flow cytometry sorting  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85

    Structured Review

    Thermo Fisher flow cytometry sorting
    Nlrc3 -/- mice are protected from M . tuberculosis infection. WT and Nlrc3 -/- mice infected with approximately 200 colony-forming units (c.f.u.) of M . tuberculosis were monitored. (A) Bacterial burdens were determined after infection at 3 and 6 w.p.i.. (B) Bacterial burdens were determined after infection at 3 w.p.i.. (C) Frequencies of lung-infiltrating cells that are neutrophils (CD11b + Gr-1 + ) or monocyte-macrophages (CD11b + Gr-1 - ) at 3 w.p.i.. (D) Expressions of CD86, MHC-II and CD206 were detected on monocyte-macrophages (CD11b + Gr-1 - ) via flow <t>cytometry</t> at 3 w.p.i.. (E) ROS production by monocyte-macrophages (CD11b + Gr-1 - ) were detected assessed as mean fluorescence intensity (MFI) of intracellular CFDA. (F) Concentrations of nitrate were measured by nitrate reductase assay and concentrations of IL-6 and IL-1β in lungs (homogenized in 2 ml PBS and 0.05% Tween 80) were detected by ELISA at 3 w.p.i.. Data shown in are the mean ±SD. * P
    Flow Cytometry Sorting, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 20499 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flow cytometry sorting/product/Thermo Fisher
    Average 85 stars, based on 20499 article reviews
    Price from $9.99 to $1999.99
    flow cytometry sorting - by Bioz Stars, 2020-07
    85/100 stars

    Images

    1) Product Images from "NLRC3 negatively regulates CD4+ T cells and impacts protective immunity during Mycobacterium tuberculosis infection"

    Article Title: NLRC3 negatively regulates CD4+ T cells and impacts protective immunity during Mycobacterium tuberculosis infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007266

    Nlrc3 -/- mice are protected from M . tuberculosis infection. WT and Nlrc3 -/- mice infected with approximately 200 colony-forming units (c.f.u.) of M . tuberculosis were monitored. (A) Bacterial burdens were determined after infection at 3 and 6 w.p.i.. (B) Bacterial burdens were determined after infection at 3 w.p.i.. (C) Frequencies of lung-infiltrating cells that are neutrophils (CD11b + Gr-1 + ) or monocyte-macrophages (CD11b + Gr-1 - ) at 3 w.p.i.. (D) Expressions of CD86, MHC-II and CD206 were detected on monocyte-macrophages (CD11b + Gr-1 - ) via flow cytometry at 3 w.p.i.. (E) ROS production by monocyte-macrophages (CD11b + Gr-1 - ) were detected assessed as mean fluorescence intensity (MFI) of intracellular CFDA. (F) Concentrations of nitrate were measured by nitrate reductase assay and concentrations of IL-6 and IL-1β in lungs (homogenized in 2 ml PBS and 0.05% Tween 80) were detected by ELISA at 3 w.p.i.. Data shown in are the mean ±SD. * P
    Figure Legend Snippet: Nlrc3 -/- mice are protected from M . tuberculosis infection. WT and Nlrc3 -/- mice infected with approximately 200 colony-forming units (c.f.u.) of M . tuberculosis were monitored. (A) Bacterial burdens were determined after infection at 3 and 6 w.p.i.. (B) Bacterial burdens were determined after infection at 3 w.p.i.. (C) Frequencies of lung-infiltrating cells that are neutrophils (CD11b + Gr-1 + ) or monocyte-macrophages (CD11b + Gr-1 - ) at 3 w.p.i.. (D) Expressions of CD86, MHC-II and CD206 were detected on monocyte-macrophages (CD11b + Gr-1 - ) via flow cytometry at 3 w.p.i.. (E) ROS production by monocyte-macrophages (CD11b + Gr-1 - ) were detected assessed as mean fluorescence intensity (MFI) of intracellular CFDA. (F) Concentrations of nitrate were measured by nitrate reductase assay and concentrations of IL-6 and IL-1β in lungs (homogenized in 2 ml PBS and 0.05% Tween 80) were detected by ELISA at 3 w.p.i.. Data shown in are the mean ±SD. * P

    Techniques Used: Mouse Assay, Infection, Flow Cytometry, Cytometry, Fluorescence, Reductase Assay, Enzyme-linked Immunosorbent Assay

    2) Product Images from "Direct and indirect pro-inflammatory cytokine response resulting from TC-83 infection of glial cells"

    Article Title: Direct and indirect pro-inflammatory cytokine response resulting from TC-83 infection of glial cells

    Journal: Virulence

    doi: 10.1080/21505594.2018.1509668

    IL-1β released by infected microglial cells prime astrocytes for increased TC-83 uptake. (a) Experimental workflow indicating the pre-treatment and infection schematic for HMC3 cells. Supernatants were removed from TC-83 inoculated HMC3 cells and overlaid onto naïve U-87 MG cells prior to inoculation of the astrocytes with TC-83 (MOI:2). (b) Production of infectious virus in these treatment conditions was measured at 6, 12, and 18hpi by plaque assay. (c-d) To determine which pro-inflammatory cytokines are contributing to the increase in TC-83 uptake, astrocytes were pre-treated with biologically relevant doses of IL-1α, IL-1β, IL-6, or IL-8 and production of infectious virus measured at 6, 12, and 18hpi by plaque assay. (e) Supernatants from HMC3 cells pre-treated with 100pg/mL IL-1β were overlaid on naïve U-87 MG astrocytes prior to TC-83 infection (MOI:2). Plaque assay measured production of infectious virus at 6, 12, and 18hpi. (f) The infection scheme from (a) was repeated in the context of 100µM Pirfenidone, a potent IL-1β inhibitor. (g) IL-1β gene expression was measured in the context of direct infected U-87 MG and HMC3 cells, and 1h supernatants from infected U-87 MG cells overlaid on naïve HMC3 microglia. Gene induction was determined via the ΔΔCτ method with 18S as the endogenous control. The quantitative data are depicted as the means of three independent experiments ± SD. For all images, representative data were selectively obtained from three individual experiments. * p
    Figure Legend Snippet: IL-1β released by infected microglial cells prime astrocytes for increased TC-83 uptake. (a) Experimental workflow indicating the pre-treatment and infection schematic for HMC3 cells. Supernatants were removed from TC-83 inoculated HMC3 cells and overlaid onto naïve U-87 MG cells prior to inoculation of the astrocytes with TC-83 (MOI:2). (b) Production of infectious virus in these treatment conditions was measured at 6, 12, and 18hpi by plaque assay. (c-d) To determine which pro-inflammatory cytokines are contributing to the increase in TC-83 uptake, astrocytes were pre-treated with biologically relevant doses of IL-1α, IL-1β, IL-6, or IL-8 and production of infectious virus measured at 6, 12, and 18hpi by plaque assay. (e) Supernatants from HMC3 cells pre-treated with 100pg/mL IL-1β were overlaid on naïve U-87 MG astrocytes prior to TC-83 infection (MOI:2). Plaque assay measured production of infectious virus at 6, 12, and 18hpi. (f) The infection scheme from (a) was repeated in the context of 100µM Pirfenidone, a potent IL-1β inhibitor. (g) IL-1β gene expression was measured in the context of direct infected U-87 MG and HMC3 cells, and 1h supernatants from infected U-87 MG cells overlaid on naïve HMC3 microglia. Gene induction was determined via the ΔΔCτ method with 18S as the endogenous control. The quantitative data are depicted as the means of three independent experiments ± SD. For all images, representative data were selectively obtained from three individual experiments. * p

    Techniques Used: Infection, Plaque Assay, Expressing

    3) Product Images from "miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway"

    Article Title: miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway

    Journal: Biological Research

    doi: 10.1186/s40659-018-0179-2

    miR-214 attenuated NRK-52E cell apoptosis under hypoxia. a qRT-PCR analysis of miR-214 expression in kidney cortex of I/R-induced rat AKI models after 12, 48 and 72 h of reperfusion. n = 6 rats/group. b qRT-PCR analysis of miR-214 expression at 0, 3 h, 6 h, and 9 h during hypoxic incubation of cultured NRK-52E cells. c qRT-PCR analysis of miR-214 expression in NRK-52E cells after transfection with miR-214, anti-miR-214, or matched controls. d–e Flow cytometry was used to analyze the apoptotic rate of NRK-52E cells after transfection with miR-214, anti-miR-214, or respective controls during hypoxia incubation. f Western blot was performed to detect the protein levels of Bcl-2 and Bax in NRK-52E cells after introduction with miR-214, anti-miR-214, or corresponding controls under hypoxia. Each experiment was independently repeated 3 times. * P
    Figure Legend Snippet: miR-214 attenuated NRK-52E cell apoptosis under hypoxia. a qRT-PCR analysis of miR-214 expression in kidney cortex of I/R-induced rat AKI models after 12, 48 and 72 h of reperfusion. n = 6 rats/group. b qRT-PCR analysis of miR-214 expression at 0, 3 h, 6 h, and 9 h during hypoxic incubation of cultured NRK-52E cells. c qRT-PCR analysis of miR-214 expression in NRK-52E cells after transfection with miR-214, anti-miR-214, or matched controls. d–e Flow cytometry was used to analyze the apoptotic rate of NRK-52E cells after transfection with miR-214, anti-miR-214, or respective controls during hypoxia incubation. f Western blot was performed to detect the protein levels of Bcl-2 and Bax in NRK-52E cells after introduction with miR-214, anti-miR-214, or corresponding controls under hypoxia. Each experiment was independently repeated 3 times. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Incubation, Cell Culture, Transfection, Flow Cytometry, Cytometry, Western Blot

    Dkk3 was a target of miR-214 in NRK-52E cells. a The predictive wild-type miR-214 binding sites in the 3′UTR of Dkk3 and the corresponding mutant binding sites were displayed. b The relative luciferase activity was measured by luciferase reporter assay after NRK-52E cells were cotransfected with Dkk3 -WT or Dkk3 -MUT and miR-214 or miR-con. The mRNA ( c ) and protein ( d , e ) levels of DKK3 were examined by qRT-PCR and western blot in NRK-52E cells introduced with miR-214, anti-miR-214, or matched controls. Each experiment was independently repeated 3 times. * P
    Figure Legend Snippet: Dkk3 was a target of miR-214 in NRK-52E cells. a The predictive wild-type miR-214 binding sites in the 3′UTR of Dkk3 and the corresponding mutant binding sites were displayed. b The relative luciferase activity was measured by luciferase reporter assay after NRK-52E cells were cotransfected with Dkk3 -WT or Dkk3 -MUT and miR-214 or miR-con. The mRNA ( c ) and protein ( d , e ) levels of DKK3 were examined by qRT-PCR and western blot in NRK-52E cells introduced with miR-214, anti-miR-214, or matched controls. Each experiment was independently repeated 3 times. * P

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

    Evaluation of rat AKI model following I/R surgery and NRK-52E cell model following hypoxia treatment. The serum levels of SCr ( a ), BUN ( b ), and urine Kim-1 ( c ) in I/R-induced rat AKI models at 24 h after surgery were measured. d The mRNA expressions of TNF-α, IL-1β, and IL-6 in hypoxia-induced NRK-52E cell I/R model were detected by qRT-PCR. e The protein levels of Bcl-2 and Bax in hypoxia-induced NRK-52E cell I/R models were assessed by western blot. n = 6 rats/group. * P
    Figure Legend Snippet: Evaluation of rat AKI model following I/R surgery and NRK-52E cell model following hypoxia treatment. The serum levels of SCr ( a ), BUN ( b ), and urine Kim-1 ( c ) in I/R-induced rat AKI models at 24 h after surgery were measured. d The mRNA expressions of TNF-α, IL-1β, and IL-6 in hypoxia-induced NRK-52E cell I/R model were detected by qRT-PCR. e The protein levels of Bcl-2 and Bax in hypoxia-induced NRK-52E cell I/R models were assessed by western blot. n = 6 rats/group. * P

