and IKOβ before and after mice were dosed with tamoxifen starting at 19 weeks, b tumor incidence, and c tumor volume remaining (Cre mice n = 10, IKOβ mice n = 11). d Representative photographs of skin tumors before and after tamoxifen on the same mouse. e Tumor multiplicity in Cre and IKOβ mice before and after mice were dosed with tamoxifen starting at 34 weeks, and f tumor incidence (Cre mice n = 15, IKOβ mice n = 18). Data are expressed as means. *indicates significantly different from IKOβ controls p
    Figure Legend Snippet: Oncogenic Ras skin tumors depend on C/EBPβ for survival. a Tumor multiplicity in Cre and IKOβ before and after mice were dosed with tamoxifen starting at 19 weeks, b tumor incidence, and c tumor volume remaining (Cre mice n = 10, IKOβ mice n = 11). d Representative photographs of skin tumors before and after tamoxifen on the same mouse. e Tumor multiplicity in Cre and IKOβ mice before and after mice were dosed with tamoxifen starting at 34 weeks, and f tumor incidence (Cre mice n = 15, IKOβ mice n = 18). Data are expressed as means. *indicates significantly different from IKOβ controls p

    Techniques Used: Mouse Assay

    Skin tumors exhibit DNA damage and regressing C/EBPβ-deficient tumors do not display differences in proliferation, senescence, differentiation or inflammation. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen and tumors were collected two weeks after the initial vehicle or tamoxifen dose. a Quantification of IHC staining for Phospho-Histone H2A.X (γH2AX) in adjacent normal epidermis (vehicle n = 4 mice, tamoxifen n = 4 mice). b Quantification of γH2AX in tumor (vehicle n = 13 tumors, tamoxifen n = 8 tumors). c Quantification of IHC staining for BrdU incorporation in tumors (vehicle n = 17 tumors, tamoxifen n = 9 tumors). d Quantification of IHC staining for Ki67 in tumors (vehicle n = 13 tumors, tamoxifen n = 9 tumors). e Photograph of IHC staining for Keratin 5 (left) and Keratin 10 (right). Quantification of f neutrophil infiltration, g mononuclear leukocyte infiltration, and h total inflammation (vehicle n = 18 tumors, tamoxifen n = 18 tumors) from H E-stained tumor sections. i Quantification of CD4 IHC staining in tumor parenchyma and stroma (vehicle n = 7 tumors, tamoxifen n = 7 tumors). j Quantification of CD8 IHC staining in tumor parenchyma and stroma (vehicle n = 10 tumors, tamoxifen n = 9 tumors). k Quantification of F4/80 IHC staining in tumor parenchyma and stroma (vehicle n = 11 tumors, tamoxifen n = 9 tumors). Data are expressed as mean ± SD or percent counts. *indicates significantly different from controls p
    Figure Legend Snippet: Skin tumors exhibit DNA damage and regressing C/EBPβ-deficient tumors do not display differences in proliferation, senescence, differentiation or inflammation. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen and tumors were collected two weeks after the initial vehicle or tamoxifen dose. a Quantification of IHC staining for Phospho-Histone H2A.X (γH2AX) in adjacent normal epidermis (vehicle n = 4 mice, tamoxifen n = 4 mice). b Quantification of γH2AX in tumor (vehicle n = 13 tumors, tamoxifen n = 8 tumors). c Quantification of IHC staining for BrdU incorporation in tumors (vehicle n = 17 tumors, tamoxifen n = 9 tumors). d Quantification of IHC staining for Ki67 in tumors (vehicle n = 13 tumors, tamoxifen n = 9 tumors). e Photograph of IHC staining for Keratin 5 (left) and Keratin 10 (right). Quantification of f neutrophil infiltration, g mononuclear leukocyte infiltration, and h total inflammation (vehicle n = 18 tumors, tamoxifen n = 18 tumors) from H E-stained tumor sections. i Quantification of CD4 IHC staining in tumor parenchyma and stroma (vehicle n = 7 tumors, tamoxifen n = 7 tumors). j Quantification of CD8 IHC staining in tumor parenchyma and stroma (vehicle n = 10 tumors, tamoxifen n = 9 tumors). k Quantification of F4/80 IHC staining in tumor parenchyma and stroma (vehicle n = 11 tumors, tamoxifen n = 9 tumors). Data are expressed as mean ± SD or percent counts. *indicates significantly different from controls p

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining, BrdU Incorporation Assay

    Regressing C/EBPβ-deficient tumors display tumor-specific elevations in apoptosis and p53 protein, whereas adjacent C/EBPβ-depleted skin is unaffected. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen at 19 weeks and tumors were collected 2 weeks later. a Deletion of C/EBPβ was confirmed by IHC staining. b Photograph displaying apoptotic cells (left) and quantification of apoptosis in H E-stained tumors (right) (vehicle n = 18 tumors, tamoxifen n = 10 tumors). c Photograph displaying p53 IHC staining (left) and quantification of IHC staining for p53 (right) (vehicle n = 15 tumors, tamoxifen n = 12 tumors). d Quantification of apoptosis in H E-stained adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). e Quantification of IHC staining for p53 in adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). f Quantification of p53 mRNA from tumors (vehicle n = 3 tumors, tamoxifen n = 3 tumor). g Quantification of IHC staining for p53 phosphorylated on serine 18 (vehicle n = 9 tumors, tamoxifen n = 11 tumors). Data are expressed as mean ± SD. *indicates significantly different from controls p
    Figure Legend Snippet: Regressing C/EBPβ-deficient tumors display tumor-specific elevations in apoptosis and p53 protein, whereas adjacent C/EBPβ-depleted skin is unaffected. Tumor bearing IKOβ mice were dosed with either vehicle control or tamoxifen at 19 weeks and tumors were collected 2 weeks later. a Deletion of C/EBPβ was confirmed by IHC staining. b Photograph displaying apoptotic cells (left) and quantification of apoptosis in H E-stained tumors (right) (vehicle n = 18 tumors, tamoxifen n = 10 tumors). c Photograph displaying p53 IHC staining (left) and quantification of IHC staining for p53 (right) (vehicle n = 15 tumors, tamoxifen n = 12 tumors). d Quantification of apoptosis in H E-stained adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). e Quantification of IHC staining for p53 in adjacent normal epidermis (vehicle n = 5 mice, tamoxifen n = 8 mice). f Quantification of p53 mRNA from tumors (vehicle n = 3 tumors, tamoxifen n = 3 tumor). g Quantification of IHC staining for p53 phosphorylated on serine 18 (vehicle n = 9 tumors, tamoxifen n = 11 tumors). Data are expressed as mean ± SD. *indicates significantly different from controls p

