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    Thermo Fisher gene exp b2m hs00187842 m1
    Effect of tocilizumab on the expression of C5aR1, C5aR2, and C3aR in NSTEMI patients. The effect of tocilizumab, a monoclonal antibody inhibiting interleukin 6 receptor (IL-6R), on the expression of the three complement anaphylatoxin receptors [C5aR1 (A) , C5aR2 (B) , and C3aR (C) ] was investigated in patients with non-ST-elevation myocardial infarction (NSTEMI). mRNA levels were quantified by qPCR and related to the reference gene <t>beta-2-microglobulin.</t> The tocilizumab group (gray bars, n = 28) and the placebo group (white bars, n = 32) are presented at four different time-points. Baseline levels show the receptor expression at inclusion, i.e., after hospital admission, before treatment was given. Follow-up time points were day 2 and 3, and 6 months. A group of healthy individuals ( n = 15) were included as controls. The qPCR results were quantified using the 2 −ΔΔCT method, normalized to reference genes and presented as fold change with the healthy controls as calibrator. Data are given as median and 95% CI. * P
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    1) Product Images from "IL-6 Receptor Inhibition by Tocilizumab Attenuated Expression of C5a Receptor 1 and 2 in Non-ST-Elevation Myocardial Infarction"

    Article Title: IL-6 Receptor Inhibition by Tocilizumab Attenuated Expression of C5a Receptor 1 and 2 in Non-ST-Elevation Myocardial Infarction

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02035

    Effect of tocilizumab on the expression of C5aR1, C5aR2, and C3aR in NSTEMI patients. The effect of tocilizumab, a monoclonal antibody inhibiting interleukin 6 receptor (IL-6R), on the expression of the three complement anaphylatoxin receptors [C5aR1 (A) , C5aR2 (B) , and C3aR (C) ] was investigated in patients with non-ST-elevation myocardial infarction (NSTEMI). mRNA levels were quantified by qPCR and related to the reference gene beta-2-microglobulin. The tocilizumab group (gray bars, n = 28) and the placebo group (white bars, n = 32) are presented at four different time-points. Baseline levels show the receptor expression at inclusion, i.e., after hospital admission, before treatment was given. Follow-up time points were day 2 and 3, and 6 months. A group of healthy individuals ( n = 15) were included as controls. The qPCR results were quantified using the 2 −ΔΔCT method, normalized to reference genes and presented as fold change with the healthy controls as calibrator. Data are given as median and 95% CI. * P
    Figure Legend Snippet: Effect of tocilizumab on the expression of C5aR1, C5aR2, and C3aR in NSTEMI patients. The effect of tocilizumab, a monoclonal antibody inhibiting interleukin 6 receptor (IL-6R), on the expression of the three complement anaphylatoxin receptors [C5aR1 (A) , C5aR2 (B) , and C3aR (C) ] was investigated in patients with non-ST-elevation myocardial infarction (NSTEMI). mRNA levels were quantified by qPCR and related to the reference gene beta-2-microglobulin. The tocilizumab group (gray bars, n = 28) and the placebo group (white bars, n = 32) are presented at four different time-points. Baseline levels show the receptor expression at inclusion, i.e., after hospital admission, before treatment was given. Follow-up time points were day 2 and 3, and 6 months. A group of healthy individuals ( n = 15) were included as controls. The qPCR results were quantified using the 2 −ΔΔCT method, normalized to reference genes and presented as fold change with the healthy controls as calibrator. Data are given as median and 95% CI. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    2) Product Images from "Inhibition of Plk1 and Cyclin B1 Expression Results in Panobinostat-Induced G2 Delay and Mitotic Defects"

    Article Title: Inhibition of Plk1 and Cyclin B1 Expression Results in Panobinostat-Induced G2 Delay and Mitotic Defects

    Journal: Scientific Reports

    doi: 10.1038/srep02640

    Reduction of G2-specific Cyclin B1 expression following panobinostat treatment. FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA (A) and protein (B and C) harvested as described in the materials and methods. (A) Cyclin B1 mRNA expression was assessed by qRT-PCR. The data shown represent Cyclin B1 mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B) 60 μg protein from each extract was separated by SDS-PAGE on two separate gels that were run using identical conditions. Cyclin B1 (upper panel) and β-actin (lower panel) protein levels were assessed by Western blot. Full-length digital images of these western blots can be viewed in the supplementary information ( supplementary figure 5 ). (C) Cyclin B1 protein levels from three independent experiments were quantified using ImageGauge v4.21 software and normalised to β-actin levels from the same experiment. The data shown represent the mean and standard deviation.
    Figure Legend Snippet: Reduction of G2-specific Cyclin B1 expression following panobinostat treatment. FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA (A) and protein (B and C) harvested as described in the materials and methods. (A) Cyclin B1 mRNA expression was assessed by qRT-PCR. The data shown represent Cyclin B1 mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B) 60 μg protein from each extract was separated by SDS-PAGE on two separate gels that were run using identical conditions. Cyclin B1 (upper panel) and β-actin (lower panel) protein levels were assessed by Western blot. Full-length digital images of these western blots can be viewed in the supplementary information ( supplementary figure 5 ). (C) Cyclin B1 protein levels from three independent experiments were quantified using ImageGauge v4.21 software and normalised to β-actin levels from the same experiment. The data shown represent the mean and standard deviation.

    Techniques Used: Expressing, Blocking Assay, Quantitative RT-PCR, Standard Deviation, SDS Page, Western Blot, Software

    Reduction of G2-specific Plk1 expression following panobinostat treatment. FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA (A) and protein (B and C) harvested as described in the materials and methods. (A) PLK1 mRNA expression was assessed by qRT-PCR. The data shown represent PLK1 mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B) 60 μg protein from each extract was separated by SDS-PAGE on two separate gels run using identical conditions. The upper panel was probed with Plk1 antibody and the lower panel probed with β-actin antibody. Full-length digital images of these western blots can be viewed in the supplementary information ( supplementary figure 5 ). (C) Plk1 protein levels from three independent experiments were quantified using ImageGauge v4.21 software and normalised to β-actin levels from the same experiment. The data shown represent the mean and standard deviation.
    Figure Legend Snippet: Reduction of G2-specific Plk1 expression following panobinostat treatment. FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA (A) and protein (B and C) harvested as described in the materials and methods. (A) PLK1 mRNA expression was assessed by qRT-PCR. The data shown represent PLK1 mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B) 60 μg protein from each extract was separated by SDS-PAGE on two separate gels run using identical conditions. The upper panel was probed with Plk1 antibody and the lower panel probed with β-actin antibody. Full-length digital images of these western blots can be viewed in the supplementary information ( supplementary figure 5 ). (C) Plk1 protein levels from three independent experiments were quantified using ImageGauge v4.21 software and normalised to β-actin levels from the same experiment. The data shown represent the mean and standard deviation.

    Techniques Used: Expressing, Blocking Assay, Quantitative RT-PCR, Standard Deviation, SDS Page, Western Blot, Software

    Panobinostat induced p21 Waf1/Cip1 expression. FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA (A) and protein (B and C) harvested as described in the materials and methods. (A) p21 Waf1/Cip1 mRNA expression was assessed by qRT-PCR. The data shown represent p21 Waf1/Cip1 mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B) 60 μg protein from each extract was separated by SDS-PAGE on two separate gels runs using identical conditions and p21 Waf1/Cip1 (upper panel) and β-actin (lower panel) protein levels were assessed by Western blot. Full-length digital images of these western blots can be viewed in the supplementary information ( supplementary figure 5 ). (C) p21 Waf1/Cip1 protein levels from three independent experiments were quantified using ImageGauge v4.21 software and normalised to β-actin levels from the same experiment. The data shown represent the mean and standard deviation.
    Figure Legend Snippet: Panobinostat induced p21 Waf1/Cip1 expression. FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA (A) and protein (B and C) harvested as described in the materials and methods. (A) p21 Waf1/Cip1 mRNA expression was assessed by qRT-PCR. The data shown represent p21 Waf1/Cip1 mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B) 60 μg protein from each extract was separated by SDS-PAGE on two separate gels runs using identical conditions and p21 Waf1/Cip1 (upper panel) and β-actin (lower panel) protein levels were assessed by Western blot. Full-length digital images of these western blots can be viewed in the supplementary information ( supplementary figure 5 ). (C) p21 Waf1/Cip1 protein levels from three independent experiments were quantified using ImageGauge v4.21 software and normalised to β-actin levels from the same experiment. The data shown represent the mean and standard deviation.

    Techniques Used: Expressing, Blocking Assay, Quantitative RT-PCR, Standard Deviation, SDS Page, Western Blot, Software

    Loss of E2F1 recruitment to the Plk1 and Cyclin B1 promoters in response to panobinostat. (A) FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA. E2F1, E2F2, E2F3 and E2F5 mRNA expression was assessed by qRT-PCR. The data shown represent mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B and C) Asynchronous FaDu cells were incubated with 100 nM panobinostat or excipient control for 12 hours and crosslinked. Chromatin was then extracted and sheared and clarified chromatin preparations were immunoprecipitated with anti-FLAG (negative), E2F1, E2F2 or E2F3 antibody. The percentage of bound PLK1 promoter (B) or Cyclin B1 promoter (C) was then assessed by qRT-PCR and shown as the mean and standard deviation of three independent experiments.
    Figure Legend Snippet: Loss of E2F1 recruitment to the Plk1 and Cyclin B1 promoters in response to panobinostat. (A) FaDu cells were synchronised by double thymidine block and released in medium containing 100 nM panobinostat or excipient control. Cells were harvested at the stated time points post release and RNA. E2F1, E2F2, E2F3 and E2F5 mRNA expression was assessed by qRT-PCR. The data shown represent mRNA levels normalised to 0 hour time point and are shown as the mean and standard deviation of three independent experiments. (B and C) Asynchronous FaDu cells were incubated with 100 nM panobinostat or excipient control for 12 hours and crosslinked. Chromatin was then extracted and sheared and clarified chromatin preparations were immunoprecipitated with anti-FLAG (negative), E2F1, E2F2 or E2F3 antibody. The percentage of bound PLK1 promoter (B) or Cyclin B1 promoter (C) was then assessed by qRT-PCR and shown as the mean and standard deviation of three independent experiments.

    Techniques Used: Blocking Assay, Expressing, Quantitative RT-PCR, Standard Deviation, Incubation, Immunoprecipitation

    3) Product Images from "Adiponectin Induces Oncostatin M Expression in Osteoblasts through the PI3K/Akt Signaling Pathway"

    Article Title: Adiponectin Induces Oncostatin M Expression in Osteoblasts through the PI3K/Akt Signaling Pathway

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17010029

    Adiponectin increases OSM expression through nuclear factor-κB (NF-κB) pathway. ( A ) Osteoblasts were pretreated with pyrrolidine dithiocarbamate (PDTC) (10 μM) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (10 μM) or transfected with p65 siRNA (0.5 nM) followed by stimulation with adiponectin (100 ng/mL), the mRNA expression of OSM were analyzed by qPCR ( n = 5); ( B ) Cells were transfected with p65 siRNA for 24 h, the protein level of p65 was measured by Western blot (upper-panel), and supernatant medium was collected to measure OSM expression by ELISA assay (lower-panel) ( n = 5); ( C ) Cells were pretreated with PDTC and TPCK for 30 min followed by stimulation with adiponectin (100 ng/mL), the protein level of OSM was measured by Western blot ( n = 6); ( D ) Cells were incubated with adiponectin in time intervals, and phosphate-IKK, -IκB, and -p65 expression were investigated by Western blot ( n = 5); ( E ) Cells were pretreated with PI3K inhibitor, LY294002 (10 μM), or Akt inhibitor (20 μM) for 30 min followed by stimulation with adiponectin (100 ng/mL), phosphate-IKK, -IκB, and -p65 expression were investigated by Western blot ( n = 6). Results are expressed as mean ± standard error of mean S.E.M. *, p
    Figure Legend Snippet: Adiponectin increases OSM expression through nuclear factor-κB (NF-κB) pathway. ( A ) Osteoblasts were pretreated with pyrrolidine dithiocarbamate (PDTC) (10 μM) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (10 μM) or transfected with p65 siRNA (0.5 nM) followed by stimulation with adiponectin (100 ng/mL), the mRNA expression of OSM were analyzed by qPCR ( n = 5); ( B ) Cells were transfected with p65 siRNA for 24 h, the protein level of p65 was measured by Western blot (upper-panel), and supernatant medium was collected to measure OSM expression by ELISA assay (lower-panel) ( n = 5); ( C ) Cells were pretreated with PDTC and TPCK for 30 min followed by stimulation with adiponectin (100 ng/mL), the protein level of OSM was measured by Western blot ( n = 6); ( D ) Cells were incubated with adiponectin in time intervals, and phosphate-IKK, -IκB, and -p65 expression were investigated by Western blot ( n = 5); ( E ) Cells were pretreated with PI3K inhibitor, LY294002 (10 μM), or Akt inhibitor (20 μM) for 30 min followed by stimulation with adiponectin (100 ng/mL), phosphate-IKK, -IκB, and -p65 expression were investigated by Western blot ( n = 6). Results are expressed as mean ± standard error of mean S.E.M. *, p

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation

    Involvement of Akt in adiponectin-induced OSM expression in osteoblasts. ( A ) Osteoblasts were pretreated with Akt inhibitors (Akti) (20 μM) or transfected with Akt siRNA (0.5 nM) for 24 h followed by stimulation with adiponectin (100 ng/mL), OSM expression was measured by qPCR ( n = 6); ( B ) Cells were transfected with Akt siRNA (0.5 nM) for 24h, the protein level of Akt was measured by Western blot ( upper-panel ), and supernatant medium was collected to measure OSM expression by ELISA assay ( lower-panel ) ( n = 5); ( C ) Cells were pretreated with Akti (20 μM) for 30 min followed by stimulation with adiponectin (100 ng/mL), the protein level of OSM was measured by Western blot ( n = 5); ( D ) Osteoblasts were incubated with adiponectin (100 ng/mL) in time intervals, and phosphate-PI3K expression was investigated by Western blot ( n = 4); ( E ) Cells were pretreated with PI3K inhibitor, LY294002 (10 μM), for 30 min followed by stimulation with adiponectin (100 ng/mL), phosphate-Akt expression was investigated by Western blot ( n = 5). Results are expressed as mean ± S.E.M. *, p
    Figure Legend Snippet: Involvement of Akt in adiponectin-induced OSM expression in osteoblasts. ( A ) Osteoblasts were pretreated with Akt inhibitors (Akti) (20 μM) or transfected with Akt siRNA (0.5 nM) for 24 h followed by stimulation with adiponectin (100 ng/mL), OSM expression was measured by qPCR ( n = 6); ( B ) Cells were transfected with Akt siRNA (0.5 nM) for 24h, the protein level of Akt was measured by Western blot ( upper-panel ), and supernatant medium was collected to measure OSM expression by ELISA assay ( lower-panel ) ( n = 5); ( C ) Cells were pretreated with Akti (20 μM) for 30 min followed by stimulation with adiponectin (100 ng/mL), the protein level of OSM was measured by Western blot ( n = 5); ( D ) Osteoblasts were incubated with adiponectin (100 ng/mL) in time intervals, and phosphate-PI3K expression was investigated by Western blot ( n = 4); ( E ) Cells were pretreated with PI3K inhibitor, LY294002 (10 μM), for 30 min followed by stimulation with adiponectin (100 ng/mL), phosphate-Akt expression was investigated by Western blot ( n = 5). Results are expressed as mean ± S.E.M. *, p

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation

    Signaling pathways of phosphatidylinositol 3-kinase (PI3K) involved in potentiating action of adiponectin. ( A ) Osteoblasts were pretreated with PI3K inhibitors, LY294002 (10 μM) or Wortmannin (5 μM), or transfected with p85 short interference RNA (siRNA) (0.5 nM) for 24 h followed by stimulation with adiponectin (100 ng/mL), OSM expression was measured by qPCR ( n = 6); ( B ) Cells were transfected with p85 siRNA (0.5 nM) for 24 h, the protein level of PI3K was measured by Western blot ( upper-panel ), and supernatant medium was collected to measure OSM expression by ELISAassay ( lower-panel ) ( n = 4); ( C ) Cells were pretreated with PI3K inhibitors, LY294002 (10 μM) or Wortmannin (5 μM), for 30 min followed by stimulation with adiponectin (100 ng/mL), the protein level of OSM was measured by Western blot ( n = 5); ( D ) Osteoblasts were incubated with adiponectin (100 ng/mL) in time intervals, and phosphate-PI3K expression was investigated by Western blot ( n = 6). Results are expressed as mean ± S.E.M. *, p
    Figure Legend Snippet: Signaling pathways of phosphatidylinositol 3-kinase (PI3K) involved in potentiating action of adiponectin. ( A ) Osteoblasts were pretreated with PI3K inhibitors, LY294002 (10 μM) or Wortmannin (5 μM), or transfected with p85 short interference RNA (siRNA) (0.5 nM) for 24 h followed by stimulation with adiponectin (100 ng/mL), OSM expression was measured by qPCR ( n = 6); ( B ) Cells were transfected with p85 siRNA (0.5 nM) for 24 h, the protein level of PI3K was measured by Western blot ( upper-panel ), and supernatant medium was collected to measure OSM expression by ELISAassay ( lower-panel ) ( n = 4); ( C ) Cells were pretreated with PI3K inhibitors, LY294002 (10 μM) or Wortmannin (5 μM), for 30 min followed by stimulation with adiponectin (100 ng/mL), the protein level of OSM was measured by Western blot ( n = 5); ( D ) Osteoblasts were incubated with adiponectin (100 ng/mL) in time intervals, and phosphate-PI3K expression was investigated by Western blot ( n = 6). Results are expressed as mean ± S.E.M. *, p

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

    Adiponectin increases oncostatin M (OSM) production in human osteoblasts. ( A ) Osteoblastic cells were incubated with various concentrations of adiponectin (3–200 ng/mL) in OSM mRNA expression and were measured by quantitative polymerase chain reaction (qPCR) ( n = 6); ( B ) Cells were incubated with various concentrations of adiponectin (3–200 ng/mL) and OSM protein levels were measured by Western blot ( n = 4); ( C ) Osteoblasts were stimulated with adiponectin (3–200 ng/mL) for 24 h and the supernatant medium was collected and analyzed by enzyme-linked immunosorbent assay (ELISA) ( n = 5). Results are expressed as mean ± standard error of mean S.E.M. *, p
    Figure Legend Snippet: Adiponectin increases oncostatin M (OSM) production in human osteoblasts. ( A ) Osteoblastic cells were incubated with various concentrations of adiponectin (3–200 ng/mL) in OSM mRNA expression and were measured by quantitative polymerase chain reaction (qPCR) ( n = 6); ( B ) Cells were incubated with various concentrations of adiponectin (3–200 ng/mL) and OSM protein levels were measured by Western blot ( n = 4); ( C ) Osteoblasts were stimulated with adiponectin (3–200 ng/mL) for 24 h and the supernatant medium was collected and analyzed by enzyme-linked immunosorbent assay (ELISA) ( n = 5). Results are expressed as mean ± standard error of mean S.E.M. *, p

    Techniques Used: Incubation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

    4) Product Images from "Systemic Delivery of Anti-miRNA for Suppression of Triple Negative Breast Cancer Utilizing RNA Nanotechnology"

    Article Title: Systemic Delivery of Anti-miRNA for Suppression of Triple Negative Breast Cancer Utilizing RNA Nanotechnology

    Journal: ACS Nano

    doi: 10.1021/acsnano.5b02471

    Evaluation of targeting and therapeutic effects of 3WJ-EGFRapt/anti-miR-21 nanoparticles in vitro . (A) Confocal images showing efficient binding and internalization into MDA-MB-231 cells. Green: cytoplasm; blue: nuclei; and red: RNA nanoparticles. (B) Flow cytometry assay showing the binding to MDA-MB-231 cells. (C) Dual-luciferase assay demonstrating in vitro delivery of anti-miR-21 LNA into MDA-MB-231 cells. (D) qRT-PCR assay depicting the effect of miR-21 knockdown on target gene expression level of PTEN and PDCD4 after treatment. RQ: relative quantification. (E) Caspase-3 assay showing the cellular apoptotic effects of MDA-MB-231 cells after treatment.
    Figure Legend Snippet: Evaluation of targeting and therapeutic effects of 3WJ-EGFRapt/anti-miR-21 nanoparticles in vitro . (A) Confocal images showing efficient binding and internalization into MDA-MB-231 cells. Green: cytoplasm; blue: nuclei; and red: RNA nanoparticles. (B) Flow cytometry assay showing the binding to MDA-MB-231 cells. (C) Dual-luciferase assay demonstrating in vitro delivery of anti-miR-21 LNA into MDA-MB-231 cells. (D) qRT-PCR assay depicting the effect of miR-21 knockdown on target gene expression level of PTEN and PDCD4 after treatment. RQ: relative quantification. (E) Caspase-3 assay showing the cellular apoptotic effects of MDA-MB-231 cells after treatment.