    Techniques Used: Quantitative RT-PCR, Western Blot

    miR-214 protected against AKI in vivo through targeting Dkk3 and activating Wnt/β-catenin pathway. miR-214 or miR-con was intraperitoneally injected into mice, followed by ischemic surgery. a qRT-PCR analysis of miR-214 expression in I/R-induced rat AKI model and sham mice. The serum levels of BUN ( b ), SCr ( c ), and Urine Kim-1 ( d ) level in I/R-induced rat AKI model and sham mice. e Western blot analysis of Bcl-2, Bax and fibronectin in I/R-induced rat AKI model and sham group. f The protein levels of DKK3, β-catenin, c-myc, and cyclin D1 in I/R-induced rat AKI model and sham group. Each experiment was independently repeated 3 times. * P
    Figure Legend Snippet: miR-214 protected against AKI in vivo through targeting Dkk3 and activating Wnt/β-catenin pathway. miR-214 or miR-con was intraperitoneally injected into mice, followed by ischemic surgery. a qRT-PCR analysis of miR-214 expression in I/R-induced rat AKI model and sham mice. The serum levels of BUN ( b ), SCr ( c ), and Urine Kim-1 ( d ) level in I/R-induced rat AKI model and sham mice. e Western blot analysis of Bcl-2, Bax and fibronectin in I/R-induced rat AKI model and sham group. f The protein levels of DKK3, β-catenin, c-myc, and cyclin D1 in I/R-induced rat AKI model and sham group. Each experiment was independently repeated 3 times. * P

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

    4) Product Images from "Phosphatase activity of the control of virulence sensor kinase CovS is critical for the pathogenesis of group A streptococcus"

    Article Title: Phosphatase activity of the control of virulence sensor kinase CovS is critical for the pathogenesis of group A streptococcus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007354

    Effect of CovRS mutations selected during skin/soft tissue infection on CovR regulatory function. ( A ) CovR~P levels of indicated strains grown to mid-exponential phase in THY measured by Phostag-Western blot analysis (n = 2). (B-D) Transcript levels (means ± standard deviations; n = 4) of the indicated genes in the CovR/S-deleted or mutated strains relative to those of the wild type, as measured by TaqMan qRT-PCR. Strains were grown in THY to mid-exponential phase. * = P
    Figure Legend Snippet: Effect of CovRS mutations selected during skin/soft tissue infection on CovR regulatory function. ( A ) CovR~P levels of indicated strains grown to mid-exponential phase in THY measured by Phostag-Western blot analysis (n = 2). (B-D) Transcript levels (means ± standard deviations; n = 4) of the indicated genes in the CovR/S-deleted or mutated strains relative to those of the wild type, as measured by TaqMan qRT-PCR. Strains were grown in THY to mid-exponential phase. * = P

    Techniques Used: Infection, Western Blot, Quantitative RT-PCR

    Influence of CovR~P on transcript levels of genes representing distinct classes. ( A ) Schematic depiction of CovR~P levels in the indicated GAS strains. Values were derived from previous Phostag-Western blot analyses of CovR~P status. ( B-D ) Transcript levels (means ± standard deviations; n = 4) of indicated genes that are representative for distinct gene classes in the isoallelic GAS strains relative to those of the wild type, as measured by TaqMan qRT-PCR. Strains were grown in THY to late-exponential phase.
    Figure Legend Snippet: Influence of CovR~P on transcript levels of genes representing distinct classes. ( A ) Schematic depiction of CovR~P levels in the indicated GAS strains. Values were derived from previous Phostag-Western blot analyses of CovR~P status. ( B-D ) Transcript levels (means ± standard deviations; n = 4) of indicated genes that are representative for distinct gene classes in the isoallelic GAS strains relative to those of the wild type, as measured by TaqMan qRT-PCR. Strains were grown in THY to late-exponential phase.

    Techniques Used: Derivative Assay, Western Blot, Quantitative RT-PCR

    5) Product Images from "Deciphering the complex role of thrombospondin-1 in glioblastoma development"

    Article Title: Deciphering the complex role of thrombospondin-1 in glioblastoma development

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08480-y

    THBS1/CD47 interaction in P3 tumour invasion and growth. a P3 cells were included into collagen I gels and then incubated in normoxia (21 % O 2 ) or hypoxia (1 % O 2 ). P3 spheroid invasion was measured in collagen I gels after 24 h. Scale: 50 µm. The graph represents the results as means ± s.d. of three independent experiments, each done in 6–8 replicates for each condition. * P
    Figure Legend Snippet: THBS1/CD47 interaction in P3 tumour invasion and growth. a P3 cells were included into collagen I gels and then incubated in normoxia (21 % O 2 ) or hypoxia (1 % O 2 ). P3 spheroid invasion was measured in collagen I gels after 24 h. Scale: 50 µm. The graph represents the results as means ± s.d. of three independent experiments, each done in 6–8 replicates for each condition. * P

    Techniques Used: Incubation

    Proposed model for GBM invasion TGFβ1 is expressed in both the core and the invasive areas in GBM. THBS1 is transcriptionally regulated via SMAD3, which binds to regulatory elements in the THBS1 gene. THBS1 will then be released and act on tumour cell invasion and expansion. The interaction with CD47 is critical in this process
    Figure Legend Snippet: Proposed model for GBM invasion TGFβ1 is expressed in both the core and the invasive areas in GBM. THBS1 is transcriptionally regulated via SMAD3, which binds to regulatory elements in the THBS1 gene. THBS1 will then be released and act on tumour cell invasion and expansion. The interaction with CD47 is critical in this process

    Techniques Used: Activated Clotting Time Assay

    Tumour-associated CD47 controls glioma cell invasion and motility. a Immunoblots of protein extracts from control or CD47-1/-2 shRNA-transduced P3 cells probed with anti-CD47 or anti-Tubulin antibodies. b P3 cells were transduced with control or CD47 (−1 and −2) shRNAs. P3 spheroid invasion was measured in collagen I gels after 24 h. Scale: 50 µm. The graph below represents the results as means ± s.d. of three independent experiments each done in 6–8 replicates for each condition. ** P
    Figure Legend Snippet: Tumour-associated CD47 controls glioma cell invasion and motility. a Immunoblots of protein extracts from control or CD47-1/-2 shRNA-transduced P3 cells probed with anti-CD47 or anti-Tubulin antibodies. b P3 cells were transduced with control or CD47 (−1 and −2) shRNAs. P3 spheroid invasion was measured in collagen I gels after 24 h. Scale: 50 µm. The graph below represents the results as means ± s.d. of three independent experiments each done in 6–8 replicates for each condition. ** P

    Techniques Used: Western Blot, shRNA, Transduction

    6) Product Images from "MicroRNA-23a-3p Inhibits Mucosal Melanoma Growth and Progression through Targeting Adenylate Cyclase 1 and Attenuating cAMP and MAPK Pathways"

    Article Title: MicroRNA-23a-3p Inhibits Mucosal Melanoma Growth and Progression through Targeting Adenylate Cyclase 1 and Attenuating cAMP and MAPK Pathways

    Journal: Theranostics

    doi: 10.7150/thno.30516

    ADCY1 knockdown phenocopies the effects of miR-23a-3p overexpression in vitro . (A) HMVII and VMRC-MELG cells were transfected with two ADCY1 siRNAs, and cell viability was measured using the CellTiter-Glo assay. (B) Photos of colony formation of HMVII and VMRC-MELG cells infected with ADCY1-specific shRNA (Lenti-shADCY1) or control construct (Lenti-shCtrl). Data are presented as the mean ± SD (n = 3). (C) HMVII and VMRC-MELG cells were stained with PI at 24 and 48 h after transfection with ADCY1 siRNAs, and cell cycle distribution was analyzed by flow cytometry. (D-E) Migration and invasion abilities of HMVII and VMRC-MELG cells transfected with two ADCY1-specific siRNAs were assessed using wound-healing (D) and Transwell (E) assays. (F-G) HMVII cells stably transfected with ADCY1-expression plasmid were transfected with miR-23a-3p mimics or miR-Ctrl, and cell viability was analyzed by CellTiter-Glo assay (F) and cell cycle distribution (G) was analyzed by flow cytometry. * P
    Figure Legend Snippet: ADCY1 knockdown phenocopies the effects of miR-23a-3p overexpression in vitro . (A) HMVII and VMRC-MELG cells were transfected with two ADCY1 siRNAs, and cell viability was measured using the CellTiter-Glo assay. (B) Photos of colony formation of HMVII and VMRC-MELG cells infected with ADCY1-specific shRNA (Lenti-shADCY1) or control construct (Lenti-shCtrl). Data are presented as the mean ± SD (n = 3). (C) HMVII and VMRC-MELG cells were stained with PI at 24 and 48 h after transfection with ADCY1 siRNAs, and cell cycle distribution was analyzed by flow cytometry. (D-E) Migration and invasion abilities of HMVII and VMRC-MELG cells transfected with two ADCY1-specific siRNAs were assessed using wound-healing (D) and Transwell (E) assays. (F-G) HMVII cells stably transfected with ADCY1-expression plasmid were transfected with miR-23a-3p mimics or miR-Ctrl, and cell viability was analyzed by CellTiter-Glo assay (F) and cell cycle distribution (G) was analyzed by flow cytometry. * P

    Techniques Used: Over Expression, In Vitro, Transfection, Glo Assay, Infection, shRNA, Construct, Staining, Flow Cytometry, Cytometry, Migration, Stable Transfection, Expressing, Plasmid Preparation

    miR-23a-3p inhibits the cAMP and MAPK pathways in mucosal melanoma through ADCY1. HMVII and VMRC-MELG cells were transfected with miR-23a-3p mimics or two ADCY1-specific siRNAs and their corresponding controls as described. (A) Intracellular cAMP content was measured using the Cyclic AMP XP Chemiluminescent Assay and was normalized to the level in control-treated cells. (B-C) PKA activity was detected using a PepTag assay. Representative photographs are shown and PKA activities were normalized to those in control-treated cells. (D-E) Immunoblot analysis of the phosphorylation and expression of cAMP and MAPK pathway-associated downstream factors 48 h after treatment with miR-23a-3p mimic (D) or ADCY1 siRNAs (E). GAPDH served as a loading control.
    Figure Legend Snippet: miR-23a-3p inhibits the cAMP and MAPK pathways in mucosal melanoma through ADCY1. HMVII and VMRC-MELG cells were transfected with miR-23a-3p mimics or two ADCY1-specific siRNAs and their corresponding controls as described. (A) Intracellular cAMP content was measured using the Cyclic AMP XP Chemiluminescent Assay and was normalized to the level in control-treated cells. (B-C) PKA activity was detected using a PepTag assay. Representative photographs are shown and PKA activities were normalized to those in control-treated cells. (D-E) Immunoblot analysis of the phosphorylation and expression of cAMP and MAPK pathway-associated downstream factors 48 h after treatment with miR-23a-3p mimic (D) or ADCY1 siRNAs (E). GAPDH served as a loading control.