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining

    57) Product Images from "n3 and n6 polyunsaturated fatty acids differentially modulate prostaglandin E secretion but not markers of lipogenesis in adipocytes"

    Article Title: n3 and n6 polyunsaturated fatty acids differentially modulate prostaglandin E secretion but not markers of lipogenesis in adipocytes

    Journal: Nutrition & Metabolism

    doi: 10.1186/1743-7075-6-5

    Effects of n3 and n6 PUFA and COX-2 inhibition on FAS mRNA expression . 3T3 L-1 adipocytes were grown 6–7 days post-confluence, and starved for 24 h in serum-free media containing FA-free BSA. Fatty acids were incubated in media containing FA-free BSA for two hours at 37°C in a shaking water bath prior to treatment. Treatment consisted of CI (1 μM), EPA and AA (150 μM each treatment), and AA + EPA (75 μM each FA). After 48 hours, cells were scraped using 350 μL of Qiazol lysis reagent, and total RNA was extracted using the RNeasy™ lipid tissue midi kit (Qiagen) following the manufacturer's protocol. RNA was stored at -80° for further analysis. Real time RT-PCR analysis was performed as described in the materials and methods. For all treatments n = 6. Results represent the mean ± SEM. All values were significantly different than control, and values labeled with different letters are significantly different (p
    Figure Legend Snippet: Effects of n3 and n6 PUFA and COX-2 inhibition on FAS mRNA expression . 3T3 L-1 adipocytes were grown 6–7 days post-confluence, and starved for 24 h in serum-free media containing FA-free BSA. Fatty acids were incubated in media containing FA-free BSA for two hours at 37°C in a shaking water bath prior to treatment. Treatment consisted of CI (1 μM), EPA and AA (150 μM each treatment), and AA + EPA (75 μM each FA). After 48 hours, cells were scraped using 350 μL of Qiazol lysis reagent, and total RNA was extracted using the RNeasy™ lipid tissue midi kit (Qiagen) following the manufacturer's protocol. RNA was stored at -80° for further analysis. Real time RT-PCR analysis was performed as described in the materials and methods. For all treatments n = 6. Results represent the mean ± SEM. All values were significantly different than control, and values labeled with different letters are significantly different (p

    Techniques Used: Inhibition, Expressing, Incubation, Lysis, Quantitative RT-PCR, Labeling

    58) Product Images from "Flow-dependent regulation of genome-wide mRNA and microRNA expression in endothelial cells in vivo"

    Article Title: Flow-dependent regulation of genome-wide mRNA and microRNA expression in endothelial cells in vivo

    Journal: Scientific Data

    doi: 10.1038/sdata.2014.39

    ( a ) The diagram shows naturally occurring d-flow (lesser curvature, LC) and s-flow regions (greater curvature, GC) in the aortic arch. Also shown is the surgically induced d-flow in the partial carotid ligation model in which three of the four caudal branches of the left common carotid artery (LCA) are ligated, while the contralateral right common carotid artery (RCA) remains untouched in order to serve as an internal control. ( b ) The scheme shows the intimal RNA extraction step that enables collection of endothelial-enriched RNAs from the mouse carotids. Using an insulin syringe with a 29G needle and 150 μl QIAzol lysis buffer (Qiagen) and carefully flushing the intima with QIAzol lysis buffer into an Eppendorf tube. Intima eluate or the leftover (media+adventitia) are then used for total RNA isolation. ( c ) Representative image of the ApoE −/− mouse carotids showing plaque in the LCA after 2 weeks of high-fat diet confirming disease status, while the RCA remains plaque-free. ( d ) The scheme shows our approach to discover potential, flow-sensitive miRNAs-target gene interactions. Using a simplified hypothesis that the upregulated miRNA will negatively regulate its target gene and vice versa, we initially generated a predicted target gene list for highly differentially regulated miRNAs and then compared this putative target gene list to the list of flow-sensitive mRNAs (that followed an inverse relation with the miRNAs) in the mRNA array. Using this approach, we identified two potential targets for miR-712 as TIMP3 and RECK.
    Figure Legend Snippet: ( a ) The diagram shows naturally occurring d-flow (lesser curvature, LC) and s-flow regions (greater curvature, GC) in the aortic arch. Also shown is the surgically induced d-flow in the partial carotid ligation model in which three of the four caudal branches of the left common carotid artery (LCA) are ligated, while the contralateral right common carotid artery (RCA) remains untouched in order to serve as an internal control. ( b ) The scheme shows the intimal RNA extraction step that enables collection of endothelial-enriched RNAs from the mouse carotids. Using an insulin syringe with a 29G needle and 150 μl QIAzol lysis buffer (Qiagen) and carefully flushing the intima with QIAzol lysis buffer into an Eppendorf tube. Intima eluate or the leftover (media+adventitia) are then used for total RNA isolation. ( c ) Representative image of the ApoE −/− mouse carotids showing plaque in the LCA after 2 weeks of high-fat diet confirming disease status, while the RCA remains plaque-free. ( d ) The scheme shows our approach to discover potential, flow-sensitive miRNAs-target gene interactions. Using a simplified hypothesis that the upregulated miRNA will negatively regulate its target gene and vice versa, we initially generated a predicted target gene list for highly differentially regulated miRNAs and then compared this putative target gene list to the list of flow-sensitive mRNAs (that followed an inverse relation with the miRNAs) in the mRNA array. Using this approach, we identified two potential targets for miR-712 as TIMP3 and RECK.