    Techniques Used: In Vitro, Binding Assay, Multiple Displacement Amplification, Flow Cytometry, Cytometry, Luciferase, Quantitative RT-PCR, Expressing, Caspase-3 Assay

    5) Product Images from "MicroRNA-125a influences breast cancer stem cells by targeting leukemia inhibitory factor receptor which regulates the hippo signaling pathway"

    Article Title: MicroRNA-125a influences breast cancer stem cells by targeting leukemia inhibitory factor receptor which regulates the hippo signaling pathway

    Journal: Oncotarget

    doi:

    miR-125a modulation affects malignant and non-malignant breast epithelial stem cells A. qRT-PCR data shows effective inhibition of miR-125a in MCF7 cells using 100nM of antagomirs. B. qRT-PCR data shows effective over expression of miR-125a in MCF12A cells with miR-125a mimic. C. Immunoblotting analysis for LIFR post miR-125a inhibition in MCF7 CSCs. D. Immunoblotting analysis for LIFR in miR-125a over expressing MCF12A stem cells. E. Flow cytometric analysis shows increased percentage of stem cells with miR-125a over expression in MCF12A cells. F. Sphere forming assay demonstrates higher percentage of 3D sphere forming cells in miR-125a over expressing MCF12A. G. Flow cytometric analysis shows decreased percentage of stem cells with miR-125a inhibition in MCF7 cells. H. Sphere forming assay demonstrates lower percentage of 3D sphere forming cells in miR-125a inhibited MCF7 cells. I. Immunostaining of MCF12A spheres for SOX2 expression with over expression of miR-125a compared to mock controls.
    Figure Legend Snippet: miR-125a modulation affects malignant and non-malignant breast epithelial stem cells A. qRT-PCR data shows effective inhibition of miR-125a in MCF7 cells using 100nM of antagomirs. B. qRT-PCR data shows effective over expression of miR-125a in MCF12A cells with miR-125a mimic. C. Immunoblotting analysis for LIFR post miR-125a inhibition in MCF7 CSCs. D. Immunoblotting analysis for LIFR in miR-125a over expressing MCF12A stem cells. E. Flow cytometric analysis shows increased percentage of stem cells with miR-125a over expression in MCF12A cells. F. Sphere forming assay demonstrates higher percentage of 3D sphere forming cells in miR-125a over expressing MCF12A. G. Flow cytometric analysis shows decreased percentage of stem cells with miR-125a inhibition in MCF7 cells. H. Sphere forming assay demonstrates lower percentage of 3D sphere forming cells in miR-125a inhibited MCF7 cells. I. Immunostaining of MCF12A spheres for SOX2 expression with over expression of miR-125a compared to mock controls.

    Techniques Used: Quantitative RT-PCR, Inhibition, Over Expression, Expressing, Flow Cytometry, Immunostaining

    Inverse correlation between miR-125a and LIFR in stem cells A. qRT-PCR data shows expression of miR-125a in MCF7 and human primary breast cancer cells (BC-052) compared to MCF12A cells B. Expression levels of miR-125a in MCF7 and primary breast CSCs as compared to MCF12A stem cells. C. Pictorial representation of the relative levels of miR-125a in malignant and non-malignant stem cells and bulk cells D. Transcript level of LIFR in MCF7 and human primary breast cancer stem cells as compared to MCF12A stem cells. E. Immunoblotting analysis of LIFR in MCF7 and human primary breast cancer stem cells as compared to MCF12A stem cells. F. Transcript level of LIFR in MCF7 and human primary breast cancer cells as compared to MCF12A cells G. Immunoblotting analysis of LIFR in MCF7 and primary breast cancer cells as compared to MCF12A cells. H. Predicted interaction of miR-125a with LIFR in silico . Luciferase assay data for HEK293 cells demonstrating a decrease in the luciferase activity with addition of miR-125a mimics to wtLIFR 3′UTR-1 expressing cells but not in wtLIFR 3′UTR-2 and mutLIFR 3′UTR.
    Figure Legend Snippet: Inverse correlation between miR-125a and LIFR in stem cells A. qRT-PCR data shows expression of miR-125a in MCF7 and human primary breast cancer cells (BC-052) compared to MCF12A cells B. Expression levels of miR-125a in MCF7 and primary breast CSCs as compared to MCF12A stem cells. C. Pictorial representation of the relative levels of miR-125a in malignant and non-malignant stem cells and bulk cells D. Transcript level of LIFR in MCF7 and human primary breast cancer stem cells as compared to MCF12A stem cells. E. Immunoblotting analysis of LIFR in MCF7 and human primary breast cancer stem cells as compared to MCF12A stem cells. F. Transcript level of LIFR in MCF7 and human primary breast cancer cells as compared to MCF12A cells G. Immunoblotting analysis of LIFR in MCF7 and primary breast cancer cells as compared to MCF12A cells. H. Predicted interaction of miR-125a with LIFR in silico . Luciferase assay data for HEK293 cells demonstrating a decrease in the luciferase activity with addition of miR-125a mimics to wtLIFR 3′UTR-1 expressing cells but not in wtLIFR 3′UTR-2 and mutLIFR 3′UTR.

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

    6) Product Images from "Collagen I promotes hepatocellular carcinoma cell proliferation by regulating integrin β1/FAK signaling pathway in nonalcoholic fatty liver"

    Article Title: Collagen I promotes hepatocellular carcinoma cell proliferation by regulating integrin β1/FAK signaling pathway in nonalcoholic fatty liver

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21525

    Collagen I contributes to the NAFLD-related HCC ( A ) qRT-PCR analysis showed that Collagen I levels were 2.8 fold higher in HCC samples than those in adjacent non-tumor liver tissues. ( B ) Immunofluorescence staining confirmed the higher expression of Collagen I (green) in HCC tissues compared to normal liver tissues. ( C ) Immunofluorescence staining revealed a higher expression of Collagen I (green) in human fatty liver compared to human normal liver. ( D ) In the mouse models of NAFLD/NASH, Collagen I was found to be upregulated. ( E , F ) HCC cells cultured on different ECM or different concentration of Collagen I to determine the effect of Collagen I on HCC cell proliferation. The vitality of cells at each time point was detected by CCK-8 assay. (E) SMMC-7721 and HepG2 cells cultured on Collagen I proliferated significantly faster than those on either Collagen IV or fibronectin. (F) The effect of Collagen I on cell proliferation appeared to be dose-dependent. Scale bar: 50 μm (B); 100 μm (C, D), * P
    Figure Legend Snippet: Collagen I contributes to the NAFLD-related HCC ( A ) qRT-PCR analysis showed that Collagen I levels were 2.8 fold higher in HCC samples than those in adjacent non-tumor liver tissues. ( B ) Immunofluorescence staining confirmed the higher expression of Collagen I (green) in HCC tissues compared to normal liver tissues. ( C ) Immunofluorescence staining revealed a higher expression of Collagen I (green) in human fatty liver compared to human normal liver. ( D ) In the mouse models of NAFLD/NASH, Collagen I was found to be upregulated. ( E , F ) HCC cells cultured on different ECM or different concentration of Collagen I to determine the effect of Collagen I on HCC cell proliferation. The vitality of cells at each time point was detected by CCK-8 assay. (E) SMMC-7721 and HepG2 cells cultured on Collagen I proliferated significantly faster than those on either Collagen IV or fibronectin. (F) The effect of Collagen I on cell proliferation appeared to be dose-dependent. Scale bar: 50 μm (B); 100 μm (C, D), * P

    Techniques Used: Quantitative RT-PCR, Immunofluorescence, Staining, Expressing, Cell Culture, Concentration Assay, CCK-8 Assay

    The expression of integrin β1 in NAFLD-related HCC ( A – C ) The expression of integrin β1 in HCC. (A) qRT-PCR analysis showed that integrin β1 levels were 1.1 fold higher in HCC samples than those in adjacent non-tumor liver tissues. (B) Correlation analysis indicated that the expression level of integrin β1 were positively correlated with the level of Collagen I, r = 0.43, P = 0.004. (C) Immunohistochemical staining revealed a high expression of integrin β1 in HCC tissues. ( D ) HepG2 cells were seeded into the NLM and FLM and cultured for 15 days. Western blot indicated that cells cultured in FLM had a higher expression of integrin β1 than those in NLM. ( E , F ) HCC cells cultured on different ECM or different concentration of Collagen I. (E) For SMMC-7721 and HepG2, integrin β1 was higher expressed in Collagen I compared to Collagen IV and fibronectin. (F) Increasing Collagen I concentration could up regulated the expression of integrin β1 in both cell lines. ( G , H ) The expression of integrin β1 in mouse orthotopic tumor model. (G) Western blot indicated that tumors grown in HF- or MCD-fed mice expressed higher level of integrin β1. (H) Immunohistochemical staining of integrin β1 confirmed the same results. ( I – J ) Integrin β1 was knocked down in HCC cells by shRNA technology through lentiviral transduction. (I) Western blot confirmed the stable knock-down of integrin β1 in both cell lines. (J) HepG2 and SMMC-7721 cells cultured on Collagen I grew significantly slowly after knocking down integrin β1. Scale bar: 100 μm (C), 50 μm (H), * P
    Figure Legend Snippet: The expression of integrin β1 in NAFLD-related HCC ( A – C ) The expression of integrin β1 in HCC. (A) qRT-PCR analysis showed that integrin β1 levels were 1.1 fold higher in HCC samples than those in adjacent non-tumor liver tissues. (B) Correlation analysis indicated that the expression level of integrin β1 were positively correlated with the level of Collagen I, r = 0.43, P = 0.004. (C) Immunohistochemical staining revealed a high expression of integrin β1 in HCC tissues. ( D ) HepG2 cells were seeded into the NLM and FLM and cultured for 15 days. Western blot indicated that cells cultured in FLM had a higher expression of integrin β1 than those in NLM. ( E , F ) HCC cells cultured on different ECM or different concentration of Collagen I. (E) For SMMC-7721 and HepG2, integrin β1 was higher expressed in Collagen I compared to Collagen IV and fibronectin. (F) Increasing Collagen I concentration could up regulated the expression of integrin β1 in both cell lines. ( G , H ) The expression of integrin β1 in mouse orthotopic tumor model. (G) Western blot indicated that tumors grown in HF- or MCD-fed mice expressed higher level of integrin β1. (H) Immunohistochemical staining of integrin β1 confirmed the same results. ( I – J ) Integrin β1 was knocked down in HCC cells by shRNA technology through lentiviral transduction. (I) Western blot confirmed the stable knock-down of integrin β1 in both cell lines. (J) HepG2 and SMMC-7721 cells cultured on Collagen I grew significantly slowly after knocking down integrin β1. Scale bar: 100 μm (C), 50 μm (H), * P

    Techniques Used: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Cell Culture, Western Blot, Concentration Assay, Mouse Assay, shRNA, Transduction

    7) Product Images from "Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males"

    Article Title: Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males

    Journal: Cell Stem Cell

    doi: 10.1016/j.stem.2018.05.001

    Xist Expression Is Abolished by a Robust Naive Pluripotent Network (A) Schematic illustrating the experiment performed to evaluate the impact of the nPSC culture conditions on the expression of Xist . (B) qRT-PCR analysis of Nanog and Xist in XX1, XX2, XY1, and XY2 ESC lines in SL versus 2iL. P indicates number of passages in 2iL. Error bars represent ± SD. (C) Flow cytometry analysis of male SL Nanog -GFP ESCs and subsequent sorting into three Nanog -GFP populations: low, medium, and high. (D) qRT-PCR analysis of Nanog , Klf4 , Oct4 , and Xist in low, medium, and high Nanog -GFP ESCs. Error bars represent ± SD. (E) Strand-specific RNA-seq showing expression of the positive and negative strands at the Xist locus in male 2iL ESCs. The double-strand Xist probe used in (F) is represented in red. (F) RNA FISH in male and female 2iL ESCs with a double-strand (ds) probe (left) or with a single-strand (ss) probe detecting only Xist (right). The percentage of cells with probe signal is indicated. Female EpiSCs were used as a control for the ss probe. The scale bar represents 5 μm. (G) qRT-PCR analysis of Nanog and Xist in female and male Nanog flox/− , Rosa26-CreERT2 ESCs in 2iL at indicated time points following treatment with 4-OHT. Error bars represent ± SD. (H) qRT-PCR analysis of Xist in XX3 and XY1 ESCs in 2iL, 2i or after 3 and 5 days in 1 μM JAKi + 2i. Error bars represent ± SD. (I) qRT-PCR analysis of Oct4 and Xist in female and male Oct4 flox/− , Rosa26-CreERT2 ESCs in 2iL at indicated time points following treatment with 4-OHT. Female somatic cells were used as control for Xist expression. Error bars represent ± SD.
    Figure Legend Snippet: Xist Expression Is Abolished by a Robust Naive Pluripotent Network (A) Schematic illustrating the experiment performed to evaluate the impact of the nPSC culture conditions on the expression of Xist . (B) qRT-PCR analysis of Nanog and Xist in XX1, XX2, XY1, and XY2 ESC lines in SL versus 2iL. P indicates number of passages in 2iL. Error bars represent ± SD. (C) Flow cytometry analysis of male SL Nanog -GFP ESCs and subsequent sorting into three Nanog -GFP populations: low, medium, and high. (D) qRT-PCR analysis of Nanog , Klf4 , Oct4 , and Xist in low, medium, and high Nanog -GFP ESCs. Error bars represent ± SD. (E) Strand-specific RNA-seq showing expression of the positive and negative strands at the Xist locus in male 2iL ESCs. The double-strand Xist probe used in (F) is represented in red. (F) RNA FISH in male and female 2iL ESCs with a double-strand (ds) probe (left) or with a single-strand (ss) probe detecting only Xist (right). The percentage of cells with probe signal is indicated. Female EpiSCs were used as a control for the ss probe. The scale bar represents 5 μm. (G) qRT-PCR analysis of Nanog and Xist in female and male Nanog flox/− , Rosa26-CreERT2 ESCs in 2iL at indicated time points following treatment with 4-OHT. Error bars represent ± SD. (H) qRT-PCR analysis of Xist in XX3 and XY1 ESCs in 2iL, 2i or after 3 and 5 days in 1 μM JAKi + 2i. Error bars represent ± SD. (I) qRT-PCR analysis of Oct4 and Xist in female and male Oct4 flox/− , Rosa26-CreERT2 ESCs in 2iL at indicated time points following treatment with 4-OHT. Female somatic cells were used as control for Xist expression. Error bars represent ± SD.

    Techniques Used: Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, RNA Sequencing Assay, Fluorescence In Situ Hybridization

    Xist Is Transiently and Rapidly Upregulated in Male nPSC Differentiation and Male EpiSC Reprogramming (A) Schematic illustrating three conditions employed to differentiate 2iL and SL nPSCs: suspension culture in serum to generate EBs or adherent monolayer culture in serum-free media ± Fgf2+ActivinA (FA). (B) qRT-PCR analysis of Xist during differentiation of male ESCs in three different conditions. Before differentiation, ESCs were maintained in 2iL or SL conditions, as indicated. Error bars represent ± SD. (C) Strand-specific RNA-seq (negative strand only) showing expression of Xist during differentiation of male 2iL ESCs in three different conditions. Scale represents reads per million (RPM). (D) Heatmap showing expression profile of Xist , differentiation markers, and naive markers during differentiation of male 2iL ESCs, as indicated. Scale represents Z scores of log2-transformed expression values. (E) qRT-PCR analysis of Xist during EB differentiation of male versus female 2iL ESCs. Error bars represent ± SD. (F) Flow cytometry analysis of male GY118F Rex1 +/dGFP EpiSCs following reprogramming induction with GCSF in 2iL. Cells were sorted at different time points, with Rex1 -dGFP reporter activation indicating the subset of cells successfully transitioning to the naive identity. A representative plot from day 3 is shown. (G) qRT-PCR analysis of Xist and naive markers ( Oct4 , Tfcp2l1 , and Esrrb ) in male Rex1 -positive reprogramming intermediates at different time points after induction of reprogramming with 2iL+GCSF/GY118F. Parental EpiSCs (day 0) and ESCs in 2iL were used as controls. Error bars represent ± SD.
    Figure Legend Snippet: Xist Is Transiently and Rapidly Upregulated in Male nPSC Differentiation and Male EpiSC Reprogramming (A) Schematic illustrating three conditions employed to differentiate 2iL and SL nPSCs: suspension culture in serum to generate EBs or adherent monolayer culture in serum-free media ± Fgf2+ActivinA (FA). (B) qRT-PCR analysis of Xist during differentiation of male ESCs in three different conditions. Before differentiation, ESCs were maintained in 2iL or SL conditions, as indicated. Error bars represent ± SD. (C) Strand-specific RNA-seq (negative strand only) showing expression of Xist during differentiation of male 2iL ESCs in three different conditions. Scale represents reads per million (RPM). (D) Heatmap showing expression profile of Xist , differentiation markers, and naive markers during differentiation of male 2iL ESCs, as indicated. Scale represents Z scores of log2-transformed expression values. (E) qRT-PCR analysis of Xist during EB differentiation of male versus female 2iL ESCs. Error bars represent ± SD. (F) Flow cytometry analysis of male GY118F Rex1 +/dGFP EpiSCs following reprogramming induction with GCSF in 2iL. Cells were sorted at different time points, with Rex1 -dGFP reporter activation indicating the subset of cells successfully transitioning to the naive identity. A representative plot from day 3 is shown. (G) qRT-PCR analysis of Xist and naive markers ( Oct4 , Tfcp2l1 , and Esrrb ) in male Rex1 -positive reprogramming intermediates at different time points after induction of reprogramming with 2iL+GCSF/GY118F. Parental EpiSCs (day 0) and ESCs in 2iL were used as controls. Error bars represent ± SD.

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

    8) Product Images from "Convergent analysis of genome-wide genotyping and transcriptomic data suggests association of zinc finger genes with lithium response in bipolar disorder"

    Article Title: Convergent analysis of genome-wide genotyping and transcriptomic data suggests association of zinc finger genes with lithium response in bipolar disorder

    Journal: American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics

    doi: 10.1002/ajmg.b.32663

    Regional association plot Regional association plot of the region on chromosome 19 in which the two genes ( ZNF493 , ZNF429 ) associated with lithium response and significantly affected by lithium treatment are located. The index SNP is the top-SNP (rs12975981) of ZNF493 and is shown as a purple diamond. Association p-values of SNPs are plotted as points. SNPs are colored based on linkage disequilibrium with the index SNP. cM, centimorgan; Mb, megabase; SNP, single nucleotide polymorphism.
    Figure Legend Snippet: Regional association plot Regional association plot of the region on chromosome 19 in which the two genes ( ZNF493 , ZNF429 ) associated with lithium response and significantly affected by lithium treatment are located. The index SNP is the top-SNP (rs12975981) of ZNF493 and is shown as a purple diamond. Association p-values of SNPs are plotted as points. SNPs are colored based on linkage disequilibrium with the index SNP. cM, centimorgan; Mb, megabase; SNP, single nucleotide polymorphism.