    Techniques Used: Transfection, Activity Assay, Expressing

    ADCY1 is a direct target of miR-23a-3p. (A) Correlation between ADCY1 mRNA and miR-23a-3p in 20 fresh-frozen MM tissues. Expression of ADCY1 mRNA and miR-23a-3p was measured by qRT-PCR and was normalized to the mean level in three normal mucosal nevi. GAPDH and U6 were used as endogenous controls, respectively. (B) Correlation between ADCY1 protein and miR-23a-3p in 117 FFPE samples. (C) Representative IHC staining images of ADCY1 in patients with low and high miR-23a-3p expression. Bar, 100 mm. (D) Putative miR-23a-3p interaction sites within the 3'-UTR of ADCY1 mRNA are indicated by boxes. Mutations were generated in the complementary site of the ADCY1 3´-UTR for the seed sequences of miR-23a-3p as shown in capital letters. (E) The full length of ADCY1 3'-UTR with putative wild-type (WT) miR-23a-3p-binding site or a binding site mutated at the 3'-UTR region (Mut) were cloned downstream of the CMV promoter in the pMIR-REPORT vector. Luciferase activity was measured after co-transfection of the reporter constructs with WT- or Mut-interacting sites of ADCY1 and miR-23a-3p mimic or miR-Ctrl in HEK293T cells. Data are presented as the mean ± SD of three independent experiments. (F) ADCY1 transcripts level at 48 h after transfection with miR-23a-3p and control as determined by qRT-PCR. GAPDH was used as an internal control. (G) HMVII and VMRC-MELG cells were transfected with miR-23a-3p mimic or inhibitor and the corresponding controls. ADCY1 protein expression after 48 h of transfection was detected by western blot analysis. Band intensities were quantified using ImageJ. (H-J) Tissue sections from subcutaneous xenografts (H), lung metastasis (I), and intra-abdominal metastasis (J) were stained with H E, ADCY1 and Ki-67. Bar, 50 μm. * P
    Figure Legend Snippet: ADCY1 is a direct target of miR-23a-3p. (A) Correlation between ADCY1 mRNA and miR-23a-3p in 20 fresh-frozen MM tissues. Expression of ADCY1 mRNA and miR-23a-3p was measured by qRT-PCR and was normalized to the mean level in three normal mucosal nevi. GAPDH and U6 were used as endogenous controls, respectively. (B) Correlation between ADCY1 protein and miR-23a-3p in 117 FFPE samples. (C) Representative IHC staining images of ADCY1 in patients with low and high miR-23a-3p expression. Bar, 100 mm. (D) Putative miR-23a-3p interaction sites within the 3'-UTR of ADCY1 mRNA are indicated by boxes. Mutations were generated in the complementary site of the ADCY1 3´-UTR for the seed sequences of miR-23a-3p as shown in capital letters. (E) The full length of ADCY1 3'-UTR with putative wild-type (WT) miR-23a-3p-binding site or a binding site mutated at the 3'-UTR region (Mut) were cloned downstream of the CMV promoter in the pMIR-REPORT vector. Luciferase activity was measured after co-transfection of the reporter constructs with WT- or Mut-interacting sites of ADCY1 and miR-23a-3p mimic or miR-Ctrl in HEK293T cells. Data are presented as the mean ± SD of three independent experiments. (F) ADCY1 transcripts level at 48 h after transfection with miR-23a-3p and control as determined by qRT-PCR. GAPDH was used as an internal control. (G) HMVII and VMRC-MELG cells were transfected with miR-23a-3p mimic or inhibitor and the corresponding controls. ADCY1 protein expression after 48 h of transfection was detected by western blot analysis. Band intensities were quantified using ImageJ. (H-J) Tissue sections from subcutaneous xenografts (H), lung metastasis (I), and intra-abdominal metastasis (J) were stained with H E, ADCY1 and Ki-67. Bar, 50 μm. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Staining, Generated, Binding Assay, Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Cotransfection, Construct, Transfection, Western Blot

    ADCY1 knockdown phenocopies the effects of miR-23a-3p overexpression in vivo . (A) HMVII cells stably transfected with shADCY1 were subcutaneously injected into NOD/SCID mice, the tumor volumes were monitored, and tumor weights were measured after the mice were sacrificed on day 30. (B-D) HMVII cells stably transfected with shADCY1 were injected via the lateral tail vein into NOD/SCID mice, metastatic colonies were counted and quantitatively analyzed, and representative lung (B) and intra-abdominal (D) metastases images are shown. * P
    Figure Legend Snippet: ADCY1 knockdown phenocopies the effects of miR-23a-3p overexpression in vivo . (A) HMVII cells stably transfected with shADCY1 were subcutaneously injected into NOD/SCID mice, the tumor volumes were monitored, and tumor weights were measured after the mice were sacrificed on day 30. (B-D) HMVII cells stably transfected with shADCY1 were injected via the lateral tail vein into NOD/SCID mice, metastatic colonies were counted and quantitatively analyzed, and representative lung (B) and intra-abdominal (D) metastases images are shown. * P

    Techniques Used: Over Expression, In Vivo, Stable Transfection, Transfection, Injection, Mouse Assay

    7) Product Images from "Arx Expression Suppresses Ventralization of the Developing Dorsal Forebrain"

    Article Title: Arx Expression Suppresses Ventralization of the Developing Dorsal Forebrain

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36194-6

    GLI3R can repress Olig2 induction in Arx cKO/y cortex. ( a ) Representative images of the E14.5 Arx cKO/y cortex electroporated with a control GFP ( pCIG )or Gli3R expression construct ( pCIG-Gli3R ) at E12.5 and double immunolabeled with GFP (green, EPed cells) and OLIG2 (red). The lower panels are magnified images of the boxed areas in the upper panels. Dotted lines mark the outlines of the sections. ( b ) Quantification of results in ( a ). The ratio of OLIG2+ GFP+ cells over GFP+ cells was plotted for both GFP and Gli3R electroporated samples. Error bars: mean ± s.d (n = 4 sections from two embryos per genotype; **P = 0.0034; unpaired t-test). CTX, embryonic neocortex; GE, ganglionic eminence.
    Figure Legend Snippet: GLI3R can repress Olig2 induction in Arx cKO/y cortex. ( a ) Representative images of the E14.5 Arx cKO/y cortex electroporated with a control GFP ( pCIG )or Gli3R expression construct ( pCIG-Gli3R ) at E12.5 and double immunolabeled with GFP (green, EPed cells) and OLIG2 (red). The lower panels are magnified images of the boxed areas in the upper panels. Dotted lines mark the outlines of the sections. ( b ) Quantification of results in ( a ). The ratio of OLIG2+ GFP+ cells over GFP+ cells was plotted for both GFP and Gli3R electroporated samples. Error bars: mean ± s.d (n = 4 sections from two embryos per genotype; **P = 0.0034; unpaired t-test). CTX, embryonic neocortex; GE, ganglionic eminence.

    Techniques Used: Expressing, Construct, Immunolabeling

    ARX represses OLIG2 expression. ( a ) Representative images of embryonic neocortex (E14.5) of the Arx cKO /+ female (Arx Het) double immunolabeled with OLIG2 and ARX antibodies ( a’ – a”’ : magnified images of the boxed area in left panel). ARX+ cells are OLIG2−, and OLIG2+ cells are Arx−. Dotted lines mark the ventricular surface. Longer brackets indicate example areas with high number of ARX+ cells, while shorter brackets denote areas with high number of OLIG2+ cells. ( b ) Representative images of the immunofluorescent labeling (GFP and OLIG2) of the Arx cKO/y cortex electroporated with either pCIG (encoding GFP only) or pCIG-Arx (encoding ARX-IRES-GFP) (EP at E13.5 and harvested at E14.5). GFP antibody was used to label electroporated (EPed) cells (green, cytoplasmic staining pattern). Right most images are magnified images of the boxed areas. Long arrows indicate examples of GFP electroporated cells expressing OLIG2 and short arrows indicate examples of ARX electroporated cells not expressing OLIG2. ( c ) Quantification of results in B. The ratio of OLIG2+ GFP+ cells over GFP+ cells was plotted for both GFP ( pCIG ) and Arx ( pCIP-Arx ) electroporated samples. Error bars: mean ± s.d (n = 3 for GFP EP and n = 4 for Arx EP; ***P = 0.0008; unpaired t-test).
    Figure Legend Snippet: ARX represses OLIG2 expression. ( a ) Representative images of embryonic neocortex (E14.5) of the Arx cKO /+ female (Arx Het) double immunolabeled with OLIG2 and ARX antibodies ( a’ – a”’ : magnified images of the boxed area in left panel). ARX+ cells are OLIG2−, and OLIG2+ cells are Arx−. Dotted lines mark the ventricular surface. Longer brackets indicate example areas with high number of ARX+ cells, while shorter brackets denote areas with high number of OLIG2+ cells. ( b ) Representative images of the immunofluorescent labeling (GFP and OLIG2) of the Arx cKO/y cortex electroporated with either pCIG (encoding GFP only) or pCIG-Arx (encoding ARX-IRES-GFP) (EP at E13.5 and harvested at E14.5). GFP antibody was used to label electroporated (EPed) cells (green, cytoplasmic staining pattern). Right most images are magnified images of the boxed areas. Long arrows indicate examples of GFP electroporated cells expressing OLIG2 and short arrows indicate examples of ARX electroporated cells not expressing OLIG2. ( c ) Quantification of results in B. The ratio of OLIG2+ GFP+ cells over GFP+ cells was plotted for both GFP ( pCIG ) and Arx ( pCIP-Arx ) electroporated samples. Error bars: mean ± s.d (n = 3 for GFP EP and n = 4 for Arx EP; ***P = 0.0008; unpaired t-test).

    Techniques Used: Expressing, Immunolabeling, Labeling, Staining

    Forced expression of Olig2 represses Pax6 and Tbr2 expression. ( a,b) Representative images of the immunofluorescent labeling of the WT embryonic cortex electroporated with a control GFP ( pCIG ) or Olig2 expression construct ( pCIG-Olig2 ) (E13.5 → E14.5). Antibodies against GFP (green) and TBR2 (red) ( a ) or PAX6 (red) ( b ) were used. The number of PAX6+ and TBR2+ cells are reduced in the Olig2 electroporated cortices compared to the control. ( c,d) Quantification of results in a ( c ) and b ( d ). The ratio of TBR2+ GFP+ or PAX6+ GFP+ cells over GFP+ cells was plotted for GFP or Olig2 electroporated brains. Error bars: mean ± s.d (For Tbr2, n = 3 for each sample; ***P = 0.0007; For Pax6, n = 3 for GFP; n = 5 for Olig2; **P = 0.0012; unpaired t-test).
    Figure Legend Snippet: Forced expression of Olig2 represses Pax6 and Tbr2 expression. ( a,b) Representative images of the immunofluorescent labeling of the WT embryonic cortex electroporated with a control GFP ( pCIG ) or Olig2 expression construct ( pCIG-Olig2 ) (E13.5 → E14.5). Antibodies against GFP (green) and TBR2 (red) ( a ) or PAX6 (red) ( b ) were used. The number of PAX6+ and TBR2+ cells are reduced in the Olig2 electroporated cortices compared to the control. ( c,d) Quantification of results in a ( c ) and b ( d ). The ratio of TBR2+ GFP+ or PAX6+ GFP+ cells over GFP+ cells was plotted for GFP or Olig2 electroporated brains. Error bars: mean ± s.d (For Tbr2, n = 3 for each sample; ***P = 0.0007; For Pax6, n = 3 for GFP; n = 5 for Olig2; **P = 0.0012; unpaired t-test).

    Techniques Used: Expressing, Labeling, Construct

    8) Product Images from "Red Grape Skin Polyphenols Blunt Matrix Metalloproteinase-2 and -9 Activity and Expression in Cell Models of Vascular Inflammation: Protective Role in Degenerative and Inflammatory Diseases"

    Article Title: Red Grape Skin Polyphenols Blunt Matrix Metalloproteinase-2 and -9 Activity and Expression in Cell Models of Vascular Inflammation: Protective Role in Degenerative and Inflammatory Diseases

    Journal: Molecules

    doi: 10.3390/molecules21091147

    Inhibitory effects of red grape skin polyphenol extracts on MMP-2 and MMP-9 activity and expression and endothelial invasion. HUVEC were incubated with NSPE and PSPE (0.5, 5, 25 µg/mL) or ethanol vehicle (E, 0.025% v / v ) for 1 h and then stimulated with 20 nmol/L PMA, after which culture media were collected and analysed by gelatin zymography ( A ) or by ELISA ( B , C ); invasiveness was performed by transwell cell invasion assay ( D ); mRNA levels of MMP-9, MMP-2 and TIMP-1, TIMP-2 were assessed by qRT-PCR ( E , F ). Results are expressed as mean ± SD and are representative of four separate experiments yielding similar results. # p
    Figure Legend Snippet: Inhibitory effects of red grape skin polyphenol extracts on MMP-2 and MMP-9 activity and expression and endothelial invasion. HUVEC were incubated with NSPE and PSPE (0.5, 5, 25 µg/mL) or ethanol vehicle (E, 0.025% v / v ) for 1 h and then stimulated with 20 nmol/L PMA, after which culture media were collected and analysed by gelatin zymography ( A ) or by ELISA ( B , C ); invasiveness was performed by transwell cell invasion assay ( D ); mRNA levels of MMP-9, MMP-2 and TIMP-1, TIMP-2 were assessed by qRT-PCR ( E , F ). Results are expressed as mean ± SD and are representative of four separate experiments yielding similar results. # p