    Techniques Used: Flow Cytometry, Ligation, RNA Extraction, Lysis, Isolation, Generated

    59) Product Images from "Activated kinase screening identifies the IKBKE oncogene as a positive regulator of autophagy"

    Article Title: Activated kinase screening identifies the IKBKE oncogene as a positive regulator of autophagy

    Journal: Autophagy

    doi: 10.1080/15548627.2018.1517855

    IKBKE is required for autophagy induced by the ERBB2 breast oncogene. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon pharmacological inhibition of IKBKE activity by CYT387 (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P
    Figure Legend Snippet: IKBKE is required for autophagy induced by the ERBB2 breast oncogene. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells overexpressing an activated form of ERBB2 (ERBB2 [CA]) upon pharmacological inhibition of IKBKE activity by CYT387 (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, ERBB2 (CA) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P

    Techniques Used: Confocal Microscopy, Multiple Displacement Amplification, Expressing, Immunofluorescence, Transfection, Negative Control, Staining, Software, Inhibition, Activity Assay

    The catalytic activity of IKBKE is required for its ability to control autophagy. (a) WB analysis to evaluate autophagic flux of MDA-MB-231 cells stably expressing IKBKE (WT) and IKBKE kinase dead (KD). Cells stably expressing an empty vector were used as a negative control. Where indicated, 4-h treatment with 400 nM BAF was performed. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels (used as loading control), is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Confocal microscopy analysis of MDA-MB-231 cells expressing IKBKE (WT) and IKBKE (KD), to evaluate autophagic flux. Cells were subjected to immunofluorescence analysis. Where indicated, 4-h treatment with 400 nM BAF was performed. In these representative images, LC3B is visualized in green, IKBKE (WT or KD) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. (c) Time course experiment to analyze the autophagic flux in MDA-MB-231 treated with the CYT387 drug, by WB analysis. Cells were treated with different concentrations of the drug (2, 5 and 10 μM) and treated or not with 400 nM BAF for 2 h. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels (used as loading control), is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Confocal microscopy analysis of MDA-MB-231 cells treated with CYT387, to evaluate the autophagic flux. Cells were treated with different concentrations of the drug (2, 5 and 10 μM) and treated or not with 400 nM BAF for 2 h. Then, cells were subjected to immunofluorescence analysis. In these representative images, LC3B is visualized in green, endogenous IKBKE in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P
    Figure Legend Snippet: The catalytic activity of IKBKE is required for its ability to control autophagy. (a) WB analysis to evaluate autophagic flux of MDA-MB-231 cells stably expressing IKBKE (WT) and IKBKE kinase dead (KD). Cells stably expressing an empty vector were used as a negative control. Where indicated, 4-h treatment with 400 nM BAF was performed. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels (used as loading control), is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Confocal microscopy analysis of MDA-MB-231 cells expressing IKBKE (WT) and IKBKE (KD), to evaluate autophagic flux. Cells were subjected to immunofluorescence analysis. Where indicated, 4-h treatment with 400 nM BAF was performed. In these representative images, LC3B is visualized in green, IKBKE (WT or KD) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. (c) Time course experiment to analyze the autophagic flux in MDA-MB-231 treated with the CYT387 drug, by WB analysis. Cells were treated with different concentrations of the drug (2, 5 and 10 μM) and treated or not with 400 nM BAF for 2 h. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels (used as loading control), is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Confocal microscopy analysis of MDA-MB-231 cells treated with CYT387, to evaluate the autophagic flux. Cells were treated with different concentrations of the drug (2, 5 and 10 μM) and treated or not with 400 nM BAF for 2 h. Then, cells were subjected to immunofluorescence analysis. In these representative images, LC3B is visualized in green, endogenous IKBKE in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P

    Techniques Used: Activity Assay, Western Blot, Multiple Displacement Amplification, Stable Transfection, Expressing, Plasmid Preparation, Negative Control, Confocal Microscopy, Immunofluorescence, Staining, Software

    A role for autophagy in IKBKE-dependent normal breast epithelial cell transformation and TNBC proliferation. (a) Proliferation assay of MDA-MB-231 cells upon downregulation of endogenous IKBKE, ATG5, ULK1 protein levels by transfection with the indicated siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679, #680 and siRNA for ATG5 and ULK1 ). Cell viability was evaluated 72 h post-transfection by counting cells in triplicate with a Z2 Coulter Counter. Data were processed in Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Same as in (a), but using MDA-MB-468 TNBC cells. (c) Cell count of 1-7HB2 cell stably expressing IKBKE (WT), evaluating cell proliferation, by counting cells in triplicate with a Z2 Coulter Counter, upon downregulation (72 h post-transfection) of endogenous ATG5 and ULK1 with specific siRNA. A scrambled siRNA was used as a negative control. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Proliferation assay of 1-7HB2 cell stably expressing IKBKE (WT) upon pharmacological inhibition of autophagic activity by SAR-405 (10 μM) and Spautin-1 (100 μM). Cell proliferation was evaluated after 72-h treatment by counting cells in triplicate with a Z2 Coulter Counter. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.
    Figure Legend Snippet: A role for autophagy in IKBKE-dependent normal breast epithelial cell transformation and TNBC proliferation. (a) Proliferation assay of MDA-MB-231 cells upon downregulation of endogenous IKBKE, ATG5, ULK1 protein levels by transfection with the indicated siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679, #680 and siRNA for ATG5 and ULK1 ). Cell viability was evaluated 72 h post-transfection by counting cells in triplicate with a Z2 Coulter Counter. Data were processed in Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Same as in (a), but using MDA-MB-468 TNBC cells. (c) Cell count of 1-7HB2 cell stably expressing IKBKE (WT), evaluating cell proliferation, by counting cells in triplicate with a Z2 Coulter Counter, upon downregulation (72 h post-transfection) of endogenous ATG5 and ULK1 with specific siRNA. A scrambled siRNA was used as a negative control. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Proliferation assay of 1-7HB2 cell stably expressing IKBKE (WT) upon pharmacological inhibition of autophagic activity by SAR-405 (10 μM) and Spautin-1 (100 μM). Cell proliferation was evaluated after 72-h treatment by counting cells in triplicate with a Z2 Coulter Counter. Data were processed with Prism 6 software. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.