    Techniques Used:

    9) Product Images from "Paracoccidioides brasiliensis Enolase Is a Surface Protein That Binds Plasminogen and Mediates Interaction of Yeast Forms with Host Cells ▿"

    Article Title: Paracoccidioides brasiliensis Enolase Is a Surface Protein That Binds Plasminogen and Mediates Interaction of Yeast Forms with Host Cells ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00221-10

    Analysis of enolase transcripts by quantitative real time RT-PCR. qRT-PCR plot of Pb Eno expression levels of transcripts from yeast cells of P. brasiliensis derived from lungs, livers, and spleens of mice after 7 days of infection. Control systems consisted
    Figure Legend Snippet: Analysis of enolase transcripts by quantitative real time RT-PCR. qRT-PCR plot of Pb Eno expression levels of transcripts from yeast cells of P. brasiliensis derived from lungs, livers, and spleens of mice after 7 days of infection. Control systems consisted

    Techniques Used: Quantitative RT-PCR, Expressing, Derivative Assay, Mouse Assay, Infection

    10) Product Images from "Metabolic network rewiring of propionate flux compensates vitamin B12 deficiency in C. elegans"

    Article Title: Metabolic network rewiring of propionate flux compensates vitamin B12 deficiency in C. elegans

    Journal: eLife

    doi: 10.7554/eLife.17670

    Comparison between putative human and C. elegans propionate shunts. ( A ) Comparison between C. elegans propionate shunt genes (red) and candidate human shunt genes (green, gray). Green text indicates higher confidence annotations based on patient mutations and metabolomics, or in the case of ADHFE1 one-to-one orthology of unique enzymes in both genomes. 3-HP is marked in magenta to indicate that it is a biomarker for impaired flux in the canonical, vitamin B12-dependent propionate breakdown pathway, such as occurs in patients with propionic or methylmalonic acidemia. ( B ) C. elegans propionate shunt genes and orthologs in mouse and humans are strongly co-expressed as a group compared to 10,000 random permutations of five genes from either the whole genome, a subset of only metabolic genes, or a subset of related metabolic genes from connected pathways, including BCAA breakdown and the TCA cycle. The expression data used for this analysis was compiled and weighted using the SEEK and modSEEK databases. Distributions of co-expression scores are shown for each set of randomizations, and vertical dashed lines indicate actual weighted co-expression score for propionate shunt genes and orthologs in human, mouse and C. elegans . ( C ) The expression of several candidate human genes is activated in response to propionate in HepG2 liver carcinoma cells. qRT-PCR experiment showing the average of four replicate experiments, each containing three technical replicates. DOI: http://dx.doi.org/10.7554/eLife.17670.010
    Figure Legend Snippet: Comparison between putative human and C. elegans propionate shunts. ( A ) Comparison between C. elegans propionate shunt genes (red) and candidate human shunt genes (green, gray). Green text indicates higher confidence annotations based on patient mutations and metabolomics, or in the case of ADHFE1 one-to-one orthology of unique enzymes in both genomes. 3-HP is marked in magenta to indicate that it is a biomarker for impaired flux in the canonical, vitamin B12-dependent propionate breakdown pathway, such as occurs in patients with propionic or methylmalonic acidemia. ( B ) C. elegans propionate shunt genes and orthologs in mouse and humans are strongly co-expressed as a group compared to 10,000 random permutations of five genes from either the whole genome, a subset of only metabolic genes, or a subset of related metabolic genes from connected pathways, including BCAA breakdown and the TCA cycle. The expression data used for this analysis was compiled and weighted using the SEEK and modSEEK databases. Distributions of co-expression scores are shown for each set of randomizations, and vertical dashed lines indicate actual weighted co-expression score for propionate shunt genes and orthologs in human, mouse and C. elegans . ( C ) The expression of several candidate human genes is activated in response to propionate in HepG2 liver carcinoma cells. qRT-PCR experiment showing the average of four replicate experiments, each containing three technical replicates. DOI: http://dx.doi.org/10.7554/eLife.17670.010

    Techniques Used: Biomarker Assay, Expressing, Quantitative RT-PCR

    11) Product Images from "SMG7 is a critical regulator of p53 stability and function in DNA damage stress response"

    Article Title: SMG7 is a critical regulator of p53 stability and function in DNA damage stress response

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2015.42

    SMG7 regulates p21 activation and cell cycle arrest upon DNA damage. ( a ) Total cell extracts from control or irradiated cells (at indicated time points) were analyzed with western blot analysis using antibodies against SMG7, p53, Mdm2, p21 and actin. ( b ) Control or irradiated cells (16 h) were treated with 5-ethynyl-2′-deoxyuridine (EDU) (10 μ M ) for 1 h and then processed for EDU and 7-AAD staining, followed by flow cytometry analysis. ( c ) Total cell extracts from control or Doxorubicin-treated cell (200 ng ml −1 for various time points) were analyzed by western blot as in a . ( d ) Control or Doxorubicin-treated (200 ng ml −1 , 16 h) cells were fed with EDU (10 μ M ) for 1 h and then processed for EDU (green) and DAPI (blue) staining, followed by fluorescence microscopy. ( e ) Quantitation of EDU-positive cells from d . Error bars indicate s.d. calculated from three independent experiments.
    Figure Legend Snippet: SMG7 regulates p21 activation and cell cycle arrest upon DNA damage. ( a ) Total cell extracts from control or irradiated cells (at indicated time points) were analyzed with western blot analysis using antibodies against SMG7, p53, Mdm2, p21 and actin. ( b ) Control or irradiated cells (16 h) were treated with 5-ethynyl-2′-deoxyuridine (EDU) (10 μ M ) for 1 h and then processed for EDU and 7-AAD staining, followed by flow cytometry analysis. ( c ) Total cell extracts from control or Doxorubicin-treated cell (200 ng ml −1 for various time points) were analyzed by western blot as in a . ( d ) Control or Doxorubicin-treated (200 ng ml −1 , 16 h) cells were fed with EDU (10 μ M ) for 1 h and then processed for EDU (green) and DAPI (blue) staining, followed by fluorescence microscopy. ( e ) Quantitation of EDU-positive cells from d . Error bars indicate s.d. calculated from three independent experiments.

    Techniques Used: Activation Assay, Irradiation, Western Blot, Staining, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Quantitation Assay

    In vitro and in vivo interactions between SMG7 and p53. ( a ) Scheme of human SMG7 protein containing the N-terminal 14-3-3-like Tetratricopeptide repeat (TPR) and C-terminal low complex region. ( b ) Immunofluorescence microscopy of H1299 cells transiently expressing FH-SMG7 stained with α-HA antibody (green) and 4,6-diamidino-2-phenylindole (DAPI; blue). ( c ) Cell extracts from H1299 cells transiently expressing p53 with or without FH-SMG7 and the α-Flag immunoprecipitates were analyzed using western blot using α-SMG7 and α-p53 antibodies. ( d , e ) SMG7 binding to p53 in vitro . GST or GST-p53 fusion proteins (full length or various fragments) were used in pulldown assays with purified FH-SMG7 ( d ) or in vitro translated FH-SMG7 ( e ). FH-SMG7 was detected by western blot and GST proteins stained with Ponceau S. ( f , g ) Endogenous interaction of SMG7 with p53 in HCT116 ( f ) and U2OS cells ( g ). Cell extracts and the α-normal mouse IgG or α-p53 immunoprecipitates were analyzed with western blot analysis using α-SMG7 and α-p53 antibodies. ( h ) Cell extracts from control or irradiated cells (at various time points) and the α-Flag immunoprecipitates were analyzed with western blot analysis using antibodies as indicated.
    Figure Legend Snippet: In vitro and in vivo interactions between SMG7 and p53. ( a ) Scheme of human SMG7 protein containing the N-terminal 14-3-3-like Tetratricopeptide repeat (TPR) and C-terminal low complex region. ( b ) Immunofluorescence microscopy of H1299 cells transiently expressing FH-SMG7 stained with α-HA antibody (green) and 4,6-diamidino-2-phenylindole (DAPI; blue). ( c ) Cell extracts from H1299 cells transiently expressing p53 with or without FH-SMG7 and the α-Flag immunoprecipitates were analyzed using western blot using α-SMG7 and α-p53 antibodies. ( d , e ) SMG7 binding to p53 in vitro . GST or GST-p53 fusion proteins (full length or various fragments) were used in pulldown assays with purified FH-SMG7 ( d ) or in vitro translated FH-SMG7 ( e ). FH-SMG7 was detected by western blot and GST proteins stained with Ponceau S. ( f , g ) Endogenous interaction of SMG7 with p53 in HCT116 ( f ) and U2OS cells ( g ). Cell extracts and the α-normal mouse IgG or α-p53 immunoprecipitates were analyzed with western blot analysis using α-SMG7 and α-p53 antibodies. ( h ) Cell extracts from control or irradiated cells (at various time points) and the α-Flag immunoprecipitates were analyzed with western blot analysis using antibodies as indicated.

    Techniques Used: In Vitro, In Vivo, Immunofluorescence, Microscopy, Expressing, Staining, Western Blot, Binding Assay, Purification, Irradiation

    Knockout of SMG7 abrogates DNA damage-induced p53 stabilization. ( a ) Scheme of AAV-mediated knockout of SMG7 : shown from top to bottom are AAV- SMG7 targeting vector, wild-type SMG7 allele, knockout SMG7 allele containing the IRES-neo cassette and knockout SMG7 allele after removal of the IRES-neo cassette. See details of generation of HCT116 SMG7 −/− cells in Materials and Methods. ( b ) Western blot analysis of SMG7 in HCT116 and two SMG7 −/− clones. Asterisk indicates a nonspecific band. ( c ) Cell extracts from control, irradiated (10 Gy, 4 h) and Doxorubicin-treated cells (200 ng ml −1 , 4 h) were analyzed using western blot analysis with α-SMG7, α-p53 and α-actin antibodies. ( d ) Cell extracts from control and MG132-treated (20 μM, 4 h) cells were analyzed using western blot analysis as in c . ( e ) Control or irradiated cells were harvested at various time points after Cycloheximide (CHX, 50 μg ml −1 ) treatment and cell extracts were analyzed using western blot analysis with α-SMG7, α-p53, α-Mdm2 and α-actin antibodies.
    Figure Legend Snippet: Knockout of SMG7 abrogates DNA damage-induced p53 stabilization. ( a ) Scheme of AAV-mediated knockout of SMG7 : shown from top to bottom are AAV- SMG7 targeting vector, wild-type SMG7 allele, knockout SMG7 allele containing the IRES-neo cassette and knockout SMG7 allele after removal of the IRES-neo cassette. See details of generation of HCT116 SMG7 −/− cells in Materials and Methods. ( b ) Western blot analysis of SMG7 in HCT116 and two SMG7 −/− clones. Asterisk indicates a nonspecific band. ( c ) Cell extracts from control, irradiated (10 Gy, 4 h) and Doxorubicin-treated cells (200 ng ml −1 , 4 h) were analyzed using western blot analysis with α-SMG7, α-p53 and α-actin antibodies. ( d ) Cell extracts from control and MG132-treated (20 μM, 4 h) cells were analyzed using western blot analysis as in c . ( e ) Control or irradiated cells were harvested at various time points after Cycloheximide (CHX, 50 μg ml −1 ) treatment and cell extracts were analyzed using western blot analysis with α-SMG7, α-p53, α-Mdm2 and α-actin antibodies.

    Techniques Used: Knock-Out, Plasmid Preparation, Western Blot, Clone Assay, Irradiation

    Identification of SMG7 as a p53-associated protein. ( a ) Scheme of AAV-mediated epitagging of p53 : (1) AAV- p53 targeting vector; (2) wild-type p53 allele; (3) modified p53 allele containing the Flag-HA cassette; (4) FH-tagged p53 polypeptide. See details of generation of HCT116 p53 +/FH cells in Materials and Methods. ( b ) Cell extracts from control or irradiated cells (10 Gy, 4 h) and the corresponding α-Flag immunoprecipitates were analyzed with western blot analysis using antibodies as indicated. ( c ) Silver staining of affinity-purified p53-containing protein complex from irradiated cells (upper panel). Cell extracts and the Flag/HA immunoprecipitates were analyzed with western blot analysis using antibodies as indicated. ( d ) Several representative SMG7 peptide sequences identified using mass spectrometric analysis. Numbers indicate the overlapping position of the peptides with SMG7 sequence (NP_963862).
    Figure Legend Snippet: Identification of SMG7 as a p53-associated protein. ( a ) Scheme of AAV-mediated epitagging of p53 : (1) AAV- p53 targeting vector; (2) wild-type p53 allele; (3) modified p53 allele containing the Flag-HA cassette; (4) FH-tagged p53 polypeptide. See details of generation of HCT116 p53 +/FH cells in Materials and Methods. ( b ) Cell extracts from control or irradiated cells (10 Gy, 4 h) and the corresponding α-Flag immunoprecipitates were analyzed with western blot analysis using antibodies as indicated. ( c ) Silver staining of affinity-purified p53-containing protein complex from irradiated cells (upper panel). Cell extracts and the Flag/HA immunoprecipitates were analyzed with western blot analysis using antibodies as indicated. ( d ) Several representative SMG7 peptide sequences identified using mass spectrometric analysis. Numbers indicate the overlapping position of the peptides with SMG7 sequence (NP_963862).

    Techniques Used: Plasmid Preparation, Modification, Irradiation, Western Blot, Silver Staining, Affinity Purification, Sequencing

    12) Product Images from "Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability"

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv933

    Copy-numbers of amplified gene existing in HSRs are decreased by cohesin reduction. ( A ) COLO 320-HSR cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P
    Figure Legend Snippet: Copy-numbers of amplified gene existing in HSRs are decreased by cohesin reduction. ( A ) COLO 320-HSR cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P

    Techniques Used: Amplification, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, CNV Assay

    Copy-numbers of amplified APIP/PDHX/CD44 locus are decreased by cohesin reduction in SNU16 cells. ( A ) High-resolution array-CGH analysis was performed with the control GFP-KD or RAD21-KD SNU16 cells on day 60 after RAD21-KD. Chromosome 11 is represented by ideograms showing G-banding patterns (left ideogram, RAD21-KD SNU16 cells compared with the control GFP-KD cells; right ideogram, parental SNU16 cells compared with normal gastric cells). Gains/amplifications (red) are shown on the right side of each ideogram, while losses (blue) appear on the left side. APIP/PDHX/CD44 locus for further analyses are indicated by yellow boxes. ( B ) Localization of the FISH probes specific for APIP/PDHX (labeled with Cy3, red), and CD44 (labeled with FITC, green) located on chromosome 11p13 is depicted. Metaphase FISH analysis of the APIP/PDHX/CD44 locus on chromosome 11p13 in SNU16 cells revealed that two different probes co-localized on the same locus and exist in three types of amplification: DMs ( ), HSRs ( ) and distributed insertions ( ). ( C ) Cohesin-mediated focal copy-number changes of APIP and CD44 were evaluated using a TaqMan-quantitative PCR-based CNV assay with control GFP-KD or RAD21-KD SNU16 cells on day 60 after viral transduction. Data are presented as averages ± SD of biological triplicate independent viral transduction experiments. * P
    Figure Legend Snippet: Copy-numbers of amplified APIP/PDHX/CD44 locus are decreased by cohesin reduction in SNU16 cells. ( A ) High-resolution array-CGH analysis was performed with the control GFP-KD or RAD21-KD SNU16 cells on day 60 after RAD21-KD. Chromosome 11 is represented by ideograms showing G-banding patterns (left ideogram, RAD21-KD SNU16 cells compared with the control GFP-KD cells; right ideogram, parental SNU16 cells compared with normal gastric cells). Gains/amplifications (red) are shown on the right side of each ideogram, while losses (blue) appear on the left side. APIP/PDHX/CD44 locus for further analyses are indicated by yellow boxes. ( B ) Localization of the FISH probes specific for APIP/PDHX (labeled with Cy3, red), and CD44 (labeled with FITC, green) located on chromosome 11p13 is depicted. Metaphase FISH analysis of the APIP/PDHX/CD44 locus on chromosome 11p13 in SNU16 cells revealed that two different probes co-localized on the same locus and exist in three types of amplification: DMs ( ), HSRs ( ) and distributed insertions ( ). ( C ) Cohesin-mediated focal copy-number changes of APIP and CD44 were evaluated using a TaqMan-quantitative PCR-based CNV assay with control GFP-KD or RAD21-KD SNU16 cells on day 60 after viral transduction. Data are presented as averages ± SD of biological triplicate independent viral transduction experiments. * P

    Techniques Used: Amplification, Fluorescence In Situ Hybridization, Labeling, Real-time Polymerase Chain Reaction, CNV Assay, Transduction

    Copy-numbers of amplified gene existing in DMs are decreased by cohesin reduction. ( A ) COLO 320-DM cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P
    Figure Legend Snippet: Copy-numbers of amplified gene existing in DMs are decreased by cohesin reduction. ( A ) COLO 320-DM cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P

    Techniques Used: Amplification, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, CNV Assay

    13) Product Images from "Viral proteins as a potential driver of histone depletion in dinoflagellates"

    Article Title: Viral proteins as a potential driver of histone depletion in dinoflagellates

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03993-4

    DVNP expression results in transcriptional impairment in S. cerevisiae . a Spike-in normalized average gene plot showing the enrichment of two biological replicates of Rpb3 ChIPs from DVNP-3HA-NLS-expressing and control cells. b Differences in Rpb3 following DVNP expression. Values represent the mean Rpb3 occupancy calculated over gene bodies after averaging biological replicates ( n . The P -value was calculated using a two sided Welch’s t -test. Box plot notches represent an estimate of the 95% confidence interval of the median
    Figure Legend Snippet: DVNP expression results in transcriptional impairment in S. cerevisiae . a Spike-in normalized average gene plot showing the enrichment of two biological replicates of Rpb3 ChIPs from DVNP-3HA-NLS-expressing and control cells. b Differences in Rpb3 following DVNP expression. Values represent the mean Rpb3 occupancy calculated over gene bodies after averaging biological replicates ( n . The P -value was calculated using a two sided Welch’s t -test. Box plot notches represent an estimate of the 95% confidence interval of the median

    Techniques Used: Expressing

    14) Product Images from "Compartment-Specific Modulation of GABAergic Synaptic Transmission by TRPV1 Channels in the Dentate Gyrus"

    Article Title: Compartment-Specific Modulation of GABAergic Synaptic Transmission by TRPV1 Channels in the Dentate Gyrus

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3635-14.2014

    TRPV1 activation depresses inhibitory transmission in a CB1 receptor-independent manner. A , Representative traces (left) and summary data (right) showing that bath application of 2 μ m CAP, while blocking CB1R with AM251 (4 μ m ), depresses
    Figure Legend Snippet: TRPV1 activation depresses inhibitory transmission in a CB1 receptor-independent manner. A , Representative traces (left) and summary data (right) showing that bath application of 2 μ m CAP, while blocking CB1R with AM251 (4 μ m ), depresses

    Techniques Used: Activation Assay, Transmission Assay, Blocking Assay

    Mechanism underlying TRPV1-mediated depression of inhibitory transmission. A , Representative traces (left) and summary plot (right) showing that loading DGCs with BAPTA (20 m m ) completely eliminated, whereas pre-incubation with 30 μ m CPA significantly
    Figure Legend Snippet: Mechanism underlying TRPV1-mediated depression of inhibitory transmission. A , Representative traces (left) and summary plot (right) showing that loading DGCs with BAPTA (20 m m ) completely eliminated, whereas pre-incubation with 30 μ m CPA significantly

    Techniques Used: Transmission Assay, Incubation

    TRPV1-mediated depression of inhibition is postsynaptic. A , Top, Representative traces of two consecutive GABAergic IPSCs (100 ms interstimulus interval) before (black) and after (gray) bath application of WIN, AEA, and CAP. Bottom, Summary plots showing
    Figure Legend Snippet: TRPV1-mediated depression of inhibition is postsynaptic. A , Top, Representative traces of two consecutive GABAergic IPSCs (100 ms interstimulus interval) before (black) and after (gray) bath application of WIN, AEA, and CAP. Bottom, Summary plots showing

    Techniques Used: Inhibition, Mass Spectrometry

    In vivo knockdown of TRPV1 abolishes TRPV1-mediated depression of synaptic transmission in the DG. A , Differential interference contrast (top) and fluorescence (bottom) images showing whole-cell recordings from Trpv1 shRNA-expressing (GFP + ) DGCs. R, recording
    Figure Legend Snippet: In vivo knockdown of TRPV1 abolishes TRPV1-mediated depression of synaptic transmission in the DG. A , Differential interference contrast (top) and fluorescence (bottom) images showing whole-cell recordings from Trpv1 shRNA-expressing (GFP + ) DGCs. R, recording