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

    Differential effects of pure polyphenols on the stimulated release of gelatinases in endothelial cells. HUVEC were pre-treated with increasing concentrations of each polyphenol (0.5, 5 and 25 μmol/L) or vehicle (ethanol, E, 0.025% v / v ; DMSO, D, 0.025% v / v ) for 1 h and then stimulated with 20 nmol/L PMA, after which culture media were collected and analysed by gelatin zymography. Bar graphs show the quantification of MMP-9 levels ( A ) and MMP-2/pro-MMP-2 levels ( B ) of four independent experiments (mean ± SD). # p
    Figure Legend Snippet: Differential effects of pure polyphenols on the stimulated release of gelatinases in endothelial cells. HUVEC were pre-treated with increasing concentrations of each polyphenol (0.5, 5 and 25 μmol/L) or vehicle (ethanol, E, 0.025% v / v ; DMSO, D, 0.025% v / v ) for 1 h and then stimulated with 20 nmol/L PMA, after which culture media were collected and analysed by gelatin zymography. Bar graphs show the quantification of MMP-9 levels ( A ) and MMP-2/pro-MMP-2 levels ( B ) of four independent experiments (mean ± SD). # p

    Techniques Used: Zymography

    Differential effects of pure polyphenols on the stimulated release of MMP-9 in monocytes. U937 were pre-treated with increasing concentrations of each polyphenol (0.5, 5 and 25 μmol/L) or vehicle (ethanol, E, 0.025% v / v ; DMSO, D, 0.025% v / v ) for 1 h and then stimulated with 20 nmol/L PMA, after which culture media were collected and analysed by gelatin zymography. Bar graphs show the quantification of MMP-9 levels of four independent experiments (mean ± SD). PMA release specified by dashed line. * p
    Figure Legend Snippet: Differential effects of pure polyphenols on the stimulated release of MMP-9 in monocytes. U937 were pre-treated with increasing concentrations of each polyphenol (0.5, 5 and 25 μmol/L) or vehicle (ethanol, E, 0.025% v / v ; DMSO, D, 0.025% v / v ) for 1 h and then stimulated with 20 nmol/L PMA, after which culture media were collected and analysed by gelatin zymography. Bar graphs show the quantification of MMP-9 levels of four independent experiments (mean ± SD). PMA release specified by dashed line. * p

    Techniques Used: Zymography

    Inhibitory effects of red grape skin polyphenol extracts on MMP-9 activity and expression in inflamed monocytes. U937 were incubated with NSPE and PSPE (0.5, 5, 25 µg/mL) or ethanol vehicle (E, 0.025% v / v ) for 1 h and then stimulated with 20 nmol/L PMA. Media were collected and analysed by gelatin zymography ( A ) or ELISA ( B ); mRNA levels of MMP-9, MMP-2, TIMP-1 and TIMP-2 were assessed by qRT-PCR ( C – E ); cell viability was determined by the MTT assay ( F ). Results expressed as mean ± SD are representative of four separate experiments yielding similar results. # p
    Figure Legend Snippet: Inhibitory effects of red grape skin polyphenol extracts on MMP-9 activity and expression in inflamed monocytes. U937 were incubated with NSPE and PSPE (0.5, 5, 25 µg/mL) or ethanol vehicle (E, 0.025% v / v ) for 1 h and then stimulated with 20 nmol/L PMA. Media were collected and analysed by gelatin zymography ( A ) or ELISA ( B ); mRNA levels of MMP-9, MMP-2, TIMP-1 and TIMP-2 were assessed by qRT-PCR ( C – E ); cell viability was determined by the MTT assay ( F ). Results expressed as mean ± SD are representative of four separate experiments yielding similar results. # p

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

    9) Product Images from "Epigenetic and Transcriptome Profiling Identifies a Population of Visceral Adipose-Derived Progenitor Cells with the Potential to Differentiate into an Endocrine Pancreatic Lineage"

    Article Title: Epigenetic and Transcriptome Profiling Identifies a Population of Visceral Adipose-Derived Progenitor Cells with the Potential to Differentiate into an Endocrine Pancreatic Lineage

    Journal: Cell Transplantation

    doi: 10.1177/0963689718808472

    Differentiation and function of AMCs. (A) Visceral adipose-derived AMCs from Pdx1-GFP mice were exposed to various growth and differentiation factors. Panel (A) represents the experimental setup where GFP reporter (Pdx1-GFP) AMCs were plated in (black) 96-well plates and fluorescent intensities recorded as detailed in methods. Note that some (minimal) level of GFP seen in the cells (wells 1 and 2), represented as ‘0’ intensity, diminished when exposed to the GLP1 antagonist (exendin 9-39; wells 3 and 4) or to increase in intensity when exposed to an agonist (wells 5 and 6). All conditions were set up in triplicate. (B) Cultures were stimulated to differentiate in the presence of the small molecules described in Supplementary Table 2 and the intensity of GFP fluorescence was measured at the same time of the day and at regular (24 h) intervals. Data were corrected with respect to the blank (well containing media but no cells) and presented relative to the GFP signal measured on day 1. Values generated for day 1 are therefore presented as ‘0’ fluorescence, while subsequent measurements are presented relative to day 1. AMCs generated from Pdx1-GFP transgenic mice do not show GFP expression as monolayers (C). However, when stimulated to differentiate into an endocrine pancreatic lineage, AMCs formed multiple, small ICAs that express GFP in presence of exendin-4 (D) or FGF2 (E). Bar = 20μm. GFP-expressing ICAs were analyzed using TaqMan-based quantitative PCR for the expression of endocrine pancreatic gene transcripts. Data are presented as fold difference over the detectable Ct value was calculated using the ΔΔCt method (Hardikar A et al. 29 ) (F). Human AMCs were induced to differentiate into endocrine pancreatic lineage using FGF2 and exendin-4 agonist and transplanted in NOD/SCID mice using TheraCyte ™ devices. Human AMC-derived ICAs showed detectable expression of pro-endocrine gene transcripts (G). Box and whisker plot showing the abundance of transcript at day 90 relative to expression in day 0 (undifferentiated) AMCs. The line in the box represents the median and the box represents the upper and lower quartile ( N =4 mice). ICA-containing TheraCytes ™ showed evidence of vascularization (H), showed detectable levels of human insulin (mean ± SD; N =3) in mouse circulation (I) and contained insulin-producing cells after 90 days of in-vivo studies (J); red: insulin, blue: DAPI: nuclei; bar = 20μm. AMC: adipose tissue-derived mesenchymal cell; ICA: islet-like cell aggregate; AMC: Adipose-derived mesenchymal cells; ICAs: Islet-like Cell Aggregates; Pdx1: Pancreatic and duodenal homeobox 1; GLP1: Glucagon-like Peptide 1; GFP: Green Fluoroscence Protein; FGF2: Fibroblast Growth Factor 2; PCR: Polymerase Chain Reaction; Ct: Cycle threshold; NOD/SCID: Non-obese Diabetic/Severe Combined Immunodeficiency; DAPI: 4′,6-diamidino-2-phenylindole; SD: Standard Deviation.
    Figure Legend Snippet: Differentiation and function of AMCs. (A) Visceral adipose-derived AMCs from Pdx1-GFP mice were exposed to various growth and differentiation factors. Panel (A) represents the experimental setup where GFP reporter (Pdx1-GFP) AMCs were plated in (black) 96-well plates and fluorescent intensities recorded as detailed in methods. Note that some (minimal) level of GFP seen in the cells (wells 1 and 2), represented as ‘0’ intensity, diminished when exposed to the GLP1 antagonist (exendin 9-39; wells 3 and 4) or to increase in intensity when exposed to an agonist (wells 5 and 6). All conditions were set up in triplicate. (B) Cultures were stimulated to differentiate in the presence of the small molecules described in Supplementary Table 2 and the intensity of GFP fluorescence was measured at the same time of the day and at regular (24 h) intervals. Data were corrected with respect to the blank (well containing media but no cells) and presented relative to the GFP signal measured on day 1. Values generated for day 1 are therefore presented as ‘0’ fluorescence, while subsequent measurements are presented relative to day 1. AMCs generated from Pdx1-GFP transgenic mice do not show GFP expression as monolayers (C). However, when stimulated to differentiate into an endocrine pancreatic lineage, AMCs formed multiple, small ICAs that express GFP in presence of exendin-4 (D) or FGF2 (E). Bar = 20μm. GFP-expressing ICAs were analyzed using TaqMan-based quantitative PCR for the expression of endocrine pancreatic gene transcripts. Data are presented as fold difference over the detectable Ct value was calculated using the ΔΔCt method (Hardikar A et al. 29 ) (F). Human AMCs were induced to differentiate into endocrine pancreatic lineage using FGF2 and exendin-4 agonist and transplanted in NOD/SCID mice using TheraCyte ™ devices. Human AMC-derived ICAs showed detectable expression of pro-endocrine gene transcripts (G). Box and whisker plot showing the abundance of transcript at day 90 relative to expression in day 0 (undifferentiated) AMCs. The line in the box represents the median and the box represents the upper and lower quartile ( N =4 mice). ICA-containing TheraCytes ™ showed evidence of vascularization (H), showed detectable levels of human insulin (mean ± SD; N =3) in mouse circulation (I) and contained insulin-producing cells after 90 days of in-vivo studies (J); red: insulin, blue: DAPI: nuclei; bar = 20μm. AMC: adipose tissue-derived mesenchymal cell; ICA: islet-like cell aggregate; AMC: Adipose-derived mesenchymal cells; ICAs: Islet-like Cell Aggregates; Pdx1: Pancreatic and duodenal homeobox 1; GLP1: Glucagon-like Peptide 1; GFP: Green Fluoroscence Protein; FGF2: Fibroblast Growth Factor 2; PCR: Polymerase Chain Reaction; Ct: Cycle threshold; NOD/SCID: Non-obese Diabetic/Severe Combined Immunodeficiency; DAPI: 4′,6-diamidino-2-phenylindole; SD: Standard Deviation.

    Techniques Used: Derivative Assay, Mouse Assay, Fluorescence, Generated, Transgenic Assay, Expressing, Real-time Polymerase Chain Reaction, Whisker Assay, In Vivo, Polymerase Chain Reaction, Standard Deviation

    10) Product Images from "Methotrexate-induced apoptosis in human ovarian adenocarcinoma SKOV-3 cells via ROS-mediated bax/bcl-2-cyt-c release cascading"

    Article Title: Methotrexate-induced apoptosis in human ovarian adenocarcinoma SKOV-3 cells via ROS-mediated bax/bcl-2-cyt-c release cascading

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S178510

    RT-PCR examines the transcript levels of bax, bcl-2, and cyt-c (apoptosis marker) after treatment with MTX. ( A ) Exposure of MTX (0, 15, 25, and 50 µM) shows significant ( P
    Figure Legend Snippet: RT-PCR examines the transcript levels of bax, bcl-2, and cyt-c (apoptosis marker) after treatment with MTX. ( A ) Exposure of MTX (0, 15, 25, and 50 µM) shows significant ( P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Marker

    11) Product Images from "TyrR is involved in the transcriptional regulation of biofilm formation and D-alanine catabolism in Azospirillum brasilense Sp7."

    Article Title: TyrR is involved in the transcriptional regulation of biofilm formation and D-alanine catabolism in Azospirillum brasilense Sp7.