    Techniques Used: Transformation Assay, Proliferation Assay, Multiple Displacement Amplification, Transfection, Software, Cell Counting, Stable Transfection, Expressing, Negative Control, Inhibition, Activity Assay

    The IKBKE human oncogene induces autophagy in MDA-MB-231 breast cancer cells. (a) Endogenous IKBKE protein levels were tested in indicated cell lines, by WB analysis. (b) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and 2 unrelated specific siRNA against human IKBKE , #679 and #680). Where indicated, samples were treated with 400 nM BAF or with starvation medium (STV) for 5 h. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels, is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (c) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells by confocal microscopy analysis of MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 24 h. In these representative images, SQSTM1 is visualized in green and DAPI-stained nuclei in blue. SQSTM1-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Autophagic flux was evaluated in in MDA-MB-231 cells transfected with Scr siRNA and siRNA specific for IKBKE , Followed by rescue with IKBKE (WT) overexpression. Where indicated, 4 h treatment with 400 nM BAF was performed. In these representative images, LC3B is visualized in green, IKBKE (WT) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.
    Figure Legend Snippet: The IKBKE human oncogene induces autophagy in MDA-MB-231 breast cancer cells. (a) Endogenous IKBKE protein levels were tested in indicated cell lines, by WB analysis. (b) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and 2 unrelated specific siRNA against human IKBKE , #679 and #680). Where indicated, samples were treated with 400 nM BAF or with starvation medium (STV) for 5 h. Densitometric analysis of LC3B-II levels, normalized by the corresponding MAPK1 levels, is also shown. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (c) Autophagic flux was evaluated in MDA-MB-231 breast cancer cells by confocal microscopy analysis of MDA-MB-231 breast cancer cells, upon downregulation of endogenous IKBKE protein levels by transfection of appropriate siRNA (scrambled siRNA as negative controls, and unrelated specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 24 h. In these representative images, SQSTM1 is visualized in green and DAPI-stained nuclei in blue. SQSTM1-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (d) Autophagic flux was evaluated in in MDA-MB-231 cells transfected with Scr siRNA and siRNA specific for IKBKE , Followed by rescue with IKBKE (WT) overexpression. Where indicated, 4 h treatment with 400 nM BAF was performed. In these representative images, LC3B is visualized in green, IKBKE (WT) in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown.

    Techniques Used: Multiple Displacement Amplification, Western Blot, Transfection, Confocal Microscopy, Staining, Software, Over Expression

    IKBKE is required for autophagy induced by the AKT transforming pathway. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon pharmacological inhibition of IKBKE activity by the CYT387 drug (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars, 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P
    Figure Legend Snippet: IKBKE is required for autophagy induced by the AKT transforming pathway. (a) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon downregulation of endogenous IKBKE expression. Cells were subjected to immunofluorescence analysis. IKBKE downregulation was obtained by transfection of appropriate siRNA (scrambled siRNA as a negative control and a specific siRNA against human IKBKE , #679). Where indicated, samples were treated with 400 nM BAF for 4 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars: 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. (b) Evaluation of autophagic flux by confocal microscopy analysis of MDA-MB-231 cells over-expressing an activated form of the AKT protein (myrAKT-HA) upon pharmacological inhibition of IKBKE activity by the CYT387 drug (2 μM, 2 h). Cells were subjected to immunofluorescence analysis. Where indicated, samples were treated with 400 nM BAF for 2 h. In these representative images, LC3B is visualized in green, myrAKT-HA in red, and DAPI-stained nuclei in blue. LC3B-positive dots were counted using a specific protocol by Volocity software (see graph in the lower panel). Scale bars, 25 μm. Results from one experiment, representative of 3 independent experiments (n = 3) are shown. Asterisks were attributed as follows: * P

    Techniques Used: Confocal Microscopy, Multiple Displacement Amplification, Expressing, Immunofluorescence, Transfection, Negative Control, Staining, Software, Inhibition, Activity Assay

    60) Product Images from "Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47"