    Techniques Used: In Vivo, Transmission Assay, Fluorescence, shRNA, Expressing

    Functional expression of TRPV1 channels at inhibitory synapses in the dentate gyrus. A , Pharmacological activation of TRPV1 receptors with the agonist CAP (2 μ m ) depressed GABAergic IPSCs in a compartment-specific manner. Left, Representative
    Figure Legend Snippet: Functional expression of TRPV1 channels at inhibitory synapses in the dentate gyrus. A , Pharmacological activation of TRPV1 receptors with the agonist CAP (2 μ m ) depressed GABAergic IPSCs in a compartment-specific manner. Left, Representative

    Techniques Used: Functional Assay, Expressing, Activation Assay

    15) Product Images from "RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development"

    Article Title: RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0170464

    Regulators of spine maturation are distinct from regulators of spine precursor formation. A) Representative Images of GFP-expressing DIV-16 neurons transfected with the indicated shRNA targeting sequence for 48 hours. B) Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine density later in synaptic development (DIV-16). Spine density is expressed as the percentage of the average control spine density. n = 44 control, 16 Ophn-1 shRNA #1, 5 Ophn-1 shRNA #2, 17 β -pix shRNA #1, 7 β -pix shRNA #2, 15 Frabin shRNA #1, 8 Frabin shRNA #2 neurons (Spine density was not significantly different from control as determined by t-test, except for β -pix shRNA #1 which was determined by Mann-Whitney Rank Sum Test). C) Arhgap23 shRNAs significantly increase spine density later during synaptic development (DIV-16). n = 44 control (same as B), 22 Arhgap23 shRNA #1, and 12 Arhgap23 shRNA #2 neurons; p = 0.02 for Control vs Arhgap23 shRNA #1 (Mann-Whitney Rank Sum Test), p = 0.002 for Control vs Arhgap23 shRNA #2 (Mann-Whitney Rank Sum Test). D) Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine length later in synaptic development (DIV-16) neurons. Cumulative distribution plot of spine length in DIV-16 primary rat hippocampal neurons co-expressing GFP and the indicated shRNA targeting sequence. Spine length is expressed as a percentage of the average control spine length. n = 3273 control, 651 Ophn-1 shRNA #1, 130 Ophn-1 shRNA #2, 729 β -pix shRNA #1, 449 β -pix shRNA #2, 556 Frabin shRNA #1, 688 Frabin shRNA #2 spines (Spine length was not significantly different from control as determined by Mann-Whitney Rank Sum test). E) Arhgap23 and Vav2 shRNAs significantly increase spine length later in neuronal development (DIV-16). n = 3273 control (same as D), 1207 Arhgap23 shRNA #1, 1182 Arhgap23 shRNA #2, 962 Vav2 shRNA #1, and 551 Vav2 shRNA #2 spines; p
    Figure Legend Snippet: Regulators of spine maturation are distinct from regulators of spine precursor formation. A) Representative Images of GFP-expressing DIV-16 neurons transfected with the indicated shRNA targeting sequence for 48 hours. B) Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine density later in synaptic development (DIV-16). Spine density is expressed as the percentage of the average control spine density. n = 44 control, 16 Ophn-1 shRNA #1, 5 Ophn-1 shRNA #2, 17 β -pix shRNA #1, 7 β -pix shRNA #2, 15 Frabin shRNA #1, 8 Frabin shRNA #2 neurons (Spine density was not significantly different from control as determined by t-test, except for β -pix shRNA #1 which was determined by Mann-Whitney Rank Sum Test). C) Arhgap23 shRNAs significantly increase spine density later during synaptic development (DIV-16). n = 44 control (same as B), 22 Arhgap23 shRNA #1, and 12 Arhgap23 shRNA #2 neurons; p = 0.02 for Control vs Arhgap23 shRNA #1 (Mann-Whitney Rank Sum Test), p = 0.002 for Control vs Arhgap23 shRNA #2 (Mann-Whitney Rank Sum Test). D) Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine length later in synaptic development (DIV-16) neurons. Cumulative distribution plot of spine length in DIV-16 primary rat hippocampal neurons co-expressing GFP and the indicated shRNA targeting sequence. Spine length is expressed as a percentage of the average control spine length. n = 3273 control, 651 Ophn-1 shRNA #1, 130 Ophn-1 shRNA #2, 729 β -pix shRNA #1, 449 β -pix shRNA #2, 556 Frabin shRNA #1, 688 Frabin shRNA #2 spines (Spine length was not significantly different from control as determined by Mann-Whitney Rank Sum test). E) Arhgap23 and Vav2 shRNAs significantly increase spine length later in neuronal development (DIV-16). n = 3273 control (same as D), 1207 Arhgap23 shRNA #1, 1182 Arhgap23 shRNA #2, 962 Vav2 shRNA #1, and 551 Vav2 shRNA #2 spines; p

    Techniques Used: Expressing, Transfection, shRNA, Sequencing, MANN-WHITNEY

    16) Product Images from "R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease"

    Article Title: R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20170418

    R-Spo1 treatment promotes development of Paneth cells from ISCs and increases luminal concentrations of α-defensins. (A–E and H–P) B6D2F1 mice were i.v. injected with R-Spo1 (200 µg/d) or PBS for 6 d. 1 d later, the small intestine was harvested. (A) H E staining. Bars: (top) 100 µm; (bottom) 30 µm. Areas in the white squares are magnified and shown below the original images. (B) Numbers of Paneth cells per crypt ( n = 6 per group). (C) Crypt depth ( n = 4 per group). (D) Confocal images. Lysozyme and Crp1 are expressed by Paneth cells. Bar, 100 µm. (E) Serial sections of the small intestine. H E staining (top), confocal images of Crp1, MMP-7, and the merged images of Crp1 and MMP-7. Bars, 100 µm. (F and G) Lgr5 - EGFP - creER × R26 Tomato mice were i.p. injected with 40 mg/kg tamoxifen for 3 d to label ISCs, followed by i.v. injection of R-Spo1 (200 µg/d) for 3 d. (F) Confocal images of lineage tracing in the small intestine are shown. RFP is expressed by Lgr5 + ISCs and their progenies, and Crp1 is expressed by Paneth cells. Asterisks indicate preexisting Paneth cells. Arrowheads indicate de novo generated Paneth cells from Lgr5 + ISCs. Bars, 50 µm. (G) Numbers of preexisting and de novo Paneth cells per crypt in the ileum ( n = 3 per group). (H) Numbers of goblet cells per crypt ( n = 4 per group). (I) Confocal images. Chromogranin A is expressed by EECs. Bars, 50 µm. (D–F and I) DAPI (blue) stains the nucleus. (J) Numbers of EECs per 1 mm of ileum ( n = 5 per group). (K and L) Quantitative real-time PCR analysis of Dll1 or Dll4 transcripts in the small intestine normalized to those of 18S rRNA ( n = 10 per group). (M and N) Fecal levels of Crp1 and Crp4. (O and P) mRNA extracted from highly purified Paneth cells was subjected to quantitative PCR analysis of Defa4 and Mmp7. Relative expression of mRNA in purified Paneth cells is shown by the comparative ΔCt method ( n = 3 per group). (B, C, G, H, and J–P) Data from two independent experiments were combined and are shown as means ± SE. Student’s t tests or Mann–Whitney U tests were used to compare the data. *, P
    Figure Legend Snippet: R-Spo1 treatment promotes development of Paneth cells from ISCs and increases luminal concentrations of α-defensins. (A–E and H–P) B6D2F1 mice were i.v. injected with R-Spo1 (200 µg/d) or PBS for 6 d. 1 d later, the small intestine was harvested. (A) H E staining. Bars: (top) 100 µm; (bottom) 30 µm. Areas in the white squares are magnified and shown below the original images. (B) Numbers of Paneth cells per crypt ( n = 6 per group). (C) Crypt depth ( n = 4 per group). (D) Confocal images. Lysozyme and Crp1 are expressed by Paneth cells. Bar, 100 µm. (E) Serial sections of the small intestine. H E staining (top), confocal images of Crp1, MMP-7, and the merged images of Crp1 and MMP-7. Bars, 100 µm. (F and G) Lgr5 - EGFP - creER × R26 Tomato mice were i.p. injected with 40 mg/kg tamoxifen for 3 d to label ISCs, followed by i.v. injection of R-Spo1 (200 µg/d) for 3 d. (F) Confocal images of lineage tracing in the small intestine are shown. RFP is expressed by Lgr5 + ISCs and their progenies, and Crp1 is expressed by Paneth cells. Asterisks indicate preexisting Paneth cells. Arrowheads indicate de novo generated Paneth cells from Lgr5 + ISCs. Bars, 50 µm. (G) Numbers of preexisting and de novo Paneth cells per crypt in the ileum ( n = 3 per group). (H) Numbers of goblet cells per crypt ( n = 4 per group). (I) Confocal images. Chromogranin A is expressed by EECs. Bars, 50 µm. (D–F and I) DAPI (blue) stains the nucleus. (J) Numbers of EECs per 1 mm of ileum ( n = 5 per group). (K and L) Quantitative real-time PCR analysis of Dll1 or Dll4 transcripts in the small intestine normalized to those of 18S rRNA ( n = 10 per group). (M and N) Fecal levels of Crp1 and Crp4. (O and P) mRNA extracted from highly purified Paneth cells was subjected to quantitative PCR analysis of Defa4 and Mmp7. Relative expression of mRNA in purified Paneth cells is shown by the comparative ΔCt method ( n = 3 per group). (B, C, G, H, and J–P) Data from two independent experiments were combined and are shown as means ± SE. Student’s t tests or Mann–Whitney U tests were used to compare the data. *, P

    Techniques Used: Mouse Assay, Injection, Staining, Generated, Real-time Polymerase Chain Reaction, Purification, Expressing, MANN-WHITNEY

    17) Product Images from "REST-dependent epigenetic remodeling promotes the in vivo developmental switch in NMDA receptors"

    Article Title: REST-dependent epigenetic remodeling promotes the in vivo developmental switch in NMDA receptors

    Journal: Nature neuroscience

    doi: 10.1038/nn.3214

    Maternal deprivation disrupts the increase in REST, epigenetic remodeling and decrease in GluN2B during postnatal development a , Diagram illustrating the maternal deprivation paradigm. b , Representative Western blot of hippocampal nuclear fraction. c , Summary plot ( left panel ) showing a robust decrease in REST protein expression in the hippocampus of maternally-deprived vs . normally-reared pups (n = 3). In the dentate gyrus, whereas REST mRNA decreases (n = 3), GluN2B mRNA increases ( right panel; n = 3). d , Abundance of REST and e , H3K27me3 at the grin2b promoter are decreased relative to age-matched, normally-reared rats ( n = 3). f , Representative Western blots ( left panel ) and summary data ( right panel ), showing that whereas maternal deprivation increases GluN2B protein in total lysate, endoplasmic reticulum (ER) and postsynaptic density (PSD) fractions, GluN2A and GluA2 are unchanged in all fractions. GluN1 was reduced in the ER fraction and increased in the PSD in the hippocampus from maternally-deprived pups vs. normally-reared pups. All samples were assessed at P28–31 (n = 3). Total lysate samples were normalized to β-actin, ER samples were normalized to GRP78, an ER marker, and PSD samples were normalized to PSD-95, which is not altered after maternally-deprived ( see Supplementary Table 9 and full length blots in Supplementary Fig. 12 ).
    Figure Legend Snippet: Maternal deprivation disrupts the increase in REST, epigenetic remodeling and decrease in GluN2B during postnatal development a , Diagram illustrating the maternal deprivation paradigm. b , Representative Western blot of hippocampal nuclear fraction. c , Summary plot ( left panel ) showing a robust decrease in REST protein expression in the hippocampus of maternally-deprived vs . normally-reared pups (n = 3). In the dentate gyrus, whereas REST mRNA decreases (n = 3), GluN2B mRNA increases ( right panel; n = 3). d , Abundance of REST and e , H3K27me3 at the grin2b promoter are decreased relative to age-matched, normally-reared rats ( n = 3). f , Representative Western blots ( left panel ) and summary data ( right panel ), showing that whereas maternal deprivation increases GluN2B protein in total lysate, endoplasmic reticulum (ER) and postsynaptic density (PSD) fractions, GluN2A and GluA2 are unchanged in all fractions. GluN1 was reduced in the ER fraction and increased in the PSD in the hippocampus from maternally-deprived pups vs. normally-reared pups. All samples were assessed at P28–31 (n = 3). Total lysate samples were normalized to β-actin, ER samples were normalized to GRP78, an ER marker, and PSD samples were normalized to PSD-95, which is not altered after maternally-deprived ( see Supplementary Table 9 and full length blots in Supplementary Fig. 12 ).

    Techniques Used: Western Blot, Expressing, Marker

    RNAi-mediated depletion of REST increases GluN2B mRNA and alters NMDAR properties a , Diagram illustrating RNAi injection and time course of assays (qRT-PCR or patch recording). Lentiviral mediated RNAi directed against REST (RESTi) and nontargeting RNAi were delivered by means of sterotaxic injection into the hippocampus of rats at P10 or P24, and evaluated at indicated time points. b , Differential interference contrast (DIC) and GFP fluorescence images showing viral-mediated transduction of DGCs at P14 ( top ), and a patched DGC at P29 ( bottom ). Scale bars = 50 µm ( top panel ) and 20 µm ( bottom panel ). DGL, dentate granule cell layer; ML, molecular layer; R, recording pipette. c , RESTi delivered at P10 (RESTi-P10) effectively knocks down REST by P14 (n = 3), as assessed by qPCR. Delivery of RESTi at P10 and P24 (RESTi-P24) knocked-down REST measured at P28–32 (n = 3) in dentate granule cells (DGCs). d , RESTi injected at P10, but not P24, significantly increased GluN2B mRNA(n = 3) measured at P28–32. e , RESTi delivered at P10 does not exert any effect on GluN2A (n = 3) or GluN1 (n = 3) mRNA expression measured at P28–32. f , Representative DIC ( top left panel ) and fluorescence ( bottom left panel ) images showing simultaneous whole-cell recordings from RESTi-P10 expressing (GFP + ) and non-transduced control (GFP − ) DGCs. R1 and R2 indicate the recording pipettes from GFP − and GFP + neurons, respectively. Scale bars = 20 µm. Summary data ( middle panel ) and representative traces ( right panels ), showing that delivery of RESTi at P10 ( gray circles; 6 animals/13 cells ) increases NMDAR-mediated transmission relative to that of neighboring non-transduced (control) neurons ( white circles; 6 animals/13 cells ). g , Representative traces ( left ) and summary data ( right ) showing that the NMDAR/AMPAR ratio is higher in DGCs expressing REST RNAi delivered at P10 (6 animals/23 cells) but not at P24 (6 animals/18 cells) relative to non-transduced controls (6 animals/29 cells) or controls expressing nontargeting RNAi neurons (6 animals/18 cells). A second lentiviral mediated RNAi targeted to a different sequence in REST (RESTi*) also increased the NMDAR/AMPAR ratio (2 animals/7 cells). Summary data represent the mean ± s.e.m. *p
    Figure Legend Snippet: RNAi-mediated depletion of REST increases GluN2B mRNA and alters NMDAR properties a , Diagram illustrating RNAi injection and time course of assays (qRT-PCR or patch recording). Lentiviral mediated RNAi directed against REST (RESTi) and nontargeting RNAi were delivered by means of sterotaxic injection into the hippocampus of rats at P10 or P24, and evaluated at indicated time points. b , Differential interference contrast (DIC) and GFP fluorescence images showing viral-mediated transduction of DGCs at P14 ( top ), and a patched DGC at P29 ( bottom ). Scale bars = 50 µm ( top panel ) and 20 µm ( bottom panel ). DGL, dentate granule cell layer; ML, molecular layer; R, recording pipette. c , RESTi delivered at P10 (RESTi-P10) effectively knocks down REST by P14 (n = 3), as assessed by qPCR. Delivery of RESTi at P10 and P24 (RESTi-P24) knocked-down REST measured at P28–32 (n = 3) in dentate granule cells (DGCs). d , RESTi injected at P10, but not P24, significantly increased GluN2B mRNA(n = 3) measured at P28–32. e , RESTi delivered at P10 does not exert any effect on GluN2A (n = 3) or GluN1 (n = 3) mRNA expression measured at P28–32. f , Representative DIC ( top left panel ) and fluorescence ( bottom left panel ) images showing simultaneous whole-cell recordings from RESTi-P10 expressing (GFP + ) and non-transduced control (GFP − ) DGCs. R1 and R2 indicate the recording pipettes from GFP − and GFP + neurons, respectively. Scale bars = 20 µm. Summary data ( middle panel ) and representative traces ( right panels ), showing that delivery of RESTi at P10 ( gray circles; 6 animals/13 cells ) increases NMDAR-mediated transmission relative to that of neighboring non-transduced (control) neurons ( white circles; 6 animals/13 cells ). g , Representative traces ( left ) and summary data ( right ) showing that the NMDAR/AMPAR ratio is higher in DGCs expressing REST RNAi delivered at P10 (6 animals/23 cells) but not at P24 (6 animals/18 cells) relative to non-transduced controls (6 animals/29 cells) or controls expressing nontargeting RNAi neurons (6 animals/18 cells). A second lentiviral mediated RNAi targeted to a different sequence in REST (RESTi*) also increased the NMDAR/AMPAR ratio (2 animals/7 cells). Summary data represent the mean ± s.e.m. *p

    Techniques Used: Injection, Quantitative RT-PCR, Fluorescence, Transduction, Transferring, Real-time Polymerase Chain Reaction, Expressing, Transmission Assay, Sequencing

    REST increases transiently, is recruited to and coincides with epigenetic marks of repression at the grin2b promoter during rat hippocampal postnatal development a , Representative Western blots of whole hippocampal lysates showing that REST increases, GluN2B declines, and GluN2A increases during postnatal development (see full-length blot in Supplementary Fig. 1a ). b , Time course showing that REST protein increases transiently at P14–15 (n = 5). c , GluN2B mRNA exhibits a long-term decline during postnatal development, assessed by RT-qPCR. The decline was highly significant from P15 through P60 ( vs . P3; n = 5). d,e , Time course showing that whereas GluN2B protein declines after P21 (n = 6), GluN2A protein increases markedly from P8 to P16 and remains high up to P60 (n = 3). Data were normalized to corresponding values at P3. f , GluN1 mRNA is not altered during postnatal development (n = 6). g , Representative Western blot ( left panel ) and summary data ( right panel ) show REST protein expression in the nuclear fraction of the hippocampal cell body layer, which is enriched for neurons. Note that REST abundance in the neuronal nuclear fraction increases strikingly by P14–15 (n = 4). Data were normalized to corresponding data at P9. See full-length blot in Supplementary Fig. 12 ). h , Map of the rat grin2b gene indicating location of RE1 motifs contained within the proximal (PR1; gray box ) and distal (PR2; white box ) regions of the grin2b promoter probed by chromatin immunoprecipitation (ChIP). i,j , REST occupancy is markedly enriched at the grin2b proximal (PR1, grey bars ) (n = 6) and distal PR2 ( white bars ) (n = 3) promoters at P15 but declines by P60. k,l , REST is not enriched at RE1 sites within the grin2a (n = 9), nor grin1 (n = 6) promoters. Inset, same data depicted with expanded y-axis. m,n , CoREST (n = 3) and G9a (n = 3) are enriched at PR1 by P15. o,p , Increase in H3K9me3 (n = 3) and H3K27me3 (n = 6) (marks of repression) at P15. q , Decrease in trimethylation of H3K4 (n = 3) (mark of active transcription), at PR1. r , MeCP2 occupancy is enriched at grin2b PR1 by P15 with a sharp increase by P60 (n = 3). All samples were normalized to input and to corresponding values at P3. Summary data represent the mean ± s.e.m. *p
    Figure Legend Snippet: REST increases transiently, is recruited to and coincides with epigenetic marks of repression at the grin2b promoter during rat hippocampal postnatal development a , Representative Western blots of whole hippocampal lysates showing that REST increases, GluN2B declines, and GluN2A increases during postnatal development (see full-length blot in Supplementary Fig. 1a ). b , Time course showing that REST protein increases transiently at P14–15 (n = 5). c , GluN2B mRNA exhibits a long-term decline during postnatal development, assessed by RT-qPCR. The decline was highly significant from P15 through P60 ( vs . P3; n = 5). d,e , Time course showing that whereas GluN2B protein declines after P21 (n = 6), GluN2A protein increases markedly from P8 to P16 and remains high up to P60 (n = 3). Data were normalized to corresponding values at P3. f , GluN1 mRNA is not altered during postnatal development (n = 6). g , Representative Western blot ( left panel ) and summary data ( right panel ) show REST protein expression in the nuclear fraction of the hippocampal cell body layer, which is enriched for neurons. Note that REST abundance in the neuronal nuclear fraction increases strikingly by P14–15 (n = 4). Data were normalized to corresponding data at P9. See full-length blot in Supplementary Fig. 12 ). h , Map of the rat grin2b gene indicating location of RE1 motifs contained within the proximal (PR1; gray box ) and distal (PR2; white box ) regions of the grin2b promoter probed by chromatin immunoprecipitation (ChIP). i,j , REST occupancy is markedly enriched at the grin2b proximal (PR1, grey bars ) (n = 6) and distal PR2 ( white bars ) (n = 3) promoters at P15 but declines by P60. k,l , REST is not enriched at RE1 sites within the grin2a (n = 9), nor grin1 (n = 6) promoters. Inset, same data depicted with expanded y-axis. m,n , CoREST (n = 3) and G9a (n = 3) are enriched at PR1 by P15. o,p , Increase in H3K9me3 (n = 3) and H3K27me3 (n = 6) (marks of repression) at P15. q , Decrease in trimethylation of H3K4 (n = 3) (mark of active transcription), at PR1. r , MeCP2 occupancy is enriched at grin2b PR1 by P15 with a sharp increase by P60 (n = 3). All samples were normalized to input and to corresponding values at P3. Summary data represent the mean ± s.e.m. *p