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211904

    RT-PCR analysis of dadA , tyrR and gyrA expression from the A . brasilense Sp7, 2116, and 2117 strains. RT-PCR analysis of tyrR , dadA and gyrA expression from the A . brasilense Sp7, 2116 and 2117 strains. Total RNA (1 μg) from each strain was subjected to reverse transcription, and the obtained cDNA was amplified using pairs of primers specific for tyrR . ( A ) dadA , ( B ), and gyrA ( C ) A . brasilense strains are indicated at the top of each lane by their conventional denomination. The expected PCR products of 196 bp ( tyrR ), 145 bp ( dadA ) and 143 bp ( gyrA ) were visualized by ethidium bromide staining on a 2.5% agarose gel. A positive control (+), represented by genomic DNA. A negative reaction control is represented by (-), without reverse transcriptase, both were included in each experiment. The image is representative of three independents experiments.
    Figure Legend Snippet: RT-PCR analysis of dadA , tyrR and gyrA expression from the A . brasilense Sp7, 2116, and 2117 strains. RT-PCR analysis of tyrR , dadA and gyrA expression from the A . brasilense Sp7, 2116 and 2117 strains. Total RNA (1 μg) from each strain was subjected to reverse transcription, and the obtained cDNA was amplified using pairs of primers specific for tyrR . ( A ) dadA , ( B ), and gyrA ( C ) A . brasilense strains are indicated at the top of each lane by their conventional denomination. The expected PCR products of 196 bp ( tyrR ), 145 bp ( dadA ) and 143 bp ( gyrA ) were visualized by ethidium bromide staining on a 2.5% agarose gel. A positive control (+), represented by genomic DNA. A negative reaction control is represented by (-), without reverse transcriptase, both were included in each experiment. The image is representative of three independents experiments.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Positive Control

    12) Product Images from "Growth-inhibition of cell lines derived from B cell lymphomas through antagonism of serotonin receptor signaling"

    Article Title: Growth-inhibition of cell lines derived from B cell lymphomas through antagonism of serotonin receptor signaling

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-40825-x

    5HT1A antagonist WAY significantly inhibits the proliferation of non-BL B cell derived lymphoma cells lines. ( a ) Val, a DLBCL cell line, was treated with indicated concentrations of WAY and relative cell numbers in each culture were determined by adding a fixed number of counting beads to respective culture and calculating the ratio of Val cells to beads. ( b , c ) rtqPCR analysis of the Bcl-2 (anti-apoptotic) and Bim (pro-apoptotic) gene expression in Val cells exposed to 10 µM of WAY at indicated periods. Expression levels were normalized to Beta-actin, calculated by 2(−ΔΔct) method and 0 hr expression was set to 1 and expression change is represented as a fold change. ( d ) Different B cell derived lymphoma cell lines were exposed to repetitive treatments (WAY-3×; 50 µM/24 h) for 72 h and number of cells were counted at 72 h time point using TC20 Automated Cell Counter. Data is presented as the mean of 3–5 independent experiment ± SD. Statistical comparison of control and treated cells at 72 h is indicated ( ** P
    Figure Legend Snippet: 5HT1A antagonist WAY significantly inhibits the proliferation of non-BL B cell derived lymphoma cells lines. ( a ) Val, a DLBCL cell line, was treated with indicated concentrations of WAY and relative cell numbers in each culture were determined by adding a fixed number of counting beads to respective culture and calculating the ratio of Val cells to beads. ( b , c ) rtqPCR analysis of the Bcl-2 (anti-apoptotic) and Bim (pro-apoptotic) gene expression in Val cells exposed to 10 µM of WAY at indicated periods. Expression levels were normalized to Beta-actin, calculated by 2(−ΔΔct) method and 0 hr expression was set to 1 and expression change is represented as a fold change. ( d ) Different B cell derived lymphoma cell lines were exposed to repetitive treatments (WAY-3×; 50 µM/24 h) for 72 h and number of cells were counted at 72 h time point using TC20 Automated Cell Counter. Data is presented as the mean of 3–5 independent experiment ± SD. Statistical comparison of control and treated cells at 72 h is indicated ( ** P

    Techniques Used: Derivative Assay, Expressing

    Primary B cell-derived lymphomas and lymphoma-derived cell lines express 5HT1A. ( a ) rtqPCR analysis of 5HT1A expression in lymph node or spleen biopsies samples from different types of primary NHLs. The results are presented as a fold change relative to B cells from healthy donors, expression levels were normalized to of Beta-actin and calculated by 2(−ΔΔct) method. ( b ) rtqPCR analysis of 5HT1A expression in different types of B cell-derived lymphoma cell lines. The results are presented as a fold change relative to B cells from healthy donors, expression levels were normalized to 18 s rRNA and calculated by 2(−ΔΔct) method. Data is presented as the mean of 2 independent experiments ± SD. ( c ) Western blot analysis of 5HT1A protein expression in B cell-derived lymphoma cell lines (n = 3). The gel and blot image shown in the figure was cropped from same part of the gel with an exposure time of 30 s where Cyclophilin-A was used as a loading control. Full-length blots and gels are presented in Supplementary Figure 3.
    Figure Legend Snippet: Primary B cell-derived lymphomas and lymphoma-derived cell lines express 5HT1A. ( a ) rtqPCR analysis of 5HT1A expression in lymph node or spleen biopsies samples from different types of primary NHLs. The results are presented as a fold change relative to B cells from healthy donors, expression levels were normalized to of Beta-actin and calculated by 2(−ΔΔct) method. ( b ) rtqPCR analysis of 5HT1A expression in different types of B cell-derived lymphoma cell lines. The results are presented as a fold change relative to B cells from healthy donors, expression levels were normalized to 18 s rRNA and calculated by 2(−ΔΔct) method. Data is presented as the mean of 2 independent experiments ± SD. ( c ) Western blot analysis of 5HT1A protein expression in B cell-derived lymphoma cell lines (n = 3). The gel and blot image shown in the figure was cropped from same part of the gel with an exposure time of 30 s where Cyclophilin-A was used as a loading control. Full-length blots and gels are presented in Supplementary Figure 3.

    Techniques Used: Derivative Assay, Expressing, Western Blot

    13) Product Images from "More than just inflammation: Ureaplasma species induce apoptosis in human brain microvascular endothelial cells"

    Article Title: More than just inflammation: Ureaplasma species induce apoptosis in human brain microvascular endothelial cells

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-019-1413-8

    Ureaplasma -driven pyroptosis in HBMEC. Key genes in pyroptosis (Fig. 1 ) were assessed for mRNA responses upon stimulation of HBMEC for 4 h and 30 h. Caspase 1 mRNA expression was determined via RNA sequencing ( a ) and qRT-PCR ( b ). Similarly, RNA sequencing ( c ) and qRT-PCR ( d ) were used to assess caspase 4 mRNA levels. RNA sequencing furthermore determined mRNA expression of NLRP3 ( e ) and gasdermin D ( f ). Data are presented as means ± SD (* p
    Figure Legend Snippet: Ureaplasma -driven pyroptosis in HBMEC. Key genes in pyroptosis (Fig. 1 ) were assessed for mRNA responses upon stimulation of HBMEC for 4 h and 30 h. Caspase 1 mRNA expression was determined via RNA sequencing ( a ) and qRT-PCR ( b ). Similarly, RNA sequencing ( c ) and qRT-PCR ( d ) were used to assess caspase 4 mRNA levels. RNA sequencing furthermore determined mRNA expression of NLRP3 ( e ) and gasdermin D ( f ). Data are presented as means ± SD (* p

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

    Ureaplasma -driven apoptosis in HBMEC. Enzymes and other proteins involved in the apoptotic cascade (Fig. 1 ) were analyzed upon stimulation of HBMEC for 4 h, 24 h, or 30 h. For caspase 3, mRNA expression was determined via RNA sequencing ( a ) and qRT-PCR ( b ) and enzyme activity (cleaved caspase 3) was assessed via flow cytometry ( c ). For caspase 8, RNA sequencing ( d ) and qRT-PCR ( e ) were used to evaluate mRNA levels and flow cytometry ( f ) was employed to determine protein expression. Caspase 9 mRNA levels were also assessed via RNA sequencing ( g ) and qRT-PCR ( h ), and levels of active caspase 9 were determined using flow cytometry ( i ). RNA sequencing was used to assess mRNA expression of caspase 7 ( j ), BAK ( k ), BAX ( l ), p53 ( m ), FOS ( n ), and APAF1 ( o ). Data are shown as means ± SD (* p
    Figure Legend Snippet: Ureaplasma -driven apoptosis in HBMEC. Enzymes and other proteins involved in the apoptotic cascade (Fig. 1 ) were analyzed upon stimulation of HBMEC for 4 h, 24 h, or 30 h. For caspase 3, mRNA expression was determined via RNA sequencing ( a ) and qRT-PCR ( b ) and enzyme activity (cleaved caspase 3) was assessed via flow cytometry ( c ). For caspase 8, RNA sequencing ( d ) and qRT-PCR ( e ) were used to evaluate mRNA levels and flow cytometry ( f ) was employed to determine protein expression. Caspase 9 mRNA levels were also assessed via RNA sequencing ( g ) and qRT-PCR ( h ), and levels of active caspase 9 were determined using flow cytometry ( i ). RNA sequencing was used to assess mRNA expression of caspase 7 ( j ), BAK ( k ), BAX ( l ), p53 ( m ), FOS ( n ), and APAF1 ( o ). Data are shown as means ± SD (* p

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

    14) Product Images from "A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene"

    Article Title: A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007961

    eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 mRNA (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), RNA-sequencing ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.
    Figure Legend Snippet: eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 mRNA (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), RNA-sequencing ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.

    Techniques Used: Expressing, Labeling, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Mass Spectrometry, Polymerase Chain Reaction

    Biogenesis of microRNA-548ac and genetic variants in the CD58 gene locus. ( A ) Diagram of the processing of an mRNA and an intronic miRNA from the same transcript (adapted from [ 49 ]). Both RNA splicing by the spliceosome and miRNA stem-loop cropping by the Drosha-DGCR8 complex occur cotranscriptionally. Drosha may cleave the miRNA-harboring intron before splicing commitment of the flanking exons. The resulting precursor miRNA is subsequently processed into a mature miRNA, which is loaded into the RNA-induced silencing complex (RISC). ( B ) Annotated secondary structure of hsa-mir-548ac. Highlighted in gray is the 22 nt long sequence of the mature miRNA isoform as assigned by Jima et al . (miRBase accession MIMAT0018938) [ 43 ]. The red circle marks the only common single-nucleotide polymorphism (SNP) within the stem-loop region. The G allele is overrepresented in MS patients. ( C ) Genetic variants in pairwise linkage disequilibrium (LD) with SNP rs1414273. This plot was generated using the LDproxy module of the web-based analysis tool LDlink [ 97 ]. Shown are r 2 LD values of proximal SNPs based on all subpopulations of the 1000 Genomes project (orange dots), recombination rate as estimated from HapMap data (gray line), and the position and exon-intron structure of nearby genes on chromosome 1 (chr1, GRCh37 assembly). The MS-associated SNP rs1335532 is in strong LD with SNP rs1414273 (blue) (correlated forward strand alleles: A = C, G = T). The entire block of LD spans about 50 kb but does not include the promoter region of CD58, which is encoded on the minus strand in the reference genome. ( D ) Worldwide distribution of SNP rs1335532 alleles. Global allele frequencies were visualized as two-color pie charts with the HGDP Selection Browser [ 98 ]. The disease susceptibility variant (A, blue) is the major allele in European populations and the minor allele in East Asian and Southern African populations. cM/Mb = centimorgan per megabase, HGDP = Human Genome Diversity Panel, MS = multiple sclerosis.
    Figure Legend Snippet: Biogenesis of microRNA-548ac and genetic variants in the CD58 gene locus. ( A ) Diagram of the processing of an mRNA and an intronic miRNA from the same transcript (adapted from [ 49 ]). Both RNA splicing by the spliceosome and miRNA stem-loop cropping by the Drosha-DGCR8 complex occur cotranscriptionally. Drosha may cleave the miRNA-harboring intron before splicing commitment of the flanking exons. The resulting precursor miRNA is subsequently processed into a mature miRNA, which is loaded into the RNA-induced silencing complex (RISC). ( B ) Annotated secondary structure of hsa-mir-548ac. Highlighted in gray is the 22 nt long sequence of the mature miRNA isoform as assigned by Jima et al . (miRBase accession MIMAT0018938) [ 43 ]. The red circle marks the only common single-nucleotide polymorphism (SNP) within the stem-loop region. The G allele is overrepresented in MS patients. ( C ) Genetic variants in pairwise linkage disequilibrium (LD) with SNP rs1414273. This plot was generated using the LDproxy module of the web-based analysis tool LDlink [ 97 ]. Shown are r 2 LD values of proximal SNPs based on all subpopulations of the 1000 Genomes project (orange dots), recombination rate as estimated from HapMap data (gray line), and the position and exon-intron structure of nearby genes on chromosome 1 (chr1, GRCh37 assembly). The MS-associated SNP rs1335532 is in strong LD with SNP rs1414273 (blue) (correlated forward strand alleles: A = C, G = T). The entire block of LD spans about 50 kb but does not include the promoter region of CD58, which is encoded on the minus strand in the reference genome. ( D ) Worldwide distribution of SNP rs1335532 alleles. Global allele frequencies were visualized as two-color pie charts with the HGDP Selection Browser [ 98 ]. The disease susceptibility variant (A, blue) is the major allele in European populations and the minor allele in East Asian and Southern African populations. cM/Mb = centimorgan per megabase, HGDP = Human Genome Diversity Panel, MS = multiple sclerosis.