    Article Title: Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47

    Journal: European Journal of Human Genetics

    doi: 10.1038/ejhg.2011.150

    ( a ) Expression levels of CT47 mRNA in different SCLC cell lines relative to the expression in human testis. Commercially available human total testis RNA and RNA isolated from SCLC lines were used for cDNA synthesis under identical conditions. CT47 expression levels are normalized to the expression levels measured in the testis. Different SCLC lines have different, but low levels of CT47 expression compared to the testis. Relative abundance of histone modifications and EZH2 at the CT47 promoter ( b ), exon 3 ( c ) and the distal region in SCLC cell lines ( d ). SCLC cell lines show individual variation in the abundance of different histone modifications. Generally, a loss of the repressive chromatin mark H3K9me3 can be observed in SCLCs. H3K27me3 levels are higher in SCLCs at the promoter and exon 3 region than in LCLs, but lower at the distal region. The relative abundance of the PRC2 component EZH2 responsible for generating H2K27me3 is dramatically reduced in SCLCs compared to LCLs at all region studied. ( e ) DNA methylation levels at CpGs located in the CT47 promoter region in LCL and SCLC samples. The methylation level of seven different CpGs, next to the transcriptional start site of CT47 , was determined by bisulfite sequencing and quantified by ESME program. There is a significant difference between the methylation level of LCLs and SCLCs at every CpG tested ( P
    Figure Legend Snippet: ( a ) Expression levels of CT47 mRNA in different SCLC cell lines relative to the expression in human testis. Commercially available human total testis RNA and RNA isolated from SCLC lines were used for cDNA synthesis under identical conditions. CT47 expression levels are normalized to the expression levels measured in the testis. Different SCLC lines have different, but low levels of CT47 expression compared to the testis. Relative abundance of histone modifications and EZH2 at the CT47 promoter ( b ), exon 3 ( c ) and the distal region in SCLC cell lines ( d ). SCLC cell lines show individual variation in the abundance of different histone modifications. Generally, a loss of the repressive chromatin mark H3K9me3 can be observed in SCLCs. H3K27me3 levels are higher in SCLCs at the promoter and exon 3 region than in LCLs, but lower at the distal region. The relative abundance of the PRC2 component EZH2 responsible for generating H2K27me3 is dramatically reduced in SCLCs compared to LCLs at all region studied. ( e ) DNA methylation levels at CpGs located in the CT47 promoter region in LCL and SCLC samples. The methylation level of seven different CpGs, next to the transcriptional start site of CT47 , was determined by bisulfite sequencing and quantified by ESME program. There is a significant difference between the methylation level of LCLs and SCLCs at every CpG tested ( P

    Techniques Used: Expressing, Isolation, DNA Methylation Assay, Methylation, Methylation Sequencing

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    Flow Cytometry:

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    Expressing:

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    Transferring:

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    Cytometry:

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    Blocking Assay:

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    Incubation:

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    Cell Culture:

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    Lysis:

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    Polymerase Chain Reaction:

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    Sonication:

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    Real-time Polymerase Chain Reaction:

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    Article Snippet: .. Quantitative Real-time PCR Analysis Total RNA was extracted using QIAzol lysis reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions and equal amounts of the cDNA of RNA were synthesized from 1 µg of total RNA using SuperScript II Reverse Transcriptase (Invitrogen, San Diego, CA, USA). .. Real-time PCR was performed using a Exicycler™ 96 Real-Time Quantitative Thermal Block (Bioneer Co., Daejeon, Korea) in a 20 µL reaction mixture containing 10 µL SYBR Green Premix (Bioneer Co.), 10 pmol forward primer, 10 pmol reverse primer, and 1 µg cDNA.

    Article Title: Myocyte repolarization modulates myocardial function in aging dogs
    Article Snippet: Total RNA was extracted from snap-frozen LV myocardial tissue utilizing QIAzol Lysis Reagent (Qiagen) and digested with DNase I and cleaned up with RNeasy Mini Kit (Qiagen) to eliminate genomic DNA. cDNA was obtained from 1 μg total RNA using MultiScribe reverse transcriptase kit (Applied Biosystems). .. The StepOnePlus Real-Time PCR system (Applied Biosystems) was employed for quantitative RT-PCR.

    RNA Extraction:

    Article Title: Krüppel-like Factor 3 (KLF3/BKLF) Is Required for Widespread Repression of the Inflammatory Modulator Galectin-3 (Lgals3) *
    Article Snippet: Paragraph title: RNA Extraction and cDNA Synthesis ... Tissues were harvested and suspended in TRI reagent (Sigma) or QIAzol lysis reagent (Qiagen) for lipid-rich adipose and brain samples.

    Article Title: Identification of miRNAs contributing to neuroblastoma chemoresistance
    Article Snippet: Consequently, the remaining cell pellet was treated with 700 μL of Qiazol solution [Qiagen, Germany] in order to induce cell lysis. .. Immediately after cell lysis induction, all cell lysate suspensions were snap frozen in liquid nitrogen solution and transferred to a − 80 °C freezer until RNA extraction was performed.

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    Qiagen qiazol lysis reagent
    DM suppresses the expressions of c-Fos, NFATc1, and other osteoclast marker genes. ( A ) BMMs were stimulated with RANKL (50 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of DM (50 ng/mL) for the indicated times. Total RNA was isolated from cells using <t>QIAzol</t> reagent; mRNA expression levels of c-Fos and NFATc1 were evaluated using quantitative real-time <t>RT-PCR.</t> *** p
    Qiazol Lysis Reagent, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1686 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DM suppresses the expressions of c-Fos, NFATc1, and other osteoclast marker genes. ( A ) BMMs were stimulated with RANKL (50 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of DM (50 ng/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent; mRNA expression levels of c-Fos and NFATc1 were evaluated using quantitative real-time RT-PCR. *** p

    Journal: Molecules

    Article Title: Dendrobium moniliforme Exerts Inhibitory Effects on Both Receptor Activator of Nuclear Factor Kappa-B Ligand-Mediated Osteoclast Differentiation in Vitro and Lipopolysaccharide-Induced Bone Erosion in Vivo

    doi: 10.3390/molecules21030295

    Figure Lengend Snippet: DM suppresses the expressions of c-Fos, NFATc1, and other osteoclast marker genes. ( A ) BMMs were stimulated with RANKL (50 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of DM (50 ng/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent; mRNA expression levels of c-Fos and NFATc1 were evaluated using quantitative real-time RT-PCR. *** p

    Article Snippet: Quantitative Real-Time RT-PCR Analysis Total RNA was extracted using the QIAzol lysis reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions.