    Techniques Used: Western Blot, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation

    18) Product Images from "Keratinocyte nicotinic acetylcholine receptor activation modulates early TLR2-mediated wound healing responses"

    Article Title: Keratinocyte nicotinic acetylcholine receptor activation modulates early TLR2-mediated wound healing responses

    Journal: International immunopharmacology

    doi: 10.1016/j.intimp.2015.05.047

    Nicotinic nAChR activation impairs TLR2 signaling during TLR2-mediated keratinocyte wound healing. (A, B) Western blot analyses for TLR2 signaling pathway in cytosolic and nuclear extracts from wounded NHEKs stimulated with vehicle, MALP2 (100ng ml −1
    Figure Legend Snippet: Nicotinic nAChR activation impairs TLR2 signaling during TLR2-mediated keratinocyte wound healing. (A, B) Western blot analyses for TLR2 signaling pathway in cytosolic and nuclear extracts from wounded NHEKs stimulated with vehicle, MALP2 (100ng ml −1

    Techniques Used: Activation Assay, Western Blot

    Nicotinic AChR activation dampens TLR2 agonist-mediated NHEKs migration during in vitro wound healing to suppress cathelicidin expression and pro-inflammatory cytokine production. (A) Scratch migration assay. MALP2-mediated keratinocytes migration at
    Figure Legend Snippet: Nicotinic AChR activation dampens TLR2 agonist-mediated NHEKs migration during in vitro wound healing to suppress cathelicidin expression and pro-inflammatory cytokine production. (A) Scratch migration assay. MALP2-mediated keratinocytes migration at

    Techniques Used: Activation Assay, Migration, In Vitro, Expressing

    Nicotinic nAChR activation suppresses CD14 and MyD88 expression in TLR2-mediated keratinocyte wound healing. (A) Quantitative RT-PCR analyses of TLR2 and CD14 in wounded NHEKs stimulated with nicotine in presence of MALP2 (100ng ml −1 ) ( n =3). Representative
    Figure Legend Snippet: Nicotinic nAChR activation suppresses CD14 and MyD88 expression in TLR2-mediated keratinocyte wound healing. (A) Quantitative RT-PCR analyses of TLR2 and CD14 in wounded NHEKs stimulated with nicotine in presence of MALP2 (100ng ml −1 ) ( n =3). Representative

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

    19) Product Images from "Topical nicotinic receptor activation improves wound bacterial infection outcomes and TLR2-mediated inflammation in diabetic mouse wounds"

    Article Title: Topical nicotinic receptor activation improves wound bacterial infection outcomes and TLR2-mediated inflammation in diabetic mouse wounds

    Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

    doi: 10.1111/wrr.12674

    Topical nAChR activation decreases IL-6 production in MRSA infected diabetic mouse wounds, and slightly increases IL-10 in MRSA infected control mouse wounds. The concentrations of ( A ) IL-6, ( B ) IL-17A, ( C ) TNF-α and ( D ) IL-10 at day 1 post-wounding were measured by ELISA in homogenates of MRSA infected skin wounds with or without nicotine treatment of control mice ( Lepr db/+ ) or diabetic mice ( Lepr db/db ) ( n =8 for each group). Data are presented as the mean ± sem. Statistical analyses were performed using Mann-Whitney one-tailed t-test ( * , p
    Figure Legend Snippet: Topical nAChR activation decreases IL-6 production in MRSA infected diabetic mouse wounds, and slightly increases IL-10 in MRSA infected control mouse wounds. The concentrations of ( A ) IL-6, ( B ) IL-17A, ( C ) TNF-α and ( D ) IL-10 at day 1 post-wounding were measured by ELISA in homogenates of MRSA infected skin wounds with or without nicotine treatment of control mice ( Lepr db/+ ) or diabetic mice ( Lepr db/db ) ( n =8 for each group). Data are presented as the mean ± sem. Statistical analyses were performed using Mann-Whitney one-tailed t-test ( * , p

    Techniques Used: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Mouse Assay, MANN-WHITNEY, One-tailed Test

    20) Product Images from "PDE4D regulates Spine Plasticity and Memory in the Retrosplenial Cortex"

    Article Title: PDE4D regulates Spine Plasticity and Memory in the Retrosplenial Cortex

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22193-0

    Object exploration (OE) training strongly activates RSC. ( A ) The training protocol used to test the effect of exploration on gene expression involves 2 d of habituation to the training boxes and a 15 min exposure to the objects on training day, a procedure that will produce a memory for both the objects (object recognition memory) and their spatial location (object location memory). ( B ) In RSC, IEG mRNA levels ( Fos , Arc and Npas4 ) as detected by qPCR are strongly increased 30 mins (all three genes) and 1 hr ( Fos, Arc ) after OE training, but return to baseline within 3 hrs thereby recapitulating the time course seen after cFC training (n = 8). ( C ) Correlation of RSC induction of the 66 genes shown in Fig. 1d after cFC and OE training. The OE induced gene expression changes in RSC closely recapitulate those seen 30 mins after cFC training.
    Figure Legend Snippet: Object exploration (OE) training strongly activates RSC. ( A ) The training protocol used to test the effect of exploration on gene expression involves 2 d of habituation to the training boxes and a 15 min exposure to the objects on training day, a procedure that will produce a memory for both the objects (object recognition memory) and their spatial location (object location memory). ( B ) In RSC, IEG mRNA levels ( Fos , Arc and Npas4 ) as detected by qPCR are strongly increased 30 mins (all three genes) and 1 hr ( Fos, Arc ) after OE training, but return to baseline within 3 hrs thereby recapitulating the time course seen after cFC training (n = 8). ( C ) Correlation of RSC induction of the 66 genes shown in Fig. 1d after cFC and OE training. The OE induced gene expression changes in RSC closely recapitulate those seen 30 mins after cFC training.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Blocking NMDA-R impairs memory and blocks IEG induction in HC and RSC. MK801 (0.1 mg/kg, i.p.) injected 30 mins before cFC training does not affect the response to foot-shock during conditioning ( A ), but it significantly impairs memory when tested 24 hrs but not 1 hr after training ( B ), n = 8 per treatment group and time-point). The same dose blocks training-induced IEG induction ( Fos , Arc and Npas4 ) in RSC (C, n = 8) and HC (D, n = 6). Mean ± s.e.m. are shown. Significant differences from vehicle control are indicated by an asterisk (*).
    Figure Legend Snippet: Blocking NMDA-R impairs memory and blocks IEG induction in HC and RSC. MK801 (0.1 mg/kg, i.p.) injected 30 mins before cFC training does not affect the response to foot-shock during conditioning ( A ), but it significantly impairs memory when tested 24 hrs but not 1 hr after training ( B ), n = 8 per treatment group and time-point). The same dose blocks training-induced IEG induction ( Fos , Arc and Npas4 ) in RSC (C, n = 8) and HC (D, n = 6). Mean ± s.e.m. are shown. Significant differences from vehicle control are indicated by an asterisk (*).

    Techniques Used: Blocking Assay, Injection

    Gene-responses following cFC are highly correlated in hippocampus (HC) and retrosplenial cortex (RSC). ( A ) In HC, IEG mRNA levels ( Fos , Arc and Npas4 ) as detected by qPCR are strongly increased 30 mins (all three genes) and 1 hr ( Fos , Arc ) after cFC training, and return to baseline within 3 hrs. ( B ) The time-course of IEG induction observed in HC is recapitulated in RSC (n = 8 mice per group for HC and RSC). ( C ) Venn diagram showing the number of confirmed hits of two replicate RNAseq studies comparing mRNA expression in naïve and fear conditioned mice. All but one gene ( Errf1 ) found in HC are detected in RSC. ( D ) Correlation of mRNA induction by fear conditioning in HP and RSC. The gene-responses of the 66 confirmed hits in RSC and HC are highly correlated. The greater number of calls in RSC is explained by an overall larger gene-response in this structure.
    Figure Legend Snippet: Gene-responses following cFC are highly correlated in hippocampus (HC) and retrosplenial cortex (RSC). ( A ) In HC, IEG mRNA levels ( Fos , Arc and Npas4 ) as detected by qPCR are strongly increased 30 mins (all three genes) and 1 hr ( Fos , Arc ) after cFC training, and return to baseline within 3 hrs. ( B ) The time-course of IEG induction observed in HC is recapitulated in RSC (n = 8 mice per group for HC and RSC). ( C ) Venn diagram showing the number of confirmed hits of two replicate RNAseq studies comparing mRNA expression in naïve and fear conditioned mice. All but one gene ( Errf1 ) found in HC are detected in RSC. ( D ) Correlation of mRNA induction by fear conditioning in HP and RSC. The gene-responses of the 66 confirmed hits in RSC and HC are highly correlated. The greater number of calls in RSC is explained by an overall larger gene-response in this structure.

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

    21) Product Images from "R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease"

    Article Title: R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20170418

    R-Spo1 treatment promotes development of Paneth cells from ISCs and increases luminal concentrations of α-defensins. (A–E and H–P) B6D2F1 mice were i.v. injected with R-Spo1 (200 µg/d) or PBS for 6 d. 1 d later, the small intestine was harvested. (A) H E staining. Bars: (top) 100 µm; (bottom) 30 µm. Areas in the white squares are magnified and shown below the original images. (B) Numbers of Paneth cells per crypt ( n = 6 per group). (C) Crypt depth ( n = 4 per group). (D) Confocal images. Lysozyme and Crp1 are expressed by Paneth cells. Bar, 100 µm. (E) Serial sections of the small intestine. H E staining (top), confocal images of Crp1, MMP-7, and the merged images of Crp1 and MMP-7. Bars, 100 µm. (F and G) Lgr5 - EGFP - creER × R26 Tomato mice were i.p. injected with 40 mg/kg tamoxifen for 3 d to label ISCs, followed by i.v. injection of R-Spo1 (200 µg/d) for 3 d. (F) Confocal images of lineage tracing in the small intestine are shown. RFP is expressed by Lgr5 + ISCs and their progenies, and Crp1 is expressed by Paneth cells. Asterisks indicate preexisting Paneth cells. Arrowheads indicate de novo generated Paneth cells from Lgr5 + ISCs. Bars, 50 µm. (G) Numbers of preexisting and de novo Paneth cells per crypt in the ileum ( n = 3 per group). (H) Numbers of goblet cells per crypt ( n = 4 per group). (I) Confocal images. Chromogranin A is expressed by EECs. Bars, 50 µm. (D–F and I) DAPI (blue) stains the nucleus. (J) Numbers of EECs per 1 mm of ileum ( n = 5 per group). (K and L) Quantitative real-time PCR analysis of Dll1 or Dll4 transcripts in the small intestine normalized to those of 18S rRNA ( n = 10 per group). (M and N) Fecal levels of Crp1 and Crp4. (O and P) mRNA extracted from highly purified Paneth cells was subjected to quantitative PCR analysis of Defa4 and Mmp7. Relative expression of mRNA in purified Paneth cells is shown by the comparative ΔCt method ( n = 3 per group). (B, C, G, H, and J–P) Data from two independent experiments were combined and are shown as means ± SE. Student’s t tests or Mann–Whitney U tests were used to compare the data. *, P
    Figure Legend Snippet: R-Spo1 treatment promotes development of Paneth cells from ISCs and increases luminal concentrations of α-defensins. (A–E and H–P) B6D2F1 mice were i.v. injected with R-Spo1 (200 µg/d) or PBS for 6 d. 1 d later, the small intestine was harvested. (A) H E staining. Bars: (top) 100 µm; (bottom) 30 µm. Areas in the white squares are magnified and shown below the original images. (B) Numbers of Paneth cells per crypt ( n = 6 per group). (C) Crypt depth ( n = 4 per group). (D) Confocal images. Lysozyme and Crp1 are expressed by Paneth cells. Bar, 100 µm. (E) Serial sections of the small intestine. H E staining (top), confocal images of Crp1, MMP-7, and the merged images of Crp1 and MMP-7. Bars, 100 µm. (F and G) Lgr5 - EGFP - creER × R26 Tomato mice were i.p. injected with 40 mg/kg tamoxifen for 3 d to label ISCs, followed by i.v. injection of R-Spo1 (200 µg/d) for 3 d. (F) Confocal images of lineage tracing in the small intestine are shown. RFP is expressed by Lgr5 + ISCs and their progenies, and Crp1 is expressed by Paneth cells. Asterisks indicate preexisting Paneth cells. Arrowheads indicate de novo generated Paneth cells from Lgr5 + ISCs. Bars, 50 µm. (G) Numbers of preexisting and de novo Paneth cells per crypt in the ileum ( n = 3 per group). (H) Numbers of goblet cells per crypt ( n = 4 per group). (I) Confocal images. Chromogranin A is expressed by EECs. Bars, 50 µm. (D–F and I) DAPI (blue) stains the nucleus. (J) Numbers of EECs per 1 mm of ileum ( n = 5 per group). (K and L) Quantitative real-time PCR analysis of Dll1 or Dll4 transcripts in the small intestine normalized to those of 18S rRNA ( n = 10 per group). (M and N) Fecal levels of Crp1 and Crp4. (O and P) mRNA extracted from highly purified Paneth cells was subjected to quantitative PCR analysis of Defa4 and Mmp7. Relative expression of mRNA in purified Paneth cells is shown by the comparative ΔCt method ( n = 3 per group). (B, C, G, H, and J–P) Data from two independent experiments were combined and are shown as means ± SE. Student’s t tests or Mann–Whitney U tests were used to compare the data. *, P

    Techniques Used: Mouse Assay, Injection, Staining, Generated, Real-time Polymerase Chain Reaction, Purification, Expressing, MANN-WHITNEY

    22) Product Images from "Anthropoid primate–specific retroviral element THE1B controls expression of CRH in placenta and alters gestation length"

    Article Title: Anthropoid primate–specific retroviral element THE1B controls expression of CRH in placenta and alters gestation length

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2006337

    THE1B 5′ insertion site creates novel binding site for transcription factor DLX3. (A) Top left, sequence of 5′ insertion site of THE1B near CRH ]. This DLX3 binding site is formed by the insertion of THE1B (right of black bar) into the genome (left of black bar). (C) ChIP for DLX3 (black bars) and RNA polymerase II (white bar) with quantitative real-time PCR. DLX3 is significantly associated with the 5′ end of THE1B and positive control (JRE) in human term placenta. ChIP-qPCR data were normalized to the IgG control for each target and presented relative to negative control (JRE distal). ( n = 3 for all, P
    Figure Legend Snippet: THE1B 5′ insertion site creates novel binding site for transcription factor DLX3. (A) Top left, sequence of 5′ insertion site of THE1B near CRH ]. This DLX3 binding site is formed by the insertion of THE1B (right of black bar) into the genome (left of black bar). (C) ChIP for DLX3 (black bars) and RNA polymerase II (white bar) with quantitative real-time PCR. DLX3 is significantly associated with the 5′ end of THE1B and positive control (JRE) in human term placenta. ChIP-qPCR data were normalized to the IgG control for each target and presented relative to negative control (JRE distal). ( n = 3 for all, P

    Techniques Used: Binding Assay, Sequencing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control

    BAC transgenic mice exhibit placental CRH expression and delayed parturition, which are eliminated by THE1B deletion. (A) Schematic of human BAC clone RP11-366K18 (gray) with CRH shown to scale (red). (B) Expression of human CRH measured by qPCR in adult tissues and placenta. ( n = 2–4 per group, P
    Figure Legend Snippet: BAC transgenic mice exhibit placental CRH expression and delayed parturition, which are eliminated by THE1B deletion. (A) Schematic of human BAC clone RP11-366K18 (gray) with CRH shown to scale (red). (B) Expression of human CRH measured by qPCR in adult tissues and placenta. ( n = 2–4 per group, P

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

    23) Product Images from "Merkel cell polyomavirus Tumor antigens expressed in Merkel cell carcinoma function independently of the ubiquitin ligases Fbw7 and β-TrCP"

    Article Title: Merkel cell polyomavirus Tumor antigens expressed in Merkel cell carcinoma function independently of the ubiquitin ligases Fbw7 and β-TrCP

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007543

    MCPyV LT and 57kT bind Fbw7α independently of its WD40 domain and MCPyV LT S239. (A, B) MCPyV LT was pulled-down with XT10 from whole cell lysates of 293A cells transfected with individual or combinations of full-length MCPyV LT S239A (5μg), MCPyV LT-t (10.5μg), MCPyV 57kT (5μg), Fbw7 (4.5μg), ΔFbox (3μg), or R505L (3μg), and immunoblotted with anti-FLAG to detect co-immunoprecipitated Fbw7. (B) A plasmid encoding only the sequence of MCPyV 57kT was similarly pulled-down and co-immunoprecipitated Fbw7 was detected. Asterisks (*) denote non-specific bands.
    Figure Legend Snippet: MCPyV LT and 57kT bind Fbw7α independently of its WD40 domain and MCPyV LT S239. (A, B) MCPyV LT was pulled-down with XT10 from whole cell lysates of 293A cells transfected with individual or combinations of full-length MCPyV LT S239A (5μg), MCPyV LT-t (10.5μg), MCPyV 57kT (5μg), Fbw7 (4.5μg), ΔFbox (3μg), or R505L (3μg), and immunoblotted with anti-FLAG to detect co-immunoprecipitated Fbw7. (B) A plasmid encoding only the sequence of MCPyV 57kT was similarly pulled-down and co-immunoprecipitated Fbw7 was detected. Asterisks (*) denote non-specific bands.

    Techniques Used: Transfection, Immunoprecipitation, Plasmid Preparation, Sequencing

    MCPyV LT is not bound or destabilized by the WD40 domain of Fbw7. (A) MCPyV LT was pulled-down from whole cell lysates of 293A cells transfected with MCPyV LT (5μg), LT-t (10.5μg) and either the FLAG-Fbw7α (4.5μg), or HA-Fbw7α construct (8μg). In addition, the FLAG-tagged WD40 mutant (FLAG-Fbw7α-R505L) (3μg), and the HA-tagged WD40 mutant provided by Kwun et al. (HA-Fbw7α-R465C) (8μg) were tested for their ability to co-immunoprecipitate with MCPyV LT and LT-t (17). (B) Consistent amounts of MCPyV LT (5μg) were co-expressed with increasing amounts of Fbw7 (2.5μg, 5μg, 10μg) or the degradation incompetent Fbw7 ΔFbox mutant. Immunoblotting with 2T2 was performed to compare MCPyV LT protein levels in each condition.
    Figure Legend Snippet: MCPyV LT is not bound or destabilized by the WD40 domain of Fbw7. (A) MCPyV LT was pulled-down from whole cell lysates of 293A cells transfected with MCPyV LT (5μg), LT-t (10.5μg) and either the FLAG-Fbw7α (4.5μg), or HA-Fbw7α construct (8μg). In addition, the FLAG-tagged WD40 mutant (FLAG-Fbw7α-R505L) (3μg), and the HA-tagged WD40 mutant provided by Kwun et al. (HA-Fbw7α-R465C) (8μg) were tested for their ability to co-immunoprecipitate with MCPyV LT and LT-t (17). (B) Consistent amounts of MCPyV LT (5μg) were co-expressed with increasing amounts of Fbw7 (2.5μg, 5μg, 10μg) or the degradation incompetent Fbw7 ΔFbox mutant. Immunoblotting with 2T2 was performed to compare MCPyV LT protein levels in each condition.