    Techniques Used: Sequencing, Mass Spectrometry, Generated, Blocking Assay, Selection, Variant Assay

    15) Product Images from "A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene"

    Article Title: A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007961

    eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 mRNA (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), RNA-sequencing ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.
    Figure Legend Snippet: eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 mRNA (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), RNA-sequencing ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.

    Techniques Used: Expressing, Labeling, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Mass Spectrometry, Polymerase Chain Reaction

    Biogenesis of microRNA-548ac and genetic variants in the CD58 gene locus. ( A ) Diagram of the processing of an mRNA and an intronic miRNA from the same transcript (adapted from [ 49 ]). Both RNA splicing by the spliceosome and miRNA stem-loop cropping by the Drosha-DGCR8 complex occur cotranscriptionally. Drosha may cleave the miRNA-harboring intron before splicing commitment of the flanking exons. The resulting precursor miRNA is subsequently processed into a mature miRNA, which is loaded into the RNA-induced silencing complex (RISC). ( B ) Annotated secondary structure of hsa-mir-548ac. Highlighted in gray is the 22 nt long sequence of the mature miRNA isoform as assigned by Jima et al . (miRBase accession MIMAT0018938) [ 43 ]. The red circle marks the only common single-nucleotide polymorphism (SNP) within the stem-loop region. The G allele is overrepresented in MS patients. ( C ) Genetic variants in pairwise linkage disequilibrium (LD) with SNP rs1414273. This plot was generated using the LDproxy module of the web-based analysis tool LDlink [ 97 ]. Shown are r 2 LD values of proximal SNPs based on all subpopulations of the 1000 Genomes project (orange dots), recombination rate as estimated from HapMap data (gray line), and the position and exon-intron structure of nearby genes on chromosome 1 (chr1, GRCh37 assembly). The MS-associated SNP rs1335532 is in strong LD with SNP rs1414273 (blue) (correlated forward strand alleles: A = C, G = T). The entire block of LD spans about 50 kb but does not include the promoter region of CD58, which is encoded on the minus strand in the reference genome. ( D ) Worldwide distribution of SNP rs1335532 alleles. Global allele frequencies were visualized as two-color pie charts with the HGDP Selection Browser [ 98 ]. The disease susceptibility variant (A, blue) is the major allele in European populations and the minor allele in East Asian and Southern African populations. cM/Mb = centimorgan per megabase, HGDP = Human Genome Diversity Panel, MS = multiple sclerosis.
    Figure Legend Snippet: Biogenesis of microRNA-548ac and genetic variants in the CD58 gene locus. ( A ) Diagram of the processing of an mRNA and an intronic miRNA from the same transcript (adapted from [ 49 ]). Both RNA splicing by the spliceosome and miRNA stem-loop cropping by the Drosha-DGCR8 complex occur cotranscriptionally. Drosha may cleave the miRNA-harboring intron before splicing commitment of the flanking exons. The resulting precursor miRNA is subsequently processed into a mature miRNA, which is loaded into the RNA-induced silencing complex (RISC). ( B ) Annotated secondary structure of hsa-mir-548ac. Highlighted in gray is the 22 nt long sequence of the mature miRNA isoform as assigned by Jima et al . (miRBase accession MIMAT0018938) [ 43 ]. The red circle marks the only common single-nucleotide polymorphism (SNP) within the stem-loop region. The G allele is overrepresented in MS patients. ( C ) Genetic variants in pairwise linkage disequilibrium (LD) with SNP rs1414273. This plot was generated using the LDproxy module of the web-based analysis tool LDlink [ 97 ]. Shown are r 2 LD values of proximal SNPs based on all subpopulations of the 1000 Genomes project (orange dots), recombination rate as estimated from HapMap data (gray line), and the position and exon-intron structure of nearby genes on chromosome 1 (chr1, GRCh37 assembly). The MS-associated SNP rs1335532 is in strong LD with SNP rs1414273 (blue) (correlated forward strand alleles: A = C, G = T). The entire block of LD spans about 50 kb but does not include the promoter region of CD58, which is encoded on the minus strand in the reference genome. ( D ) Worldwide distribution of SNP rs1335532 alleles. Global allele frequencies were visualized as two-color pie charts with the HGDP Selection Browser [ 98 ]. The disease susceptibility variant (A, blue) is the major allele in European populations and the minor allele in East Asian and Southern African populations. cM/Mb = centimorgan per megabase, HGDP = Human Genome Diversity Panel, MS = multiple sclerosis.

    Techniques Used: Sequencing, Mass Spectrometry, Generated, Blocking Assay, Selection, Variant Assay

    16) Product Images from "A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene"

    Article Title: A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007961

    Target gene analysis of microRNA-548ac. ( A ) Expression of hsa-miR-548ac after transient transfection of HeLa cells with a precursor miRNA expression plasmid (mir-548ac-G). The level of mature miRNA molecules was determined relative to a reference miRNA (hsa-miR-191-5p) using real-time PCR based on TaqMan miRNA assays with specific stem-loop primers. Shown are means and standard errors of 2 -ΔCt values scaled by a factor of 1000. When using a negative control vector with scrambled sequence, hsa-miR-548ac was basically not detectable. ( B ) Volcano plots of differential gene expression in HeLa cells transfected with the mir-548ac plasmid compared to cells transfected with the control plasmid. The transcriptome profiles were obtained by HTA 2.0 microarrays, which contain 67528 probe sets for human genes. Green dots indicate probe sets with p -value
    Figure Legend Snippet: Target gene analysis of microRNA-548ac. ( A ) Expression of hsa-miR-548ac after transient transfection of HeLa cells with a precursor miRNA expression plasmid (mir-548ac-G). The level of mature miRNA molecules was determined relative to a reference miRNA (hsa-miR-191-5p) using real-time PCR based on TaqMan miRNA assays with specific stem-loop primers. Shown are means and standard errors of 2 -ΔCt values scaled by a factor of 1000. When using a negative control vector with scrambled sequence, hsa-miR-548ac was basically not detectable. ( B ) Volcano plots of differential gene expression in HeLa cells transfected with the mir-548ac plasmid compared to cells transfected with the control plasmid. The transcriptome profiles were obtained by HTA 2.0 microarrays, which contain 67528 probe sets for human genes. Green dots indicate probe sets with p -value

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Negative Control, Sequencing

    17) Product Images from "Small RNA sequences derived from pre-microRNAs in the supraspliceosome"

    Article Title: Small RNA sequences derived from pre-microRNAs in the supraspliceosome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky791

    Regulation of gene expression by nuclear miR-7704. ( A ) Genome browser view for miR-7704 and the overlap with HOXD1 and HAGLR, a long non-coding RNA gene. ( B ) Average expression levels of miR-7704 from the SF based on HeLa (six samples, blue) and MCF-10A cells (three samples, orange). The expression values are based on normalizing each sample to miRNA read counts (100k per sample). The statistical bars are the calculated standard errors. ( C ) Comparison of the results of RT-PCR assays of HOXD1 and HAGLR expression in HeLa and mammary epithelial MCF-10A cells. Results represent RT-PCR from three biological repeats. The identity of the extracted bands was confirmed by DNA sequencing. (D–F) Effect of down-regulation of miR-7704 on HAGLR expression in HeLa cells. Transfection with Anti-miR-7704 inhibitor resulted in down-regulation of miR-7704 ( D ), increase in the expression level of HAGLR mRNA ( E ), and mild elevation of HOXD1 ( F ). (G–I) Effect of down-regulation of miR-7704 on HAGLR expression in MCF-10A cells. Transfection with Anti-miR-7704 inhibitor resulted in down-regulation of miR-7704 ( G ), increase in the expression level of HAGLR mRNA ( H ) and mild elevation of HOXD1 ( I ). D–I, each, show results of qPCR of three independent biological preparations. The expression levels of HAGLR and HOXD1 were normalized to β-actin expression from the same preparation.
    Figure Legend Snippet: Regulation of gene expression by nuclear miR-7704. ( A ) Genome browser view for miR-7704 and the overlap with HOXD1 and HAGLR, a long non-coding RNA gene. ( B ) Average expression levels of miR-7704 from the SF based on HeLa (six samples, blue) and MCF-10A cells (three samples, orange). The expression values are based on normalizing each sample to miRNA read counts (100k per sample). The statistical bars are the calculated standard errors. ( C ) Comparison of the results of RT-PCR assays of HOXD1 and HAGLR expression in HeLa and mammary epithelial MCF-10A cells. Results represent RT-PCR from three biological repeats. The identity of the extracted bands was confirmed by DNA sequencing. (D–F) Effect of down-regulation of miR-7704 on HAGLR expression in HeLa cells. Transfection with Anti-miR-7704 inhibitor resulted in down-regulation of miR-7704 ( D ), increase in the expression level of HAGLR mRNA ( E ), and mild elevation of HOXD1 ( F ). (G–I) Effect of down-regulation of miR-7704 on HAGLR expression in MCF-10A cells. Transfection with Anti-miR-7704 inhibitor resulted in down-regulation of miR-7704 ( G ), increase in the expression level of HAGLR mRNA ( H ) and mild elevation of HOXD1 ( I ). D–I, each, show results of qPCR of three independent biological preparations. The expression levels of HAGLR and HOXD1 were normalized to β-actin expression from the same preparation.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, DNA Sequencing, Transfection, Real-time Polymerase Chain Reaction

    18) Product Images from "CB1 receptor blockade ameliorates hepatic fat infiltration and inflammation and increases Nrf2-AMPK pathway in a rat model of severely uncontrolled diabetes"

    Article Title: CB1 receptor blockade ameliorates hepatic fat infiltration and inflammation and increases Nrf2-AMPK pathway in a rat model of severely uncontrolled diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0206152

    Effects of rimonabant on Nrf2 and its downstream gene expression and AMPK phosphorylation in rat livers. (A) Hepatic Nrf2 and antioxidant-responsive element (ARE)-mediated NQO1, HO-1, GSTA, and TRNRD1 gene expression were determined by RT-PCR, normalized for all samples to ribosomal RNA (18S) level, and expressed as fold change compared to LETO control rats (LETO Con). Representative western blots for NQO1, HO-1, and β-actin (B) and p-AMPK, AMPK, and β-actin (D). The density of signal was quantified and normalized by β-actin (C) or AMPK (E). Data are expressed as mean ± SEM (n = 4–5 per group). *P
    Figure Legend Snippet: Effects of rimonabant on Nrf2 and its downstream gene expression and AMPK phosphorylation in rat livers. (A) Hepatic Nrf2 and antioxidant-responsive element (ARE)-mediated NQO1, HO-1, GSTA, and TRNRD1 gene expression were determined by RT-PCR, normalized for all samples to ribosomal RNA (18S) level, and expressed as fold change compared to LETO control rats (LETO Con). Representative western blots for NQO1, HO-1, and β-actin (B) and p-AMPK, AMPK, and β-actin (D). The density of signal was quantified and normalized by β-actin (C) or AMPK (E). Data are expressed as mean ± SEM (n = 4–5 per group). *P

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

    19) Product Images from "PADI4-mediated epithelial-mesenchymal transition in lung cancer cells"

    Article Title: PADI4-mediated epithelial-mesenchymal transition in lung cancer cells

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2019.9968

    The expression levels of PADI4 in vitro and knockdown of PADI4 in A549 cells. (A) The statistical analysis represents the mRNA expression level of PADI4 in different lung cancer cells and normal lung epithelial cells. (B) The representative bands of western blotting and the statistical analysis. Differences among groups were analyzed by one-way ANOVA, followed by Bonferroni's multiple comparison test. *P
    Figure Legend Snippet: The expression levels of PADI4 in vitro and knockdown of PADI4 in A549 cells. (A) The statistical analysis represents the mRNA expression level of PADI4 in different lung cancer cells and normal lung epithelial cells. (B) The representative bands of western blotting and the statistical analysis. Differences among groups were analyzed by one-way ANOVA, followed by Bonferroni's multiple comparison test. *P

    Techniques Used: Expressing, In Vitro, Western Blot

    The migration and invasion features of lung cancer cells and normal lung epithelial cells. (A) The migration ability of H1299 and A549 cells, and BEAS-2B cells determined by wound-healing assay (magnification, ×200) and the statistical results are presented. (B) The invasion ability of H1299, A549 and BEAS-2B cells determined by Transwell assay (magnification, ×400) and the statistical analysis is presented. Differences between groups were analyzed by one-way analysis of variance, followed by Bonferroni's multiple comparison test. *P
    Figure Legend Snippet: The migration and invasion features of lung cancer cells and normal lung epithelial cells. (A) The migration ability of H1299 and A549 cells, and BEAS-2B cells determined by wound-healing assay (magnification, ×200) and the statistical results are presented. (B) The invasion ability of H1299, A549 and BEAS-2B cells determined by Transwell assay (magnification, ×400) and the statistical analysis is presented. Differences between groups were analyzed by one-way analysis of variance, followed by Bonferroni's multiple comparison test. *P