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

    CIE downregulates RANKL-induced early signals and marker genes during osteoclastogenesis. (a) BMMs were pretreated with DMSO (control) or CIE (50 μ g/mL) for 1 h in the presence of M-CSF (30 ng/mL) and were stimulated with RANKL (100 ng/mL) for the indicated times. Whole-cell lysates were used for western blot analysis with the specified antibodies. β -Actin served as the internal control. (b) BMMs were stimulated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of CIE (50 μ g/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent and the mRNA expression levels of OSCAR, TRAP, integrin α v, β 3, DC-STAMP, OC-STAMP, cathepsin K, and ICAM-1 were evaluated by real-time PCR. * P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Dual Effect of Chrysanthemum indicum Extract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and Resorption In Vitro

    doi: 10.1155/2014/176049

    Figure Lengend Snippet: CIE downregulates RANKL-induced early signals and marker genes during osteoclastogenesis. (a) BMMs were pretreated with DMSO (control) or CIE (50 μ g/mL) for 1 h in the presence of M-CSF (30 ng/mL) and were stimulated with RANKL (100 ng/mL) for the indicated times. Whole-cell lysates were used for western blot analysis with the specified antibodies. β -Actin served as the internal control. (b) BMMs were stimulated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of CIE (50 μ g/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent and the mRNA expression levels of OSCAR, TRAP, integrin α v, β 3, DC-STAMP, OC-STAMP, cathepsin K, and ICAM-1 were evaluated by real-time PCR. * P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (PCR) Analysis Total RNA was extracted using QIAzol lysis reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions, and equal amounts of cDNA from the RNA samples were synthesized using 1 μ g of total RNA and SuperScript II Reverse Transcriptase (Invitrogen, San Diego, CA, USA).

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

    CIE promotes ascorbic acid and β -glycerol phosphate mediated osteoblast differentiation. (a) Primary osteoblasts were treated with various concentrations of CIE for 7 days in the presence of 50 μ g/mL ascorbic acid and 10 mM β -glycerol phosphate. ALP-positive cells were stained with ALP solution. (b) Primary osteoblasts were treated with various concentrations of CIE for 21 days. Calcium accumulation within the osteoblasts was stained with ARS solution. Stained calcium deposits were dissolved by 10% CPC buffer to measure the level of staining. (c) Primary osteoblasts were stimulated with ascorbic acid (50 μ g/mL) and β -glycerol phosphate in the presence of CIE (50 μ g/mL) or DMSO. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of Runx2, ALP, Col1 α , and OPN were evaluated by real-time PCR. * P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Dual Effect of Chrysanthemum indicum Extract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and Resorption In Vitro

    doi: 10.1155/2014/176049

    Figure Lengend Snippet: CIE promotes ascorbic acid and β -glycerol phosphate mediated osteoblast differentiation. (a) Primary osteoblasts were treated with various concentrations of CIE for 7 days in the presence of 50 μ g/mL ascorbic acid and 10 mM β -glycerol phosphate. ALP-positive cells were stained with ALP solution. (b) Primary osteoblasts were treated with various concentrations of CIE for 21 days. Calcium accumulation within the osteoblasts was stained with ARS solution. Stained calcium deposits were dissolved by 10% CPC buffer to measure the level of staining. (c) Primary osteoblasts were stimulated with ascorbic acid (50 μ g/mL) and β -glycerol phosphate in the presence of CIE (50 μ g/mL) or DMSO. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of Runx2, ALP, Col1 α , and OPN were evaluated by real-time PCR. * P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (PCR) Analysis Total RNA was extracted using QIAzol lysis reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions, and equal amounts of cDNA from the RNA samples were synthesized using 1 μ g of total RNA and SuperScript II Reverse Transcriptase (Invitrogen, San Diego, CA, USA).

    Techniques: ALP Assay, Staining, Isolation, Expressing, Real-time Polymerase Chain Reaction

    CIE suppresses RANKL-induced c-Fos and NFATc1 expression. (a) Effects of CIE on levels of c-Fos and NFATc1 protein expression were evaluated using western blot analysis. β -Actin was used as the internal control. (b) BMMs were stimulated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of CIE (50 μ g/mL) for the specified times. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of c-Fos and NFATc1 were evaluated using real-time PCR. * P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Dual Effect of Chrysanthemum indicum Extract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and Resorption In Vitro

    doi: 10.1155/2014/176049

    Figure Lengend Snippet: CIE suppresses RANKL-induced c-Fos and NFATc1 expression. (a) Effects of CIE on levels of c-Fos and NFATc1 protein expression were evaluated using western blot analysis. β -Actin was used as the internal control. (b) BMMs were stimulated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of CIE (50 μ g/mL) for the specified times. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of c-Fos and NFATc1 were evaluated using real-time PCR. * P

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (PCR) Analysis Total RNA was extracted using QIAzol lysis reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions, and equal amounts of cDNA from the RNA samples were synthesized using 1 μ g of total RNA and SuperScript II Reverse Transcriptase (Invitrogen, San Diego, CA, USA).

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

    Aconitum pseudo-laeve var. erectum (APE) suppresses receptor activator of nuclear factor kappa-B ligand (RANKL)-induced c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) expression without stimulating early signal pathways. ( A ) Bone marrow macrophages (BMMs) were pretreated with DMSO (control) or APE (200 µg/mL) for 1 h in the presence of macrophage colony-stimulating factor (M-CSF; 30 ng/mL) and were stimulated with RANKL (100 ng/mL) for the indicated times. Whole-cell lysates underwent western blot analysis with the various indicated antibodies. β-actin served as the internal control; ( B ) BMMs were stimulated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of APE (200 µg/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of c-Fos and NFATc1 were evaluated using real-time PCR; ( C ) Effects of APE on protein expression levels of c-Fos and NFATc1 were evaluated using western blot analysis. β-actin was used as the internal control; ( D ) BMMs were infected with retroviruses expressing pMX-IRES-EGFP (pMX), pMX-NFATc1-EGFP, and pMX-cFos-EGFP. Infected BMMs were cultured with or without APE (200 µg/mL) in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days. After culturing, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) (left). The TRAP-positive multinucleated osteoclasts were counted (right).