    Techniques Used: Transfection, Construct, Mutagenesis

    SV40 LT, but not the MCPyV T antigens, co-immunoprecipitate with Fbw7α. (A) The SV40 LT antigen was pulled down with an anti-HA antibody from whole cell lysates of 293A cells expressing individual or combinations of HA-SV40 LT or the T701A mutant (5μg), wild-type FLAG-Fbw7 (4.5μg), or FLAG-Fbw7 ΔFbox/R505L mutants (3μg). Detection of co-immunoprecipitated Fbw7 was performed by immunoprecipitating with anti-HA, followed by immunoblotting with anti-FLAG. (B) The reciprocal IP to Fig 1A was performed with HA-SV40 LT, the MCPyV T antigens (HA-LT (5μg), HA-LT-t (10.5μg), and untagged ST (1μg)) and Fbw7, in which Fbw7 was pulled down (FLAG) and immunoblotted for interacting T antigens (anti-HA/2T2 (common-T antibody)). (C) An identical co-immunoprecipitation as Fig 1B was performed, except Fbw7 with an N-terminal Myc tag was pulled down from cellular lysates using a Myc tag specific antibody (9E10). (D) An identical co-immunoprecipitation as Fig 1B was performed with untagged SV40 and MCPyV T antigens. SV40 and MCPyV T antigens were detected by XT10 immunoblotting. (E) A co-immunoprecipitation between MCPyV T antigens (LT and ST) and Fbw7 was also performed through pull-down of the T antigens (XT10—common-T antibody) and detection of co-immunoprecipitated Fbw7 (anti-FLAG). Asterisks (*) denote non-specific bands.
    Figure Legend Snippet: SV40 LT, but not the MCPyV T antigens, co-immunoprecipitate with Fbw7α. (A) The SV40 LT antigen was pulled down with an anti-HA antibody from whole cell lysates of 293A cells expressing individual or combinations of HA-SV40 LT or the T701A mutant (5μg), wild-type FLAG-Fbw7 (4.5μg), or FLAG-Fbw7 ΔFbox/R505L mutants (3μg). Detection of co-immunoprecipitated Fbw7 was performed by immunoprecipitating with anti-HA, followed by immunoblotting with anti-FLAG. (B) The reciprocal IP to Fig 1A was performed with HA-SV40 LT, the MCPyV T antigens (HA-LT (5μg), HA-LT-t (10.5μg), and untagged ST (1μg)) and Fbw7, in which Fbw7 was pulled down (FLAG) and immunoblotted for interacting T antigens (anti-HA/2T2 (common-T antibody)). (C) An identical co-immunoprecipitation as Fig 1B was performed, except Fbw7 with an N-terminal Myc tag was pulled down from cellular lysates using a Myc tag specific antibody (9E10). (D) An identical co-immunoprecipitation as Fig 1B was performed with untagged SV40 and MCPyV T antigens. SV40 and MCPyV T antigens were detected by XT10 immunoblotting. (E) A co-immunoprecipitation between MCPyV T antigens (LT and ST) and Fbw7 was also performed through pull-down of the T antigens (XT10—common-T antibody) and detection of co-immunoprecipitated Fbw7 (anti-FLAG). Asterisks (*) denote non-specific bands.

    Techniques Used: Expressing, Mutagenesis, Immunoprecipitation

    24) Product Images from "A Mutation Deleting Sequences Encoding the Amino Terminus of Human Cytomegalovirus UL84 Impairs Interaction with UL44 and Capsid Localization"

    Article Title: A Mutation Deleting Sequences Encoding the Amino Terminus of Human Cytomegalovirus UL84 Impairs Interaction with UL44 and Capsid Localization

    Journal: Journal of Virology

    doi: 10.1128/JVI.01379-12

    Levels of viral DNA synthesis in infected cells. Viral DNA synthesis in each experiment was determined by quantitative real-time PCR at each of the time points indicated. The amount of viral DNA assayed is represented as copies of the viral gene UL83 per copy of the cellular adipsin gene.
    Figure Legend Snippet: Levels of viral DNA synthesis in infected cells. Viral DNA synthesis in each experiment was determined by quantitative real-time PCR at each of the time points indicated. The amount of viral DNA assayed is represented as copies of the viral gene UL83 per copy of the cellular adipsin gene.

    Techniques Used: DNA Synthesis, Infection, Real-time Polymerase Chain Reaction

    Levels of capsid proteins in infected cells. (A) Schematic of locus in the HCMV virus genome from which UL83 to UL85 are produced. Filled arrows above the genome represent proteins encoded within the genome. Black lines below the genome represent transcripts from which those proteins are produced. pA, polyadenylation signal. (B) Western blotting of infected cell lysate. HFF cells were infected at an MOI of 1 with the indicated viruses, and cell lysates were prepared for Western blotting at the indicated time points (indicated above the figure). (C) Determination of relative protein levels in cells. A 2-fold dilution series of protein from lane 4 of panel B (lanes 1 to 3) was analyzed by Western blotting compared to undiluted protein from lane 7 of panel B (lane 4). Proteins recognized by the antibodies used in each experiment are indicated to the right of each figure. The positions of molecular mass markers (kDa) are indicated to the left of each figure.
    Figure Legend Snippet: Levels of capsid proteins in infected cells. (A) Schematic of locus in the HCMV virus genome from which UL83 to UL85 are produced. Filled arrows above the genome represent proteins encoded within the genome. Black lines below the genome represent transcripts from which those proteins are produced. pA, polyadenylation signal. (B) Western blotting of infected cell lysate. HFF cells were infected at an MOI of 1 with the indicated viruses, and cell lysates were prepared for Western blotting at the indicated time points (indicated above the figure). (C) Determination of relative protein levels in cells. A 2-fold dilution series of protein from lane 4 of panel B (lanes 1 to 3) was analyzed by Western blotting compared to undiluted protein from lane 7 of panel B (lane 4). Proteins recognized by the antibodies used in each experiment are indicated to the right of each figure. The positions of molecular mass markers (kDa) are indicated to the left of each figure.

    Techniques Used: Infection, Produced, Western Blot

    25) Product Images from "Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts"

    Article Title: Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts

    Journal: Scientific Reports

    doi: 10.1038/srep28538

    Synergetic effect of mixed sheets consisting of PBMNCs and fibroblasts. ( a ) Comparison of VEGF secretion. The VEGF concentration in the culture medium was measured in PBMNCs, fibroblast sheets, and mixed cell sheets for 3 days. Normoxic condition: 37 °C, 20% O 2 for 3 days. Hypoxic condition: 37 °C, 20% O 2 for 2 days followed by 33 °C, 2% O 2 for 1 day. ( b ) Secretion from PBMNCs increased VEGF production by fibroblasts. Fibroblasts were cultured for 48 h with or without the PBMNC-conditioned medium, and the VEGF concentration in the supernatant was analyzed by ELISA. ( c ) TGF-β1 concentration in fibroblasts and PBMNC culture medium at 48 h. ( d ) PDGF-BB concentration in fibroblasts and PBMNC culture medium at 48 h. ( e ) Neutralizing antibody against TGF-β1 and PDGF-BB inhibited VEGF production in fibroblasts. The PBMNC-conditioned medium was co-cultured with a neutralizing antibody against TGF-β1 or PDGF-BB, and the PBMNC-conditioned medium was added to fibroblasts. After 48 h, the VEGF concentration was measured by ELISA. ( f ) TGF-β1 and PDGF-BB recombinant proteins elevated VEGF production by fibroblasts. ( g ) The PBMNC-conditioned medium increased the expression levels of VEGF, collagen I, collagen III, α-SMA, and Axin2 mRNA. Fibroblasts were cultured with the PBMNC-conditioned or control medium for 48 h. The mRNA expression levels were determined using real-time PCR. ACTB was used as an endogenous control. The expression levels were compared with that in control medium, which is presented as 1.
    Figure Legend Snippet: Synergetic effect of mixed sheets consisting of PBMNCs and fibroblasts. ( a ) Comparison of VEGF secretion. The VEGF concentration in the culture medium was measured in PBMNCs, fibroblast sheets, and mixed cell sheets for 3 days. Normoxic condition: 37 °C, 20% O 2 for 3 days. Hypoxic condition: 37 °C, 20% O 2 for 2 days followed by 33 °C, 2% O 2 for 1 day. ( b ) Secretion from PBMNCs increased VEGF production by fibroblasts. Fibroblasts were cultured for 48 h with or without the PBMNC-conditioned medium, and the VEGF concentration in the supernatant was analyzed by ELISA. ( c ) TGF-β1 concentration in fibroblasts and PBMNC culture medium at 48 h. ( d ) PDGF-BB concentration in fibroblasts and PBMNC culture medium at 48 h. ( e ) Neutralizing antibody against TGF-β1 and PDGF-BB inhibited VEGF production in fibroblasts. The PBMNC-conditioned medium was co-cultured with a neutralizing antibody against TGF-β1 or PDGF-BB, and the PBMNC-conditioned medium was added to fibroblasts. After 48 h, the VEGF concentration was measured by ELISA. ( f ) TGF-β1 and PDGF-BB recombinant proteins elevated VEGF production by fibroblasts. ( g ) The PBMNC-conditioned medium increased the expression levels of VEGF, collagen I, collagen III, α-SMA, and Axin2 mRNA. Fibroblasts were cultured with the PBMNC-conditioned or control medium for 48 h. The mRNA expression levels were determined using real-time PCR. ACTB was used as an endogenous control. The expression levels were compared with that in control medium, which is presented as 1.

    Techniques Used: Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant, Expressing, Real-time Polymerase Chain Reaction

    Immunostaining against markers of the wound healing process 3, 7, and 14 days after mixed cell sheet transplantation and trafermin treatment. ( a ) TGF-β1. ( b ) α-SMA. ( c ) Collagen III. Bar shows 200 μm.
    Figure Legend Snippet: Immunostaining against markers of the wound healing process 3, 7, and 14 days after mixed cell sheet transplantation and trafermin treatment. ( a ) TGF-β1. ( b ) α-SMA. ( c ) Collagen III. Bar shows 200 μm.

    Techniques Used: Immunostaining, Transplantation Assay

    26) Product Images from "CHD1 Contributes to Intestinal Resistance against Infection by P. aeruginosa in Drosophila melanogaster"

    Article Title: CHD1 Contributes to Intestinal Resistance against Infection by P. aeruginosa in Drosophila melanogaster

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043144

    Bacterial load is elevated in Chd1 -mutant flies. (A) Bacterial load was analyzed in isolated guts and in whole flies from which intestines had been removed. qPCR was performed with primers targeting 16S rDNA in the absence of infection (−PA) as well as 3 days and 4 days after oral infection with P. aeruginosa. (B) 251659264 P. aeruginosa titers are strongly increased in Chd1 −/− flies after infection. qPCR as in (A) with primers specific for P. aeruginosa . (C) Analysis of the gut-specific bacterium Acetobacter EW911. qPCR as in (A) with primers specific for Acetobacter EW911 . Relative differences of bacterial genes and the fly Rpl32 gene are expressed as 2 −ΔCT values. Values represent mean +/− SD of three independent experiments. Note that SD values are too small to show in the graph.
    Figure Legend Snippet: Bacterial load is elevated in Chd1 -mutant flies. (A) Bacterial load was analyzed in isolated guts and in whole flies from which intestines had been removed. qPCR was performed with primers targeting 16S rDNA in the absence of infection (−PA) as well as 3 days and 4 days after oral infection with P. aeruginosa. (B) 251659264 P. aeruginosa titers are strongly increased in Chd1 −/− flies after infection. qPCR as in (A) with primers specific for P. aeruginosa . (C) Analysis of the gut-specific bacterium Acetobacter EW911. qPCR as in (A) with primers specific for Acetobacter EW911 . Relative differences of bacterial genes and the fly Rpl32 gene are expressed as 2 −ΔCT values. Values represent mean +/− SD of three independent experiments. Note that SD values are too small to show in the graph.

    Techniques Used: Mutagenesis, Isolation, Real-time Polymerase Chain Reaction, Infection

    27) Product Images from "SOX2 as a New Regulator of HPV16 Transcription"

    Article Title: SOX2 as a New Regulator of HPV16 Transcription

    Journal: Viruses

    doi: 10.3390/v9070175

    SOX2 binds to HPV16-LCR putative binding sites in vivo. Chromatin immunoprecipitation assay (ChIP) of SOX2 were performed evaluating the HPV16-LCR region (LCR) from CaSki cells. Anti-p65 and -CTCF antibodies were employed as positive and negative controls, respectively. Non-specific IgG antibody was included as a negative control. Quantitative PCR (qPCR) analysis was performed on the DNA obtained from the ChIP assay evaluating the HPV16-LCR region (black bars), and the Lefty (gray bars) gene promoter region, as a positive control of SOX2 binding ( n = 2). The graphic represents the percentage of input recovered after immunoprecipitation with anti-SOX2, -p65 or -CTCF antibodies. Three independent biological experiments were performed. The standard error of the mean of triplicate qPCR measurements is shown. ** p
    Figure Legend Snippet: SOX2 binds to HPV16-LCR putative binding sites in vivo. Chromatin immunoprecipitation assay (ChIP) of SOX2 were performed evaluating the HPV16-LCR region (LCR) from CaSki cells. Anti-p65 and -CTCF antibodies were employed as positive and negative controls, respectively. Non-specific IgG antibody was included as a negative control. Quantitative PCR (qPCR) analysis was performed on the DNA obtained from the ChIP assay evaluating the HPV16-LCR region (black bars), and the Lefty (gray bars) gene promoter region, as a positive control of SOX2 binding ( n = 2). The graphic represents the percentage of input recovered after immunoprecipitation with anti-SOX2, -p65 or -CTCF antibodies. Three independent biological experiments were performed. The standard error of the mean of triplicate qPCR measurements is shown. ** p

    Techniques Used: Binding Assay, In Vivo, Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Positive Control, Immunoprecipitation

    28) Product Images from "MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex"

    Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0275-4

    MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in HA-PRMT5 or Myc-MEP50-expressing C3H10T1/2 cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 plasmids were transfected into C3H10T1/2 cells. After 24 h of incubation, cells were separated equally, and DMSO (−) or 300 nM SAG (+) were applied for 24 h. Protein levels are shown in Supplementary Fig. 3a . e and f qRT-PCR analysis of PRMT5 ( e ) and MEP50 ( f ) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a – c , data represent one of two independent experiments with similar results. In e and f , data represent one of three independent experiments with similar results. The source data is shown in Supplementary Data 1
    Figure Legend Snippet: MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in HA-PRMT5 or Myc-MEP50-expressing C3H10T1/2 cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 plasmids were transfected into C3H10T1/2 cells. After 24 h of incubation, cells were separated equally, and DMSO (−) or 300 nM SAG (+) were applied for 24 h. Protein levels are shown in Supplementary Fig. 3a . e and f qRT-PCR analysis of PRMT5 ( e ) and MEP50 ( f ) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a – c , data represent one of two independent experiments with similar results. In e and f , data represent one of three independent experiments with similar results. The source data is shown in Supplementary Data 1

    Techniques Used: Activity Assay, Activation Assay, Expressing, Stable Transfection, Recombinant, Binding Assay, Luciferase, Plasmid Preparation, Transfection, Incubation, Quantitative RT-PCR

    29) Product Images from "Soluble ST2 protein inhibits LPS stimulation on monocyte-derived dendritic cells"

    Article Title: Soluble ST2 protein inhibits LPS stimulation on monocyte-derived dendritic cells

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/cmi.2012.29

    TLR4 expression is suppressed by sST2. ( a ) Relative quantity of TLR4 mRNA was analyzed by real-time PCR after various stimuli (sST2, LPS, sST2/LPS) and normalized against endogenous β-actin. The data are presented as the means of three experiments±s.d.
    Figure Legend Snippet: TLR4 expression is suppressed by sST2. ( a ) Relative quantity of TLR4 mRNA was analyzed by real-time PCR after various stimuli (sST2, LPS, sST2/LPS) and normalized against endogenous β-actin. The data are presented as the means of three experiments±s.d.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    30) Product Images from "Extensive Ex Vivo Expansion of Functional Human Erythroid Precursors Established From Umbilical Cord Blood Cells by Defined Factors"

    Article Title: Extensive Ex Vivo Expansion of Functional Human Erythroid Precursors Established From Umbilical Cord Blood Cells by Defined Factors

    Journal: Molecular Therapy

    doi: 10.1038/mt.2013.201

    Gene expression studies of iE2 cells. ( a ) Expression of (mouse) transgenes in iE2 cells after long-term expansion at days (d) 118 and 207 detected by RT-PCR. P: plasmids with the transgene cDNA. ( b ) Genomic PCR detecting the mouse Sox2 and Klf4 cDNA in genomic DNA. mSox2cDNA is present in iE2 genome (upper panel) but mKlf4 cDNA is absent in iE2 genome (lower panel). iPSC is a human iPSC line derived using pMX-mSox2, mKlf4, mOct4, and mMyc vectors. ( c ) Puromycin resistance of iE2 cells during expansion suggesting continuous expression of p53shRNA from the integrated retroviral vector. ( d ) qRT-PCR analysis of human TP53 gene expression of primary day 19 culture-expanded cord blood (CB) erythroblasts (pCBE19), iE2 and iPSC cells. ( e ) iE2 cells upregulate endogenous human SOX2 gene expression and maintain c-MYC gene expression at both days 118 and 207 of expansion culture. ( f ) qRT-PCR analysis of human MYB gene expression of primary day 19 culture-expanded CB erythroblasts (pCBE19) and iE2 cells. ( g ) Dendrogram and heatmap of unsupervised hierarchical clustering analysis of global gene expression profiles of various cell types. Distance numbers on the top right represent the value of Pearson distance (1-R) determined by Pearson correlation algorithms. iE2 cells are clustering with pCBE19 cells while TF-1 cells are clustering with CD34 + HSPCs. ESC/iPSCs are clustering together and separated from all blood cells.
    Figure Legend Snippet: Gene expression studies of iE2 cells. ( a ) Expression of (mouse) transgenes in iE2 cells after long-term expansion at days (d) 118 and 207 detected by RT-PCR. P: plasmids with the transgene cDNA. ( b ) Genomic PCR detecting the mouse Sox2 and Klf4 cDNA in genomic DNA. mSox2cDNA is present in iE2 genome (upper panel) but mKlf4 cDNA is absent in iE2 genome (lower panel). iPSC is a human iPSC line derived using pMX-mSox2, mKlf4, mOct4, and mMyc vectors. ( c ) Puromycin resistance of iE2 cells during expansion suggesting continuous expression of p53shRNA from the integrated retroviral vector. ( d ) qRT-PCR analysis of human TP53 gene expression of primary day 19 culture-expanded cord blood (CB) erythroblasts (pCBE19), iE2 and iPSC cells. ( e ) iE2 cells upregulate endogenous human SOX2 gene expression and maintain c-MYC gene expression at both days 118 and 207 of expansion culture. ( f ) qRT-PCR analysis of human MYB gene expression of primary day 19 culture-expanded CB erythroblasts (pCBE19) and iE2 cells. ( g ) Dendrogram and heatmap of unsupervised hierarchical clustering analysis of global gene expression profiles of various cell types. Distance numbers on the top right represent the value of Pearson distance (1-R) determined by Pearson correlation algorithms. iE2 cells are clustering with pCBE19 cells while TF-1 cells are clustering with CD34 + HSPCs. ESC/iPSCs are clustering together and separated from all blood cells.