    Techniques Used: Migration, Wound Healing Assay, Transwell Assay

    Knockdown of PADI4 inhibits the migration and invasion ability of A549 cell. (A) The migration potential of A549 cells and shPADI4 A549 cells determined by wound-healing assay (magnification, ×200) and the statistical results are presented. (B) The invasion potential of A549 cells and shPADI4 A549 cells assessed by Transwell assay (magnification, ×400) and the statistical chart. Differences among groups were analyzed by one-way analysis of variance, followed by Bonferroni's multiple comparison test. *P
    Figure Legend Snippet: Knockdown of PADI4 inhibits the migration and invasion ability of A549 cell. (A) The migration potential of A549 cells and shPADI4 A549 cells determined by wound-healing assay (magnification, ×200) and the statistical results are presented. (B) The invasion potential of A549 cells and shPADI4 A549 cells assessed by Transwell assay (magnification, ×400) and the statistical chart. Differences among groups were analyzed by one-way analysis of variance, followed by Bonferroni's multiple comparison test. *P

    Techniques Used: Migration, Wound Healing Assay, Transwell Assay

    20) Product Images from "TRPC5 ion channel permeation promotes weight gain in hypercholesterolaemic mice"

    Article Title: TRPC5 ion channel permeation promotes weight gain in hypercholesterolaemic mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37299-8

    Increased adiponectin and reduced inflammatory gene expression in adipose tissue of DNT5 mice. All data were from quantitative real-time PCR analysis of epididymal fat pad RNA after mice had been provided with western-style diet and DOX for 12 weeks. All mice were ApoE −/− . ( A ) Comparison of adiponectin (AdipoQ) and leptin (Lep) mRNA abundance between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 10 Control, N = 14 DNT5). On the right, linear random effects model analysis showing the standardised mean difference and confidence intervals (n/N = 8/10 Control, n/N = 8/14 DNT5). Data above the zero line indicate increase in the DNT5 group. ( B ) Comparison of the inflammatory mediators tumour necrosis factor α (Tnfα), interleukin 6 (Il6), interleukin 1β (Il1β) and C-C motif chemokine ligands 2, 3, 5 and 7 (Ccl2, 3, 5 and 7) mRNA abundance between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 10 Control, N = 14 DNT5). On the right, linear random effects model analysis showing the standardised mean difference and confidence intervals. Data below the zero line indicate decrease in the DNT5 group (n/N = 8/10 Control, n/N = 8/14 DNT5).
    Figure Legend Snippet: Increased adiponectin and reduced inflammatory gene expression in adipose tissue of DNT5 mice. All data were from quantitative real-time PCR analysis of epididymal fat pad RNA after mice had been provided with western-style diet and DOX for 12 weeks. All mice were ApoE −/− . ( A ) Comparison of adiponectin (AdipoQ) and leptin (Lep) mRNA abundance between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 10 Control, N = 14 DNT5). On the right, linear random effects model analysis showing the standardised mean difference and confidence intervals (n/N = 8/10 Control, n/N = 8/14 DNT5). Data above the zero line indicate increase in the DNT5 group. ( B ) Comparison of the inflammatory mediators tumour necrosis factor α (Tnfα), interleukin 6 (Il6), interleukin 1β (Il1β) and C-C motif chemokine ligands 2, 3, 5 and 7 (Ccl2, 3, 5 and 7) mRNA abundance between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 10 Control, N = 14 DNT5). On the right, linear random effects model analysis showing the standardised mean difference and confidence intervals. Data below the zero line indicate decrease in the DNT5 group (n/N = 8/10 Control, n/N = 8/14 DNT5).

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

    Reduced hepatic gene expression for lipogenesis mediators in DNT5 mice. All data were from mice which had been provided with western-style diet and DOX for 12 weeks. All mice were ApoE −/− . ( A ) Example liver sections stained with H E. Scale bar 100 μm. ( B ) Comparison of lipid content (steatosis) between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 11 Control, N = 14 DNT5). On the right, linear random effects model showing the standardised mean difference and confidence intervals. Data below the zero line indicate decrease in the DNT5 group (n/N = 8/11 Control, n/N = 8/13 DNT5). ( C ) All data were from quantitative real-time PCR analysis of liver RNA. Comparison of expression of sterol regulatory element-binding protein 1c gene (Srebp1c), acetyl-CoA carboxylase 1 gene (Acaca), fatty acid synthase gene (Fasn) and stearoyl-CoA desaturase 1 gene (Scd1) mRNA abundance between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 11 Control, N = 14 DNT5). On the right linear random effects model showing the standardised mean difference and confidence intervals. Data below the zero line indicate decrease in the DNT5 group (n/N = 8/11 Control, n/N = 8/13 DNT5). ( D ) Comparison of non-fasting plasma concentrations of cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides between Controls and DNT5 after 12 weeks of western-style diet and DOX administration. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 11 Control, N = 14 DNT5). On the right, linear random effects model showing the standardised mean difference and confidence intervals (n/N = 8/11 Control, n/N = 8/14 DNT5).
    Figure Legend Snippet: Reduced hepatic gene expression for lipogenesis mediators in DNT5 mice. All data were from mice which had been provided with western-style diet and DOX for 12 weeks. All mice were ApoE −/− . ( A ) Example liver sections stained with H E. Scale bar 100 μm. ( B ) Comparison of lipid content (steatosis) between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 11 Control, N = 14 DNT5). On the right, linear random effects model showing the standardised mean difference and confidence intervals. Data below the zero line indicate decrease in the DNT5 group (n/N = 8/11 Control, n/N = 8/13 DNT5). ( C ) All data were from quantitative real-time PCR analysis of liver RNA. Comparison of expression of sterol regulatory element-binding protein 1c gene (Srebp1c), acetyl-CoA carboxylase 1 gene (Acaca), fatty acid synthase gene (Fasn) and stearoyl-CoA desaturase 1 gene (Scd1) mRNA abundance between Controls and DNT5. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 11 Control, N = 14 DNT5). On the right linear random effects model showing the standardised mean difference and confidence intervals. Data below the zero line indicate decrease in the DNT5 group (n/N = 8/11 Control, n/N = 8/13 DNT5). ( D ) Comparison of non-fasting plasma concentrations of cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides between Controls and DNT5 after 12 weeks of western-style diet and DOX administration. On the left, Mann-Whitney analysis showing mean and s.e.m (N = 11 Control, N = 14 DNT5). On the right, linear random effects model showing the standardised mean difference and confidence intervals (n/N = 8/11 Control, n/N = 8/14 DNT5).

    Techniques Used: Expressing, Mouse Assay, Western Blot, Staining, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Binding Assay

    21) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus-Induced Angiogenin Plays Roles in Latency via the Phospholipase C? Pathway: Blocking Angiogenin Inhibits Latent Gene Expression and Induces the Lytic Cycle ▿Kaposi's Sarcoma-Associated Herpesvirus-Induced Angiogenin Plays Roles in Latency via the Phospholipase C? Pathway: Blocking Angiogenin Inhibits Latent Gene Expression and Induces the Lytic Cycle ▿ †"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus-Induced Angiogenin Plays Roles in Latency via the Phospholipase C? Pathway: Blocking Angiogenin Inhibits Latent Gene Expression and Induces the Lytic Cycle ▿Kaposi's Sarcoma-Associated Herpesvirus-Induced Angiogenin Plays Roles in Latency via the Phospholipase C? Pathway: Blocking Angiogenin Inhibits Latent Gene Expression and Induces the Lytic Cycle ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.01532-10

    Effect of neomycin treatment on TIVE-LTC cell survival and viral gene expression. (A) TIVE-LTC cells were treated with increasing concentrations of neomycin for 3 days and assayed for cell survival by MTT assay. Paromomycin (100 μM) treatment was used as a control. (B) The trypan blue assay was done with TIVE and TIVE-LTC cells at 0, 1, 2, and 3 days posttreatment with 200 μM neomycin, and the results are represented as the percentage of cell survival by considering the number of cells in untreated cells on each day as 100%. (C and D) cDNA prepared from TIVE-LTC cells left untreated (UT) or treated with 200 μM neomycin after 24, 36, and 48 h was used for measurement of ORF73 (C) and ORF50 (D) gene expression by quantitative real-time PCR and normalized to expression of tubulin. (E) TIVE-LTC cells treated with the indicated concentrations of neomycin for 3 days were stained with antibodies against ORF73, ORF59, and gpK8.1A. Cells were washed with PBS, incubated with secondary antibody conjugated with Alexa Fluor 488 (green) for 1 h at room temperature, washed, mounted, and visualized under a fluorescence microscope. Magnification, ×20. (F) TIVE-LTC cells were left untreated or treated with 200 μM neomycin for 3 days, virus was purified from the supernatant, DNA was extracted, and ORF73 DNA copy numbers were determined. **, P
    Figure Legend Snippet: Effect of neomycin treatment on TIVE-LTC cell survival and viral gene expression. (A) TIVE-LTC cells were treated with increasing concentrations of neomycin for 3 days and assayed for cell survival by MTT assay. Paromomycin (100 μM) treatment was used as a control. (B) The trypan blue assay was done with TIVE and TIVE-LTC cells at 0, 1, 2, and 3 days posttreatment with 200 μM neomycin, and the results are represented as the percentage of cell survival by considering the number of cells in untreated cells on each day as 100%. (C and D) cDNA prepared from TIVE-LTC cells left untreated (UT) or treated with 200 μM neomycin after 24, 36, and 48 h was used for measurement of ORF73 (C) and ORF50 (D) gene expression by quantitative real-time PCR and normalized to expression of tubulin. (E) TIVE-LTC cells treated with the indicated concentrations of neomycin for 3 days were stained with antibodies against ORF73, ORF59, and gpK8.1A. Cells were washed with PBS, incubated with secondary antibody conjugated with Alexa Fluor 488 (green) for 1 h at room temperature, washed, mounted, and visualized under a fluorescence microscope. Magnification, ×20. (F) TIVE-LTC cells were left untreated or treated with 200 μM neomycin for 3 days, virus was purified from the supernatant, DNA was extracted, and ORF73 DNA copy numbers were determined. **, P

    Techniques Used: Expressing, MTT Assay, Real-time Polymerase Chain Reaction, Staining, Incubation, Fluorescence, Microscopy, Purification

    Viral gene expression in si-angiogenin-treated cells. (A) BCBL-1 cells were transduced with lentivirus expressing si-RL, si-lamin, si-ang1, and si-ang2 and assayed for cell survival by the MTT assay. (B, C, and D) BCBL-1 cells were transduced with si-RL, si-lamin, si-ang1, and si-ang2 and Western blotted for phospho- and total PLC-γ (pPLCγ and tPLCγ, respectively) (B), phospho- and total AKT (pAKT and tAKT, respectively) (C), and phospho- and total ERK (pERK and tERK, respectively) (D). cDNA prepared from BCBL-1 cells (E and F) and TIVE-LTC cells (G and H) transduced with lentivirus harboring si-ang, si-RL, or si-lamin was used for quantitative real-time PCR to measure ORF73 (E and G) and ORF50 (F and H) gene expression and normalized to expression of tubulin. (I and J) HeLa cells were transfected with si-ang, si-RL, or si-lamin and infected with KSHV (10 DNA copies/cell) 24 h posttransfection. Forty-eight hours postinfection, total RNA was collected, cDNA was prepared, and ORF73 (I) and ORF50 (J) copy numbers were estimated by quantitative real-time PCR. **, P
    Figure Legend Snippet: Viral gene expression in si-angiogenin-treated cells. (A) BCBL-1 cells were transduced with lentivirus expressing si-RL, si-lamin, si-ang1, and si-ang2 and assayed for cell survival by the MTT assay. (B, C, and D) BCBL-1 cells were transduced with si-RL, si-lamin, si-ang1, and si-ang2 and Western blotted for phospho- and total PLC-γ (pPLCγ and tPLCγ, respectively) (B), phospho- and total AKT (pAKT and tAKT, respectively) (C), and phospho- and total ERK (pERK and tERK, respectively) (D). cDNA prepared from BCBL-1 cells (E and F) and TIVE-LTC cells (G and H) transduced with lentivirus harboring si-ang, si-RL, or si-lamin was used for quantitative real-time PCR to measure ORF73 (E and G) and ORF50 (F and H) gene expression and normalized to expression of tubulin. (I and J) HeLa cells were transfected with si-ang, si-RL, or si-lamin and infected with KSHV (10 DNA copies/cell) 24 h posttransfection. Forty-eight hours postinfection, total RNA was collected, cDNA was prepared, and ORF73 (I) and ORF50 (J) copy numbers were estimated by quantitative real-time PCR. **, P