    Journal: Molecules

    Article Title: Aconitum pseudo-laeve var. erectum Inhibits Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclastogenesis via the c-Fos/nuclear Factor of Activated T-Cells, Cytoplasmic 1 Signaling Pathway and Prevents Lipopolysaccharide-Induced Bone Loss in Mice

    doi: 10.3390/molecules190811628

    Figure Lengend Snippet: Aconitum pseudo-laeve var. erectum (APE) suppresses receptor activator of nuclear factor kappa-B ligand (RANKL)-induced c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) expression without stimulating early signal pathways. ( A ) Bone marrow macrophages (BMMs) were pretreated with DMSO (control) or APE (200 µg/mL) for 1 h in the presence of macrophage colony-stimulating factor (M-CSF; 30 ng/mL) and were stimulated with RANKL (100 ng/mL) for the indicated times. Whole-cell lysates underwent western blot analysis with the various indicated antibodies. β-actin served as the internal control; ( B ) BMMs were stimulated with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the presence or absence of APE (200 µg/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of c-Fos and NFATc1 were evaluated using real-time PCR; ( C ) Effects of APE on protein expression levels of c-Fos and NFATc1 were evaluated using western blot analysis. β-actin was used as the internal control; ( D ) BMMs were infected with retroviruses expressing pMX-IRES-EGFP (pMX), pMX-NFATc1-EGFP, and pMX-cFos-EGFP. Infected BMMs were cultured with or without APE (200 µg/mL) in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days. After culturing, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) (left). The TRAP-positive multinucleated osteoclasts were counted (right).

    Article Snippet: Quantitative Real-time PCR Analysis Total RNA was extracted using QIAzol lysis reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions and equal amounts of the cDNA of RNA were synthesized from 1 µg of total RNA using SuperScript II Reverse Transcriptase (Invitrogen, San Diego, CA, USA).

    Techniques: Expressing, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Infection, Cell Culture, Staining

    Aconitum pseudo-laeve var. erectum (APE) down-regulates the expression of osteoclast-specific marker genes. Bone marrow macrophages (BMMs) were stimulated with receptor activator of nuclear factor kappa-B ligand (RANKL; 100 ng/mL) and macrophage colony-stimulating factor (M-CSF; 30 ng/mL) in the presence or absence of APE (200 µg/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of osteoclast-associated receptor (OSCAR), tartrate-resistant acid phosphatase (TRAP), Atp6v0d2, Cathepsin K, dendritic cell-specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), calcitonin receptor, and matrix metallopeptidase 9 (MMP-9) were evaluated by real-time PCR.

    Journal: Molecules

    Article Title: Aconitum pseudo-laeve var. erectum Inhibits Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclastogenesis via the c-Fos/nuclear Factor of Activated T-Cells, Cytoplasmic 1 Signaling Pathway and Prevents Lipopolysaccharide-Induced Bone Loss in Mice

    doi: 10.3390/molecules190811628

    Figure Lengend Snippet: Aconitum pseudo-laeve var. erectum (APE) down-regulates the expression of osteoclast-specific marker genes. Bone marrow macrophages (BMMs) were stimulated with receptor activator of nuclear factor kappa-B ligand (RANKL; 100 ng/mL) and macrophage colony-stimulating factor (M-CSF; 30 ng/mL) in the presence or absence of APE (200 µg/mL) for the indicated times. Total RNA was isolated from cells using QIAzol reagent and mRNA expression levels of osteoclast-associated receptor (OSCAR), tartrate-resistant acid phosphatase (TRAP), Atp6v0d2, Cathepsin K, dendritic cell-specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), calcitonin receptor, and matrix metallopeptidase 9 (MMP-9) were evaluated by real-time PCR.

    Article Snippet: Quantitative Real-time PCR Analysis Total RNA was extracted using QIAzol lysis reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions and equal amounts of the cDNA of RNA were synthesized from 1 µg of total RNA using SuperScript II Reverse Transcriptase (Invitrogen, San Diego, CA, USA).

    Techniques: Expressing, Marker, Isolation, Real-time Polymerase Chain Reaction

    Comparison of different storage temperature and trehalose for FTA Elute card stored for 2 days. ( A ) Untreated or 50 mg/ml trehalose-treated 6 mm FTA Elute card disc punch-out spotted with 20 µl serum were dried and stored at RT, 4 °C or −20 °C for 2 days before RNA was extracted using QIAzol lysis reagent. 10 miRNAs were quantified by RT-qPCR and their copy numbers were presented. Neat refers to copy number of each individual miRNA obtained from 20 µl serum extracted directly using QIAzol lysis reagent. The individual % miRNA recovery was compared between untreated and trehalose-treated FTA Elute card disc punch-out. ( B ) Average % miRNA recovery of all 10 miRNAs was calculated and plotted to compare the effect of pre-treatment of FTA Elute card disc punch-out with 50mg/ml trehalose. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data between selected pairs. Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; **P ≤ 0.01; * P ≤ 0.05, n = 3).

    Journal: Scientific Reports

    Article Title: Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix

    doi: 10.1038/s41598-017-16960-8

    Figure Lengend Snippet: Comparison of different storage temperature and trehalose for FTA Elute card stored for 2 days. ( A ) Untreated or 50 mg/ml trehalose-treated 6 mm FTA Elute card disc punch-out spotted with 20 µl serum were dried and stored at RT, 4 °C or −20 °C for 2 days before RNA was extracted using QIAzol lysis reagent. 10 miRNAs were quantified by RT-qPCR and their copy numbers were presented. Neat refers to copy number of each individual miRNA obtained from 20 µl serum extracted directly using QIAzol lysis reagent. The individual % miRNA recovery was compared between untreated and trehalose-treated FTA Elute card disc punch-out. ( B ) Average % miRNA recovery of all 10 miRNAs was calculated and plotted to compare the effect of pre-treatment of FTA Elute card disc punch-out with 50mg/ml trehalose. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data between selected pairs. Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; **P ≤ 0.01; * P ≤ 0.05, n = 3).