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

    31) Product Images from "Distinct microRNA-155 expression in the vitreous of patients with primary vitreoretinal lymphoma and uveitis"

    Article Title: Distinct microRNA-155 expression in the vitreous of patients with primary vitreoretinal lymphoma and uveitis

    Journal: American journal of ophthalmology

    doi: 10.1016/j.ajo.2013.12.014

    The Pair Plot of 110 Detectable micro Ribonucleic Acids in Vitreous Samples from Primary Vitreoretinal Lymphoma and Uveitis. The data are the average of 3 biological samples from each condition and normalized against microRNA-16. The level of the majority
    Figure Legend Snippet: The Pair Plot of 110 Detectable micro Ribonucleic Acids in Vitreous Samples from Primary Vitreoretinal Lymphoma and Uveitis. The data are the average of 3 biological samples from each condition and normalized against microRNA-16. The level of the majority

    Techniques Used:

    32) Product Images from "Overexpression of E3 ubiquitin ligase tripartite motif 32 correlates with a poor prognosis in patients with gastric cancer"

    Article Title: Overexpression of E3 ubiquitin ligase tripartite motif 32 correlates with a poor prognosis in patients with gastric cancer

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.5806

    Differential expression of TRIM32 in gastric cancer tissue specimens. Immunohistochemistry staining of TRIM32 protein in tissue specimens with (A) non-reactive (0), (B) weak positive and (C) strong positive staining. Representative images of stain in
    Figure Legend Snippet: Differential expression of TRIM32 in gastric cancer tissue specimens. Immunohistochemistry staining of TRIM32 protein in tissue specimens with (A) non-reactive (0), (B) weak positive and (C) strong positive staining. Representative images of stain in

    Techniques Used: Expressing, Immunohistochemistry, Staining

    TRIM32 mRNA expression levels in gastric cancer tissues, as detected by reverse transcription-quantitative polymerase chain reaction. (A) Relative TRIM32 expression levels of cancer tissue specimens were significantly increased compared with that of non-cancerous
    Figure Legend Snippet: TRIM32 mRNA expression levels in gastric cancer tissues, as detected by reverse transcription-quantitative polymerase chain reaction. (A) Relative TRIM32 expression levels of cancer tissue specimens were significantly increased compared with that of non-cancerous

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Prognostic value of TRIM32 expression levels in gastric cancer
    Figure Legend Snippet: Prognostic value of TRIM32 expression levels in gastric cancer

    Techniques Used: Expressing

    Downregulation of TRIM32 using siRNA inhibits the proliferation and increases the apoptosis of human gastric cancer cells. (A) MKN45 and MKN74 gastric cancer cells were transfected with control or TRIM32 siRNA. The expression levels of TRIM32 mRNA were
    Figure Legend Snippet: Downregulation of TRIM32 using siRNA inhibits the proliferation and increases the apoptosis of human gastric cancer cells. (A) MKN45 and MKN74 gastric cancer cells were transfected with control or TRIM32 siRNA. The expression levels of TRIM32 mRNA were

    Techniques Used: Transfection, Expressing

    Clinicopathological significance of TRIM32 expression levels in human gastric cancer
    Figure Legend Snippet: Clinicopathological significance of TRIM32 expression levels in human gastric cancer

    Techniques Used: Expressing

    Postoperative survival rate was significantly reduced in patients with TRIM32-high tumors compared with those with TRIM32-low tumors. (A) Overall survival rate of patients with gastric cancer (P=0.008). (B) Relapse-free survival rate of patients with
    Figure Legend Snippet: Postoperative survival rate was significantly reduced in patients with TRIM32-high tumors compared with those with TRIM32-low tumors. (A) Overall survival rate of patients with gastric cancer (P=0.008). (B) Relapse-free survival rate of patients with

    Techniques Used:

    Influence of the TRIM32 expression levels on postoperative recurrence
    Figure Legend Snippet: Influence of the TRIM32 expression levels on postoperative recurrence

    Techniques Used: Expressing

    33) Product Images from "Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR"

    Article Title: Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0200-6

    Quantitative analysis of Commd1 expression and Zrsr1 -DMR methylation in ΔPA blastocysts and the ΔPA adult brain. a Allelic expression of Commd1 was analyzed in Commd1 + (B6)/ + (BALB) (WT), Commd1 PA(B6)/ + (BALB) (PA), and Commd1 ΔPA(B6)/ + (BALB) (ΔPA) blastocysts in triplicate (shown with n) by pyrosequencing. Alleles were discriminated between using an SNP between B6 (Mat) and BALB/c (Pat) located in exon 2 (rs26846230; C in B6, T in BALB/c). The allelic expression ratios are presented relative to paternal expression in each blastocyst sample. b Methylation at Zrsr1 -DMR was analyzed in the blastocysts used in a . The 343-bp region in the DMR containing 25 CpG sites (B6; maternal allele) or 24 CpG sites (BALB/c; paternal allele) was analyzed via bisulfite sequencing. The alleles were discriminated between using an SNP (rs26846192; C in B6, A in BALB/c) located in a CpG site in the B6 sequence. Closed and open circles depict methylated and unmethylated CpGs, respectively. c Methylation at Zrsr1 -DMR was analyzed in the brains of ΔPA and WT adult F1 mice as described in b . d Allelic expression of Commd1 in the adult F1 brain was quantitatively analyzed in triplicate (shown with n) by pyrosequencing and is presented as in a . e Total Commd1 expression in an adult F1 brain of each genotype was analyzed in triplicate (shown with n) via TaqMan RT-PCR. Total expression is presented relative to the WT expression level. Asterisks (*) indicate statistical significance ( P
    Figure Legend Snippet: Quantitative analysis of Commd1 expression and Zrsr1 -DMR methylation in ΔPA blastocysts and the ΔPA adult brain. a Allelic expression of Commd1 was analyzed in Commd1 + (B6)/ + (BALB) (WT), Commd1 PA(B6)/ + (BALB) (PA), and Commd1 ΔPA(B6)/ + (BALB) (ΔPA) blastocysts in triplicate (shown with n) by pyrosequencing. Alleles were discriminated between using an SNP between B6 (Mat) and BALB/c (Pat) located in exon 2 (rs26846230; C in B6, T in BALB/c). The allelic expression ratios are presented relative to paternal expression in each blastocyst sample. b Methylation at Zrsr1 -DMR was analyzed in the blastocysts used in a . The 343-bp region in the DMR containing 25 CpG sites (B6; maternal allele) or 24 CpG sites (BALB/c; paternal allele) was analyzed via bisulfite sequencing. The alleles were discriminated between using an SNP (rs26846192; C in B6, A in BALB/c) located in a CpG site in the B6 sequence. Closed and open circles depict methylated and unmethylated CpGs, respectively. c Methylation at Zrsr1 -DMR was analyzed in the brains of ΔPA and WT adult F1 mice as described in b . d Allelic expression of Commd1 in the adult F1 brain was quantitatively analyzed in triplicate (shown with n) by pyrosequencing and is presented as in a . e Total Commd1 expression in an adult F1 brain of each genotype was analyzed in triplicate (shown with n) via TaqMan RT-PCR. Total expression is presented relative to the WT expression level. Asterisks (*) indicate statistical significance ( P

    Techniques Used: Expressing, Methylation, Methylation Sequencing, Sequencing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    Structure of the Zrsr1 / Commd1 locus and analysis of Commd1 expression and Zrsr1 -DMR methylation in the oocyte. a Zrsr1 , an approximately 2.8-kb intronless gene, and the first two exons of Commd1 are represented by gray and white boxes, respectively. Distances from the Zrsr1 gene to Commd1 exon 1 and exon 2 are indicated above the gene with double-headed arrows. The schematic is not drawn to scale. Arrows above (maternal allele) and below (paternal allele) exon 1 and the Zrsr1 gene represent the direction of transcription and the allelic expression status of the genes. The open and closed circles at the Zrsr1 promoter indicate unmethylation and methylation, respectively. A schematic of the targeting vector is shown under the gene. The closed and hatched boxes represent the truncation cassette and the neo-selection marker gene, respectively. These elements are flanked by the 5.2 kb left arm containing exon 1 and the 5.1 kb right arm, which contains part of intron 1. The truncation cassette is flanked by loxP sites, represented by gray arrowheads enclosed in open rectangles. Expected transcription patterns of the WT and PA alleles are shown above the gene schematic with thick lines and dotted lines corresponding to exons and introns, respectively. b RT-PCR analysis of Commd1 expression in growing oocytes prepared from B6 female neonates at Day 5 (D5), Day 10 (D10), and Day 15 (D15) postpartum, and fully grown MII oocytes (MII) from B6 adult females. PC: positive control for RT-PCR using adult brain cDNA. MW: molecular weight marker. c Analysis of methylation at Zrsr1 -DMR in growing and fully grown oocytes used in b . The 223-bp region in the DMR containing 14 CpGs was analyzed via bisulfite sequencing. Each row represents a dataset from one clone, and each circle represents one CpG site. Closed and open circles depict methylated and unmethylated CpGs, respectively
    Figure Legend Snippet: Structure of the Zrsr1 / Commd1 locus and analysis of Commd1 expression and Zrsr1 -DMR methylation in the oocyte. a Zrsr1 , an approximately 2.8-kb intronless gene, and the first two exons of Commd1 are represented by gray and white boxes, respectively. Distances from the Zrsr1 gene to Commd1 exon 1 and exon 2 are indicated above the gene with double-headed arrows. The schematic is not drawn to scale. Arrows above (maternal allele) and below (paternal allele) exon 1 and the Zrsr1 gene represent the direction of transcription and the allelic expression status of the genes. The open and closed circles at the Zrsr1 promoter indicate unmethylation and methylation, respectively. A schematic of the targeting vector is shown under the gene. The closed and hatched boxes represent the truncation cassette and the neo-selection marker gene, respectively. These elements are flanked by the 5.2 kb left arm containing exon 1 and the 5.1 kb right arm, which contains part of intron 1. The truncation cassette is flanked by loxP sites, represented by gray arrowheads enclosed in open rectangles. Expected transcription patterns of the WT and PA alleles are shown above the gene schematic with thick lines and dotted lines corresponding to exons and introns, respectively. b RT-PCR analysis of Commd1 expression in growing oocytes prepared from B6 female neonates at Day 5 (D5), Day 10 (D10), and Day 15 (D15) postpartum, and fully grown MII oocytes (MII) from B6 adult females. PC: positive control for RT-PCR using adult brain cDNA. MW: molecular weight marker. c Analysis of methylation at Zrsr1 -DMR in growing and fully grown oocytes used in b . The 223-bp region in the DMR containing 14 CpGs was analyzed via bisulfite sequencing. Each row represents a dataset from one clone, and each circle represents one CpG site. Closed and open circles depict methylated and unmethylated CpGs, respectively

    Techniques Used: Expressing, Methylation, Plasmid Preparation, Selection, Marker, Reverse Transcription Polymerase Chain Reaction, Positive Control, Molecular Weight, Methylation Sequencing

    34) Product Images from "MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex"

    Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0275-4

    MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 . e and f qRT-PCR analysis of PRMT5 ( e ) and MEP50 ( f ) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a – c , data represent one of two independent experiments with similar results. In e and f
    Figure Legend Snippet: MEP50/PRMT5 complex-mediated GLI1 stabilisation enhances Gli transcriptional activity and HH signalling pathway activation induces PRMT5 and MEP50 expression. a Gli transcriptional activity in PRMT5 or MEP50 knockdown cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. A multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. b qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 expression in C3H10T1/2 cells with MEP50 knockdown or PRMT5 knockdown and treated with 300 nM SAG for the indicated times. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. c Gli transcriptional activity in HA-PRMT5 or Myc-MEP50-expressing cells. HA-PRMT5, HA-PRMT5 G367A/R368A, or Myc-MEP50 and a multimerized Gli-binding site luciferase reporter plasmid and phRL-TK control reporter plasmid were transfected into C3H10T1/2 cells. After 24 h of incubation, 300 nM SAG was applied for 24 h, and then luciferase assays were performed. d qRT-PCR analysis of Ptch1 , Bcl2 , and Foxm1 . e and f qRT-PCR analysis of PRMT5 ( e ) and MEP50 ( f ) mRNA expression in C3H10T1/2 cells after 24 h of treatment with 300 nM SAG. In a – c , data represent one of two independent experiments with similar results. In e and f

    Techniques Used: Activity Assay, Activation Assay, Expressing, Stable Transfection, Recombinant, Binding Assay, Luciferase, Plasmid Preparation, Transfection, Incubation, Quantitative RT-PCR

    35) Product Images from "NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes"

    Article Title: NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19051365

    Expression of NOX isotypes in the epithelial and myeloid compartments of the small intestine. ( a ) RT-PCR. CX3CR1-GFP + and epithelial cells were sorted from the small intestine to examine their expression of NOX enzymes in the steady-state or after activation with PMA. Bar graph indicates the relative expression of Nox2 and Nox4 relative to HPRT. No significant difference was observed upon PMA treatment. Results shown are representative of three independent experiments; columns and error bars indicate mean +/− SEM. ( b ) Immunofluorescence micrograph of the small intestine from a CX3CR1-GFP mouse, immunostained for Nox4. Nox4 is expressed at high levels in GFP + and GFP − cells in the LP, and at moderate levels in epithelial cells. Scale bar is 20 µm. ( c ) Immunofluorescence micrograph of the small intestine before (top) and after (bottom) incubation with pHrodo bioparticles. At physiologic pH the pHrodo beads are non-fluorescent. pHrodo accumulation is seen as bright clusters in GFP + and GFP − cells in the LP, and diffusely throughout the epithelium. In the steady-state, no pHrodo signal is observed. pHrodo bioparticles are rapidly taken up and found in acidic compartments of GFP + and GFP − cells. Immunofluorescence images are representative of three independent experiments; scale bar is 20 µm.
    Figure Legend Snippet: Expression of NOX isotypes in the epithelial and myeloid compartments of the small intestine. ( a ) RT-PCR. CX3CR1-GFP + and epithelial cells were sorted from the small intestine to examine their expression of NOX enzymes in the steady-state or after activation with PMA. Bar graph indicates the relative expression of Nox2 and Nox4 relative to HPRT. No significant difference was observed upon PMA treatment. Results shown are representative of three independent experiments; columns and error bars indicate mean +/− SEM. ( b ) Immunofluorescence micrograph of the small intestine from a CX3CR1-GFP mouse, immunostained for Nox4. Nox4 is expressed at high levels in GFP + and GFP − cells in the LP, and at moderate levels in epithelial cells. Scale bar is 20 µm. ( c ) Immunofluorescence micrograph of the small intestine before (top) and after (bottom) incubation with pHrodo bioparticles. At physiologic pH the pHrodo beads are non-fluorescent. pHrodo accumulation is seen as bright clusters in GFP + and GFP − cells in the LP, and diffusely throughout the epithelium. In the steady-state, no pHrodo signal is observed. pHrodo bioparticles are rapidly taken up and found in acidic compartments of GFP + and GFP − cells. Immunofluorescence images are representative of three independent experiments; scale bar is 20 µm.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Immunofluorescence, Incubation

    36) Product Images from "MicroRNA-196a Is a Putative Diagnostic Biomarker and Therapeutic Target for Laryngeal Cancer"

    Article Title: MicroRNA-196a Is a Putative Diagnostic Biomarker and Therapeutic Target for Laryngeal Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071480

    Differential expression of laryngeal cancer-associated miRNAs between matched paired samples. Relative expressions of laryngeal cancer-associated miRNAs in 5 paired samples were measured by TaqMan® qRT-PCR analysis and shown in left panels . Expression levels in adjacent noncancerous counterparts (NCs) in each pair are set to be 1 in right panels for clear visualization of significant fold difference in each miRNA. Data are expressed as mean values with standard deviations. (A) Up-regulation of 2 miRNAs (miR-196a and miR-455-5p) was confirmed in cancers when 5 cancers were compared with their NCs. *, p
    Figure Legend Snippet: Differential expression of laryngeal cancer-associated miRNAs between matched paired samples. Relative expressions of laryngeal cancer-associated miRNAs in 5 paired samples were measured by TaqMan® qRT-PCR analysis and shown in left panels . Expression levels in adjacent noncancerous counterparts (NCs) in each pair are set to be 1 in right panels for clear visualization of significant fold difference in each miRNA. Data are expressed as mean values with standard deviations. (A) Up-regulation of 2 miRNAs (miR-196a and miR-455-5p) was confirmed in cancers when 5 cancers were compared with their NCs. *, p

    Techniques Used: Expressing, Quantitative RT-PCR

    37) Product Images from "Methanandamide diminish the Porphyromonas gingivalis lipopolysaccharide induced response in human periodontal ligament cells"

    Article Title: Methanandamide diminish the Porphyromonas gingivalis lipopolysaccharide induced response in human periodontal ligament cells

    Journal: BMC Oral Health

    doi: 10.1186/s12903-020-01087-6

    Effect of Meth-AEA on the gene expression of IL-6, IL-8, and MCP-1 in hPdLCs. Gene expression of IL-6, IL-8, and MCP-1 was measured in hPdLCs upon 24 h stimulation with different concentrations of Meth-AEA using qPCR method. Y-axes show n-fold expression levels (2 -ΔΔCt values) of target in relation to non-stimulated control ( n = 1). Data are shown as mean ± s.e.m. of 5 different donors. * means significantly different vs. control group ( p
    Figure Legend Snippet: Effect of Meth-AEA on the gene expression of IL-6, IL-8, and MCP-1 in hPdLCs. Gene expression of IL-6, IL-8, and MCP-1 was measured in hPdLCs upon 24 h stimulation with different concentrations of Meth-AEA using qPCR method. Y-axes show n-fold expression levels (2 -ΔΔCt values) of target in relation to non-stimulated control ( n = 1). Data are shown as mean ± s.e.m. of 5 different donors. * means significantly different vs. control group ( p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Effect of Meth-AEA on P. gingivalis LPS induced gene expression of IL-6, IL-8, and MCP-1 in hPdLCs. Gene expression of IL-6, IL-8, and MCP-1 was measured in hPdLCs after 24 h stimulation with P. gingivalis LPS and different concentrations of Meth-AEA using qPCR method. Y-axes show n-fold expression levels (2 -ΔΔCt values) of target in relation to non-stimulated control (n = 1). Data are shown as mean ± s.e.m. of 5 different donors. * means significantly different vs. control group ( p
    Figure Legend Snippet: Effect of Meth-AEA on P. gingivalis LPS induced gene expression of IL-6, IL-8, and MCP-1 in hPdLCs. Gene expression of IL-6, IL-8, and MCP-1 was measured in hPdLCs after 24 h stimulation with P. gingivalis LPS and different concentrations of Meth-AEA using qPCR method. Y-axes show n-fold expression levels (2 -ΔΔCt values) of target in relation to non-stimulated control (n = 1). Data are shown as mean ± s.e.m. of 5 different donors. * means significantly different vs. control group ( p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Effect of Meth-AEA on the production of IL-6, IL-8, and MCP-1 by hPdLCs. The content of IL-6, IL-8, and MCP-1 in conditioned media was measured after 24 h stimulation with different concentrations of Meth-AEA by commercially available ELISA. Data are shown as mean ± s.e.m. of 5 different donors
    Figure Legend Snippet: Effect of Meth-AEA on the production of IL-6, IL-8, and MCP-1 by hPdLCs. The content of IL-6, IL-8, and MCP-1 in conditioned media was measured after 24 h stimulation with different concentrations of Meth-AEA by commercially available ELISA. Data are shown as mean ± s.e.m. of 5 different donors

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Effect of Meth-AEA on P. gingivalis LPS induced production of IL-6, IL-8, and MCP-1 by hPdLCs. The content of IL-6, IL-8, and MCP-1 in conditioned media was measured after 24 h stimulation with P. gingivalis LPS and different concentrations of Meth-AEA by commercially available ELISA. Data are shown as mean ± s.e.m. of 5 different donors. * means significantly different vs. control group ( p
    Figure Legend Snippet: Effect of Meth-AEA on P. gingivalis LPS induced production of IL-6, IL-8, and MCP-1 by hPdLCs. The content of IL-6, IL-8, and MCP-1 in conditioned media was measured after 24 h stimulation with P. gingivalis LPS and different concentrations of Meth-AEA by commercially available ELISA. Data are shown as mean ± s.e.m. of 5 different donors. * means significantly different vs. control group ( p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    38) Product Images from "Rivastigmine Alleviates Experimentally Induced Colitis in Mice and Rats by Acting at Central and Peripheral Sites to Modulate Immune Responses"

    Article Title: Rivastigmine Alleviates Experimentally Induced Colitis in Mice and Rats by Acting at Central and Peripheral Sites to Modulate Immune Responses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057668