    Article Snippet: Direct isolation of serum/plasma RNA by QIAzol lysis reagent Total RNA from serum/plasma was isolated using miRNeasy serum/plasma isolation kit (Qiagen) according to manufacturer’s protocol.

    Techniques: Lysis, Quantitative RT-PCR

    miRNA expression level in GC patient and paired controls. ( A ) 20 µl of each of the 10 GC patients and 10 paired control serum were spotted on individual 6 mm FTA Elute card disc punch-out. 8 miRNAs were quantified and miRNA recovery was compared with the sample in which RNA was isolated directly from 20 µl serum using QIAzol lysis reagent (neat). ( B ) Correlation of miRNA representation from neat serum or FTA Elute card discs. Each experimental condition consisted of 10 biological replicates and data were presented as mean ± SEM. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data for selected pairs (*** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns: not significant).

    Journal: Scientific Reports

    Article Title: Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix

    doi: 10.1038/s41598-017-16960-8

    Figure Lengend Snippet: miRNA expression level in GC patient and paired controls. ( A ) 20 µl of each of the 10 GC patients and 10 paired control serum were spotted on individual 6 mm FTA Elute card disc punch-out. 8 miRNAs were quantified and miRNA recovery was compared with the sample in which RNA was isolated directly from 20 µl serum using QIAzol lysis reagent (neat). ( B ) Correlation of miRNA representation from neat serum or FTA Elute card discs. Each experimental condition consisted of 10 biological replicates and data were presented as mean ± SEM. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data for selected pairs (*** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns: not significant).

    Article Snippet: Direct isolation of serum/plasma RNA by QIAzol lysis reagent Total RNA from serum/plasma was isolated using miRNeasy serum/plasma isolation kit (Qiagen) according to manufacturer’s protocol.

    Techniques: Expressing, Isolation, Lysis

    Effect on miRNA yield during long-term FTA Elute card storage. ( A ) 20 µl serum spotted on trehalose-treated 6 mm FTA Elute card disc punch-out was subjected to accelerated aging testing of 1, 6 and 12 months before RNA was extracted using QIAzol lysis reagent. 10 miRNAs were quantified. The copy number of each miRNA was compared against the copy number obtained after storage at RT for 2 days. ( B ) Average % miRNA recovery of all 10 miRNAs for accelerated aging of 1, 6, 12 months was determined and compared with 2 days. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data. Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; **P ≤ 0.01; * P ≤ 0.05; ns: not significant, n = 3).

    Journal: Scientific Reports

    Article Title: Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix

    doi: 10.1038/s41598-017-16960-8

    Figure Lengend Snippet: Effect on miRNA yield during long-term FTA Elute card storage. ( A ) 20 µl serum spotted on trehalose-treated 6 mm FTA Elute card disc punch-out was subjected to accelerated aging testing of 1, 6 and 12 months before RNA was extracted using QIAzol lysis reagent. 10 miRNAs were quantified. The copy number of each miRNA was compared against the copy number obtained after storage at RT for 2 days. ( B ) Average % miRNA recovery of all 10 miRNAs for accelerated aging of 1, 6, 12 months was determined and compared with 2 days. Statistical analyses were performed with one-way ANOVA, followed by Bonferroni’s pairwise comparisons test data. Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; **P ≤ 0.01; * P ≤ 0.05; ns: not significant, n = 3).

    Article Snippet: Direct isolation of serum/plasma RNA by QIAzol lysis reagent Total RNA from serum/plasma was isolated using miRNeasy serum/plasma isolation kit (Qiagen) according to manufacturer’s protocol.

    Techniques: Lysis

    miRNA recovery from serum-spotted FTA Elute cards using water and heat was inefficient. ( A ) RNA was extracted directly from 20 µl serum (black) or 20 µl plasma collected in EDTA-containing tube (plasma-EDTA) (white) using QIAzol lysis reagent. 10 miRNAs were quantified by RT-qPCR based on their copy numbers. RNA was extracted from a 6 mm FTA Elute card disc punch-out spotted with 20 µl serum (black) or 20 µl plasma-EDTA (white), using water and heating at 95 °C for 30 min. 10 miRNAs were quantified by RT-qPCR and their copy numbers ( B ) and % miRNA recovery ( C ) were presented. (% recovery = (Copy number of miRNA extracted from FTA Elute card disc punch-out/Copy number of miRNA extracted directly from serum) × 100%). Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns: not significant, Student’s t -test, n = 3).

    Journal: Scientific Reports

    Article Title: Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix

    doi: 10.1038/s41598-017-16960-8

    Figure Lengend Snippet: miRNA recovery from serum-spotted FTA Elute cards using water and heat was inefficient. ( A ) RNA was extracted directly from 20 µl serum (black) or 20 µl plasma collected in EDTA-containing tube (plasma-EDTA) (white) using QIAzol lysis reagent. 10 miRNAs were quantified by RT-qPCR based on their copy numbers. RNA was extracted from a 6 mm FTA Elute card disc punch-out spotted with 20 µl serum (black) or 20 µl plasma-EDTA (white), using water and heating at 95 °C for 30 min. 10 miRNAs were quantified by RT-qPCR and their copy numbers ( B ) and % miRNA recovery ( C ) were presented. (% recovery = (Copy number of miRNA extracted from FTA Elute card disc punch-out/Copy number of miRNA extracted directly from serum) × 100%). Each experimental condition was carried out thrice and data were presented as mean ± SEM (*** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns: not significant, Student’s t -test, n = 3).

    Article Snippet: Direct isolation of serum/plasma RNA by QIAzol lysis reagent Total RNA from serum/plasma was isolated using miRNeasy serum/plasma isolation kit (Qiagen) according to manufacturer’s protocol.

    Techniques: Lysis, Quantitative RT-PCR