    Effect of rivastigmine on TNF-α and IL-6 released from peritoneal macrophages of DSS treated mice. Legend as in Figure 3 . Data are mean ± SEM and represent pooled data from 6 animals per group. Significantly different from mice with DSS-induced colitis treated with PBS, ** p
    Figure Legend Snippet: Effect of rivastigmine on TNF-α and IL-6 released from peritoneal macrophages of DSS treated mice. Legend as in Figure 3 . Data are mean ± SEM and represent pooled data from 6 animals per group. Significantly different from mice with DSS-induced colitis treated with PBS, ** p

    Techniques Used: Mouse Assay

    Effect of rivastigmine on TNF-α, MPO, TBARS and ChE activity in rats with DNBS-induced colitis. ChE = ChE activity. Data are mean ± SEM from 13 animals per group. Percent of reduction compared to values in group treated with distilled water and DNBS. Significantly different from the rats treated by rectal administration of rivastigmine (1 mg/kg) * p
    Figure Legend Snippet: Effect of rivastigmine on TNF-α, MPO, TBARS and ChE activity in rats with DNBS-induced colitis. ChE = ChE activity. Data are mean ± SEM from 13 animals per group. Percent of reduction compared to values in group treated with distilled water and DNBS. Significantly different from the rats treated by rectal administration of rivastigmine (1 mg/kg) * p

    Techniques Used: Activity Assay

    39) Product Images from "A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages"

    Article Title: A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages

    Journal: Nature cell biology

    doi: 10.1038/ncb2742

    Oct4 binding is converse to Nanog and is linked to downregulation of naive pluripotency genes. ( a , b ) qRT–PCR analysis of total Oct4, Klf4, Nanog, Tbx3, Esrrb and Rex1 expression in PB-Oct4 (Oct4-WT and -low), PB-Oct4.2A.Cherry (Oct4-WT and -low)
    Figure Legend Snippet: Oct4 binding is converse to Nanog and is linked to downregulation of naive pluripotency genes. ( a , b ) qRT–PCR analysis of total Oct4, Klf4, Nanog, Tbx3, Esrrb and Rex1 expression in PB-Oct4 (Oct4-WT and -low), PB-Oct4.2A.Cherry (Oct4-WT and -low)

    Techniques Used: Binding Assay, Quantitative RT-PCR, Expressing

    Low levels of Oct4 expression sustain self-renewal. ( a ) Phase images of ESCs and rMK+PB-Oct4 iPSCs −/− treated and untreated with 4OHT in serum/LIF culture conditions. ( b , c ) qRT–PCR analysis of Pl-1 ( b ) and pluripotency markers
    Figure Legend Snippet: Low levels of Oct4 expression sustain self-renewal. ( a ) Phase images of ESCs and rMK+PB-Oct4 iPSCs −/− treated and untreated with 4OHT in serum/LIF culture conditions. ( b , c ) qRT–PCR analysis of Pl-1 ( b ) and pluripotency markers

    Techniques Used: Expressing, Quantitative RT-PCR

    Oct4 expression at an ESC level is required for in vitro differentiation. ( a ) qRT–PCR analysis of pluripotency (total Oct4, Nanog, Rex1 ), endoderm ( FoxA1, Gata4 ), ectoderm ( Fgf5 ) and mesoderm ( T-Brachyury, Zeb2, Snai2, Nkx2.5, N-cadherin ) markers
    Figure Legend Snippet: Oct4 expression at an ESC level is required for in vitro differentiation. ( a ) qRT–PCR analysis of pluripotency (total Oct4, Nanog, Rex1 ), endoderm ( FoxA1, Gata4 ), ectoderm ( Fgf5 ) and mesoderm ( T-Brachyury, Zeb2, Snai2, Nkx2.5, N-cadherin ) markers

    Techniques Used: Expressing, In Vitro, Quantitative RT-PCR

    40) Product Images from "Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability"

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv933

    Copy-numbers of amplified gene existing in HSRs are decreased by cohesin reduction. ( A ) COLO 320-HSR cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P
    Figure Legend Snippet: Copy-numbers of amplified gene existing in HSRs are decreased by cohesin reduction. ( A ) COLO 320-HSR cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P

    Techniques Used: Amplification, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, CNV Assay

    Copy-numbers of amplified APIP/PDHX/CD44 locus are decreased by cohesin reduction in SNU16 cells. ( A ) High-resolution array-CGH analysis was performed with the control GFP-KD or RAD21-KD SNU16 cells on day 60 after RAD21-KD. Chromosome 11 is represented by ideograms showing G-banding patterns (left ideogram, RAD21-KD SNU16 cells compared with the control GFP-KD cells; right ideogram, parental SNU16 cells compared with normal gastric cells). Gains/amplifications (red) are shown on the right side of each ideogram, while losses (blue) appear on the left side. APIP/PDHX/CD44 locus for further analyses are indicated by yellow boxes. ( B ) Localization of the FISH probes specific for APIP/PDHX (labeled with Cy3, red), and CD44 (labeled with FITC, green) located on chromosome 11p13 is depicted. Metaphase FISH analysis of the APIP/PDHX/CD44 locus on chromosome 11p13 in SNU16 cells revealed that two different probes co-localized on the same locus and exist in three types of amplification: DMs ( ), HSRs ( ) and distributed insertions ( ). ( C ) Cohesin-mediated focal copy-number changes of APIP and CD44 were evaluated using a TaqMan-quantitative PCR-based CNV assay with control GFP-KD or RAD21-KD SNU16 cells on day 60 after viral transduction. Data are presented as averages ± SD of biological triplicate independent viral transduction experiments. * P
    Figure Legend Snippet: Copy-numbers of amplified APIP/PDHX/CD44 locus are decreased by cohesin reduction in SNU16 cells. ( A ) High-resolution array-CGH analysis was performed with the control GFP-KD or RAD21-KD SNU16 cells on day 60 after RAD21-KD. Chromosome 11 is represented by ideograms showing G-banding patterns (left ideogram, RAD21-KD SNU16 cells compared with the control GFP-KD cells; right ideogram, parental SNU16 cells compared with normal gastric cells). Gains/amplifications (red) are shown on the right side of each ideogram, while losses (blue) appear on the left side. APIP/PDHX/CD44 locus for further analyses are indicated by yellow boxes. ( B ) Localization of the FISH probes specific for APIP/PDHX (labeled with Cy3, red), and CD44 (labeled with FITC, green) located on chromosome 11p13 is depicted. Metaphase FISH analysis of the APIP/PDHX/CD44 locus on chromosome 11p13 in SNU16 cells revealed that two different probes co-localized on the same locus and exist in three types of amplification: DMs ( ), HSRs ( ) and distributed insertions ( ). ( C ) Cohesin-mediated focal copy-number changes of APIP and CD44 were evaluated using a TaqMan-quantitative PCR-based CNV assay with control GFP-KD or RAD21-KD SNU16 cells on day 60 after viral transduction. Data are presented as averages ± SD of biological triplicate independent viral transduction experiments. * P

    Techniques Used: Amplification, Fluorescence In Situ Hybridization, Labeling, Real-time Polymerase Chain Reaction, CNV Assay, Transduction

    Copy-numbers of amplified gene existing in DMs are decreased by cohesin reduction. ( A ) COLO 320-DM cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P
    Figure Legend Snippet: Copy-numbers of amplified gene existing in DMs are decreased by cohesin reduction. ( A ) COLO 320-DM cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. * P

    Techniques Used: Amplification, Stable Transfection, Transduction, shRNA, Real-time Polymerase Chain Reaction, CNV Assay

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Compartment-Specific Modulation of GABAergic Synaptic Transmission by TRPV1 Channels in the Dentate Gyrus
    Article Snippet: .. Real-time quantitative qPCR was performed with TaqMan probes (Applied Biosystems) for Trpv1 (Rn00583117) and hypoxanthine phosphoribosyltransferase; RN01527838_g1 ( Hprt ). .. Reactions were run in triplicate in a StepOnePlus real-time PCR system (Applied Biosystems). mRNA abundance was calculated by means of the comparative cycle threshold method at a threshold of 0.02.

    other:

    Article Title: Prenatal nicotinic exposure prolongs superior laryngeal C-fiber–mediated apnea and bradycardia through enhancing neuronal TRPV1 expression and excitation
    Article Snippet: In accord with the kit’s instruction manual, after reverse transcription, TRPV1 (Rn00583117_m1) and β-actin (Rn00667869_m1) primers were mixed for preamplification.

    Article Title: Alterations in the Anandamide Metabolism in the Development of Neuropathic Pain
    Article Snippet: The following assays were used: Rn02758689_s1 (Cnr1 ), Rn03993699_s1 (Cnr2 ), Rn00583117_m1 (Trpv1 ), Rn01786262_m1 (Napepld ), Rn00668379_g1 (Pla2g2a ), Rn01488539_m1 (Abhd4 ), Rn00583529_m1 (Gde1 ), Rn01514511_m1 (Plcb1 ), Rn01533758_m1 (Ptpn22 ), Rn01400935_m1 (Inpp5d ), Rn00577086_m1 (Faah ), Rn00568225_m1 (Ptpgs2 ), Rn01461082_m1 (Alox12 ), Rn00696151_m1 (Alox15 ), Rn01527840_m1 (Hprt1 ).

    Expressing:

    Article Title: Computational repositioning of the anticonvulsant topiramate for inflammatory bowel disease
    Article Snippet: .. Gene expression assays had the following accession numbers: 18S (Hs99999901_s1); ALOX5 (Rn00563172_m1); ALOX5AP (Rn00568506_m1); TRPV1 (Rn00583117_m1); CHRM3 (Rn00560986_s1); IFI30 (Rn01420317_m1); MAPK3 (Rn00820922_g1); IL-6 (Rn01410330_m1); IL-10 (Rn00563409_m1). .. Gene Expression levels were calculated using the ΔΔCt method.

    Article Title: Microtopographical features generated by photopolymerization recruit RhoA/ROCK through TRPV1 to direct cell and neurite growth
    Article Snippet: .. Relative standard curve method was used to detect relative mRNA expression levels with TaqMan® Gene Expression Assay for ROCK1 (Life Technologies, Rn00579490_m1), ROCK2 (Life Technologies, Rn00564633_m1), TRPV1 (Life Technologies, Rn00583117_m1), or 18s as endogenous control (Life Technologies, Rn03928990_g1). ..

    Article Title: The prophylactic effects of a traditional Japanese medicine, goshajinkigan, on paclitaxel-induced peripheral neuropathy and its mechanism of action
    Article Snippet: .. TaqMan® Gene Expression Assays (Life Technologies, Forester City, CA) was used for assay: TaqMan® probes of Trpv4 (Rn00583117_m1) and Actb (Rn00667869_m1) were used. .. Primers used for PCR were as follows: TRPV4, 5′-ACCAGTACTATGGCTTCTCC-3′ and 5′-AATTCCCTACTCTACCCTGC-3′.

    Article Title: Prenatal nicotinic exposure augments cardiorespiratory responses to activation of bronchopulmonary C-fibers
    Article Snippet: .. Expression of TRPV1, TrkA, TrkB, and p75NTR gene was detected using TaqMan Gene Expression Assays from Life Technologies (Grand Island, NY); TRPV1: Rn00583117_m1, TrkA: Rn00572130_m1, TrkB: Rn01441749_m1, and p75NTR : Rn00561634_m1. .. Endogenous GAPDH (Rn01775763_g1) was also monitored for each assay.

    Quantitative RT-PCR:

    Article Title: Effects of vitamin D on kidney histology and trpv1 channels in doxorubicin-induced nephropathy
    Article Snippet: .. Primer and probe oligonucleotides used for real-time quantitative RT-PCR were purchased Applied Biosystems (Cat no: Rn 00583117_m1) (Applied Biosystems, Foster City, CA). .. The samples were quantified for TRPV1 gene using the comparative Ct (DDCt) method, as described in the Assays-on-Demand Users Manual (Applied Biosystems, Foster City, CA).

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    Thermo Fisher real time pcr system
    Genotyping assay for the detection of the mutant allele (with the BRCA1 ex9-12del mutation) and the wild-type allele (exon 11) by real-time <t>PCR</t> and <t>TaqMan</t> ® probes. The AD plot shows 2 groups; green color samples have the allele with the deletion, samples in red color possess exon 11 or the wild-type allele (in this group all the samples are present, the 6 negative and the 4 positive ones). Samples in blue color are negative samples for the deletion, which have no fluorescence and are classified by the software as negatives. Negative controls are shown in black squares. Each sample was processed by duplicate. The axis values represent relative fluorescence (∆Rn) between both dyes (FAM ™ for the mutant allele and HEX ™ for the wild-type allele). PCR, polymerase chain reaction; AD, allelic discrimination.
    Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/real time pcr system/product/Thermo Fisher
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    real time pcr system - by Bioz Stars, 2020-07
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    93
    Thermo Fisher applied biosystemstm thermo fisher scientific steponeplustm real time pcr system
    Genotyping assay for the detection of the mutant allele (with the BRCA1 ex9-12del mutation) and the wild-type allele (exon 11) by real-time <t>PCR</t> and <t>TaqMan</t> ® probes. The AD plot shows 2 groups; green color samples have the allele with the deletion, samples in red color possess exon 11 or the wild-type allele (in this group all the samples are present, the 6 negative and the 4 positive ones). Samples in blue color are negative samples for the deletion, which have no fluorescence and are classified by the software as negatives. Negative controls are shown in black squares. Each sample was processed by duplicate. The axis values represent relative fluorescence (∆Rn) between both dyes (FAM ™ for the mutant allele and HEX ™ for the wild-type allele). PCR, polymerase chain reaction; AD, allelic discrimination.
    Applied Biosystemstm Thermo Fisher Scientific Steponeplustm Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/applied biosystemstm thermo fisher scientific steponeplustm real time pcr system/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    applied biosystemstm thermo fisher scientific steponeplustm real time pcr system - by Bioz Stars, 2020-07
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    91
    Thermo Fisher applied biosystems steponeplus
    Genotyping assay for the detection of the mutant allele (with the BRCA1 ex9-12del mutation) and the wild-type allele (exon 11) by real-time <t>PCR</t> and <t>TaqMan</t> ® probes. The AD plot shows 2 groups; green color samples have the allele with the deletion, samples in red color possess exon 11 or the wild-type allele (in this group all the samples are present, the 6 negative and the 4 positive ones). Samples in blue color are negative samples for the deletion, which have no fluorescence and are classified by the software as negatives. Negative controls are shown in black squares. Each sample was processed by duplicate. The axis values represent relative fluorescence (∆Rn) between both dyes (FAM ™ for the mutant allele and HEX ™ for the wild-type allele). PCR, polymerase chain reaction; AD, allelic discrimination.
    Applied Biosystems Steponeplus, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/applied biosystems steponeplus/product/Thermo Fisher
    Average 91 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    applied biosystems steponeplus - by Bioz Stars, 2020-07
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    Genotyping assay for the detection of the mutant allele (with the BRCA1 ex9-12del mutation) and the wild-type allele (exon 11) by real-time PCR and TaqMan ® probes. The AD plot shows 2 groups; green color samples have the allele with the deletion, samples in red color possess exon 11 or the wild-type allele (in this group all the samples are present, the 6 negative and the 4 positive ones). Samples in blue color are negative samples for the deletion, which have no fluorescence and are classified by the software as negatives. Negative controls are shown in black squares. Each sample was processed by duplicate. The axis values represent relative fluorescence (∆Rn) between both dyes (FAM ™ for the mutant allele and HEX ™ for the wild-type allele). PCR, polymerase chain reaction; AD, allelic discrimination.

    Journal: Molecular Medicine Reports

    Article Title: A novel method to detect the Mexican founder mutation BRCA1 ex9-12del associated with breast and ovarian cancer using quantitative polymerase chain reaction and TaqMan® probes

    doi: 10.3892/mmr.2018.9141

    Figure Lengend Snippet: Genotyping assay for the detection of the mutant allele (with the BRCA1 ex9-12del mutation) and the wild-type allele (exon 11) by real-time PCR and TaqMan ® probes. The AD plot shows 2 groups; green color samples have the allele with the deletion, samples in red color possess exon 11 or the wild-type allele (in this group all the samples are present, the 6 negative and the 4 positive ones). Samples in blue color are negative samples for the deletion, which have no fluorescence and are classified by the software as negatives. Negative controls are shown in black squares. Each sample was processed by duplicate. The axis values represent relative fluorescence (∆Rn) between both dyes (FAM ™ for the mutant allele and HEX ™ for the wild-type allele). PCR, polymerase chain reaction; AD, allelic discrimination.

    Article Snippet: qPCR with designed primers and TaqMan® probes Thermocycler StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Inc.) was used to perform the qPCR with TaqMan® probes.

    Techniques: Genotyping Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Fluorescence, Software, Polymerase Chain Reaction

    Detection of the BRCA1 ex9-12del mutation by real-time PCR and TaqMan ® probe. Red amplification curves represent the fluorescent signal given by the FAM ™ dye from the TaqMan ® probe detecting the variant allele in the four positive samples, which had a mean Ct of 27.46. No amplification curve is observed for the negative samples neither for negative controls (horizontal red lines). The ‘Y’ axis values represent the fluorescent signal normalized of the FAM ™ dye with the ROX, whereas the ‘X’ axis represent number of cycles. PCR. PCR, polymerase chain reaction; ROX, passive reference dye.

    Journal: Molecular Medicine Reports

    Article Title: A novel method to detect the Mexican founder mutation BRCA1 ex9-12del associated with breast and ovarian cancer using quantitative polymerase chain reaction and TaqMan® probes

    doi: 10.3892/mmr.2018.9141

    Figure Lengend Snippet: Detection of the BRCA1 ex9-12del mutation by real-time PCR and TaqMan ® probe. Red amplification curves represent the fluorescent signal given by the FAM ™ dye from the TaqMan ® probe detecting the variant allele in the four positive samples, which had a mean Ct of 27.46. No amplification curve is observed for the negative samples neither for negative controls (horizontal red lines). The ‘Y’ axis values represent the fluorescent signal normalized of the FAM ™ dye with the ROX, whereas the ‘X’ axis represent number of cycles. PCR. PCR, polymerase chain reaction; ROX, passive reference dye.

    Article Snippet: qPCR with designed primers and TaqMan® probes Thermocycler StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Inc.) was used to perform the qPCR with TaqMan® probes.

    Techniques: Mutagenesis, Real-time Polymerase Chain Reaction, Amplification, Variant Assay, Polymerase Chain Reaction

    p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Azacitidine mitigates GvHD via differential effects on the proliferation of T effectors and nTregs in vivo

    doi: 10.4049/jimmunol.1502399

    Figure Lengend Snippet: p21-/- attenuates the antiproliferative effects of AzaC (a) p21 mRNA is significantly upregulated in CD4+ and CD8+ Teff following treatment with AzaC. Teffs were isolated from the spleens of B6. Foxp3 GFP × B6.CAG DSRED and nTregs were isolated from B6. Foxp3 GFP . Cells were co-cultured at a 1:10 ratio of nTregs to Teffs for 2 days in the presence of anti-CD3/CD28 beads (bead:cell 1:1; Invitrogen) and Xcyte medium supplemented with L-glutamine (4 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and human recombinant IL-2 (hIL-2; 500 U/mL). The activated T cells were cultured with AzaC (1 μM) or PBS for an additional 2 days. Cells were sorted using FACS Aria II (BD) to isolate nTregs (CD4+DSRED-FOXP3GFP+), CD4+ Teffs (CD4+DSRed+FOXP3GFP-), and CD8+ Teffs (CD8+DSRed+FOXP3GFP-) prior to RNA extraction. QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System using pre-designed TaqMan® Gene Expression Assays (18S RNA Mm03928990 and p21 Mm04205640). Relative fold changes in expression were determined using the ΔΔCT method. AzaC treatment resulted in a 3.4 fold increase of p21 expression in CD4+ Teffs (FACS sorted to remove AzaC converted Tregs) (AzaC vs. PBS p

    Article Snippet: QPCR was performed on the Applied Biosystems StepOnePlus Real-Time System (Thermo fisher) using pre-designed TaqMan® Gene Expression Assays (Life Technologies) (18S RNA Mm03928990 and p21 Mm04205640) according to manufacturer's instructions.

    Techniques: Isolation, Cell Culture, Recombinant, FACS, RNA Extraction, Real-time Polymerase Chain Reaction, Expressing