    Techniques Used: Expressing, Transduction, MTT Assay, Western Blot, Planar Chromatography, Real-time Polymerase Chain Reaction, Transfection, Infection

    22) Product Images from "miR-491 Inhibits Osteosarcoma Lung Metastasis and Chemoresistance by Targeting αB-crystallin"

    Article Title: miR-491 Inhibits Osteosarcoma Lung Metastasis and Chemoresistance by Targeting αB-crystallin

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2017.05.018

    CRYAB Is a Target of miR-491 in OS (A) Sequence alignment of miR-491 with the 3′ UTR of CRYAB genes. (B) miR-491 negatively regulates CRYAB expression and its downstream signaling. MG63 cells that stably express miR-491 or miR-491-antisense were subjected to qRT-PCR and western blot analysis. (C) A 3′ UTR luciferase reporter assay for CRYAB. Wild-type (CRYAB-WT) or mutant 3′ UTR of CRYAB (CRYAB-MT) luciferase reporter constructs were transfected into MG63 cells that stably express miR-491 or miR-491-antisense. After 48 hr of transfection, the luciferase intensity was assessed. (D) The expression of miR-491 and CRYAB was inversely correlated in OS specimens. Using qRT-PCR, we analyzed the expression of miR-491 and CRYAB mRNA in human OS samples (n = 19). *p
    Figure Legend Snippet: CRYAB Is a Target of miR-491 in OS (A) Sequence alignment of miR-491 with the 3′ UTR of CRYAB genes. (B) miR-491 negatively regulates CRYAB expression and its downstream signaling. MG63 cells that stably express miR-491 or miR-491-antisense were subjected to qRT-PCR and western blot analysis. (C) A 3′ UTR luciferase reporter assay for CRYAB. Wild-type (CRYAB-WT) or mutant 3′ UTR of CRYAB (CRYAB-MT) luciferase reporter constructs were transfected into MG63 cells that stably express miR-491 or miR-491-antisense. After 48 hr of transfection, the luciferase intensity was assessed. (D) The expression of miR-491 and CRYAB was inversely correlated in OS specimens. Using qRT-PCR, we analyzed the expression of miR-491 and CRYAB mRNA in human OS samples (n = 19). *p

    Techniques Used: Sequencing, Expressing, Stable Transfection, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Assay, Mutagenesis, Construct, Transfection

    Inhibition of miR-491 Stimulates OS Metastasis and Chemoresistance In Vitro and In Vivo (A) Inhibition of miR-491 stimulated OS cell invasion in both U2OS and Saos-2 cells. Cells were transfected with negative control oligonucleotides (NC) or antisense oligonucleotides of miR-491 (ASO miR-491). After 24 hr of transfection, cells were subjected to an invasion assay. (B) Inhibition of miR-491 reduced E-cadherin expression and increased MMP-9 expression in OS cells. Indicated cells were transfected with NC or ASO miR-491. After 72 hr of transfection, cells were subjected to western blot analysis. (C) Inhibition of miR-491 stimulates OS lung metastasis in vivo. 143B cells were transfected with miR-491-antisense oligonucleotides expressing plasmid or empty vector. After 48 hr of transfection, cells were injected into the intramedullary cavity of the tibia of 6-week-old nude mice (n = 5 per group). The mice were sacrificed 4 weeks after cell injection, and the lung surface nodules were counted under microscopy. (D) Inhibition of miR-491 suppressed CDDP-induced cell growth inhibition in both U2OS and Saos-2 cells. After 24 hr of transfection with the indicated oligonucleotides, cells were plated into a 96-well plate. 12 hr after seeding, cells were incubated with or without CDDP (10 μM) for 48 hr and then subjected to an MTT assay. (E) Inhibition of miR-491 attenuated CDDP-induced apoptosis in U2OS and Saos-2 cells. 24 after transfection with the indicated oligonucleotides, cells were plated into a six-well plate. After 12 hr of seeding, cells were incubated with or without CDDP (10 μM) for 24 hr and then subjected to a flow cytometry assay. (F) Inhibition of miR-491 promotes tumor growth and induces resistance to CDDP in MG63 xenograft models. Stably expressing miR-491-antisense cells were injected subcutaneously into nude mice (n = 8 per group). After the tumor size reached approximately 100 mm 3 , the mice were started on a treatment of either PBS or CDDP (10 mg/kg body weight). The mice were sacrificed after 3 weeks of CDDP treatment, and the tumor weight was measured. (G) Apoptotic cells were detected using a TUNEL assay in the indicated xenograft tumor samples. (H) Inhibition of miR-491 promotes cancer cell proliferation in MG63 xenograft models. Tumor tissues from the MG63 xenograft model were stained with Ki-67. *p
    Figure Legend Snippet: Inhibition of miR-491 Stimulates OS Metastasis and Chemoresistance In Vitro and In Vivo (A) Inhibition of miR-491 stimulated OS cell invasion in both U2OS and Saos-2 cells. Cells were transfected with negative control oligonucleotides (NC) or antisense oligonucleotides of miR-491 (ASO miR-491). After 24 hr of transfection, cells were subjected to an invasion assay. (B) Inhibition of miR-491 reduced E-cadherin expression and increased MMP-9 expression in OS cells. Indicated cells were transfected with NC or ASO miR-491. After 72 hr of transfection, cells were subjected to western blot analysis. (C) Inhibition of miR-491 stimulates OS lung metastasis in vivo. 143B cells were transfected with miR-491-antisense oligonucleotides expressing plasmid or empty vector. After 48 hr of transfection, cells were injected into the intramedullary cavity of the tibia of 6-week-old nude mice (n = 5 per group). The mice were sacrificed 4 weeks after cell injection, and the lung surface nodules were counted under microscopy. (D) Inhibition of miR-491 suppressed CDDP-induced cell growth inhibition in both U2OS and Saos-2 cells. After 24 hr of transfection with the indicated oligonucleotides, cells were plated into a 96-well plate. 12 hr after seeding, cells were incubated with or without CDDP (10 μM) for 48 hr and then subjected to an MTT assay. (E) Inhibition of miR-491 attenuated CDDP-induced apoptosis in U2OS and Saos-2 cells. 24 after transfection with the indicated oligonucleotides, cells were plated into a six-well plate. After 12 hr of seeding, cells were incubated with or without CDDP (10 μM) for 24 hr and then subjected to a flow cytometry assay. (F) Inhibition of miR-491 promotes tumor growth and induces resistance to CDDP in MG63 xenograft models. Stably expressing miR-491-antisense cells were injected subcutaneously into nude mice (n = 8 per group). After the tumor size reached approximately 100 mm 3 , the mice were started on a treatment of either PBS or CDDP (10 mg/kg body weight). The mice were sacrificed after 3 weeks of CDDP treatment, and the tumor weight was measured. (G) Apoptotic cells were detected using a TUNEL assay in the indicated xenograft tumor samples. (H) Inhibition of miR-491 promotes cancer cell proliferation in MG63 xenograft models. Tumor tissues from the MG63 xenograft model were stained with Ki-67. *p

    Techniques Used: Inhibition, In Vitro, In Vivo, Transfection, Negative Control, Allele-specific Oligonucleotide, Invasion Assay, Expressing, Western Blot, Plasmid Preparation, Injection, Mouse Assay, Microscopy, Incubation, MTT Assay, Flow Cytometry, Cytometry, Stable Transfection, TUNEL Assay, Staining

    Overexpression of miR-491 Inhibits OS Metastasis and Enhances Chemosensitivity In Vitro and In Vivo (A) Overexpression of miR-491 inhibited OS cell invasion in both U2OS and Saos-2 cells. Cells were transfected with negative control oligonucleotides (NC) or miR-491 mimics. After 24 hr of transfection, cells were subjected to an invasion assay. (B) Overexpression of miR-491 increased E-cadherin expression and reduced MMP-9 expression in OS cells. Indicated cells were transfected with NC or miR-491 mimics. After 72 hr of transfection, cells were subjected to western blot analysis. (C) Overexpression of miR-491 enhanced CDDP-induced cell growth inhibition in both U2OS and Saos-2 cells. After 24 hr of transfection with the indicated oligonucleotides, cells were plated into a 96-well plate. 12 hr after of seeding, cells were incubated with or without CDDP (10 μM) for 48 hr and subjected to an MTT assay. (D) Overexpression of miR-491 enhanced the CDDP-induced apoptosis in both U2OS and Saos-2 cells. After 24 hr of transfection with the indicated oligonucleotides, cells were plated into a six-well plate. 12 hr after of seeding, cells were incubated with or without CDDP (10 μM) for 24 hr and then subjected to a flow cytometry assay. (E) Overexpression of miR-491 inhibits OS lung metastasis in vivo. 143B cells were transfected with miR-491 expression plasmid or empty vector. After 48 hr of transfection, cells were injected into the intramedullary cavity of the tibia of 6-week-old nude mice (n = 5 per group). The mice were sacrificed 4 weeks after cell injection, and the lung surface nodules were counted under microscopy. (F) Overexpression of miR-491 inhibits tumor growth and enhances CDDP-induced tumor growth inhibition in a xenograft model. Stably expressing miR-491 MG63 cells were injected subcutaneously into nude mice (n = 8 per group). After the tumor size reached approximately 100 mm 3 , the mice were started on a treatment of either PBS or CDDP (10 mg/kg body weight). The mice were sacrificed after 3 weeks of CDDP treatment, and the tumor weight was measured. (G) Overexpression of miR-491 inhibits cell proliferation in MG63 xenograft models. Tumor tissues from MG63 xenograft model were stained with Ki-67. (H) Apoptotic cells were detected using a TUNEL assay in the indicated xenograft tumor samples. *p
    Figure Legend Snippet: Overexpression of miR-491 Inhibits OS Metastasis and Enhances Chemosensitivity In Vitro and In Vivo (A) Overexpression of miR-491 inhibited OS cell invasion in both U2OS and Saos-2 cells. Cells were transfected with negative control oligonucleotides (NC) or miR-491 mimics. After 24 hr of transfection, cells were subjected to an invasion assay. (B) Overexpression of miR-491 increased E-cadherin expression and reduced MMP-9 expression in OS cells. Indicated cells were transfected with NC or miR-491 mimics. After 72 hr of transfection, cells were subjected to western blot analysis. (C) Overexpression of miR-491 enhanced CDDP-induced cell growth inhibition in both U2OS and Saos-2 cells. After 24 hr of transfection with the indicated oligonucleotides, cells were plated into a 96-well plate. 12 hr after of seeding, cells were incubated with or without CDDP (10 μM) for 48 hr and subjected to an MTT assay. (D) Overexpression of miR-491 enhanced the CDDP-induced apoptosis in both U2OS and Saos-2 cells. After 24 hr of transfection with the indicated oligonucleotides, cells were plated into a six-well plate. 12 hr after of seeding, cells were incubated with or without CDDP (10 μM) for 24 hr and then subjected to a flow cytometry assay. (E) Overexpression of miR-491 inhibits OS lung metastasis in vivo. 143B cells were transfected with miR-491 expression plasmid or empty vector. After 48 hr of transfection, cells were injected into the intramedullary cavity of the tibia of 6-week-old nude mice (n = 5 per group). The mice were sacrificed 4 weeks after cell injection, and the lung surface nodules were counted under microscopy. (F) Overexpression of miR-491 inhibits tumor growth and enhances CDDP-induced tumor growth inhibition in a xenograft model. Stably expressing miR-491 MG63 cells were injected subcutaneously into nude mice (n = 8 per group). After the tumor size reached approximately 100 mm 3 , the mice were started on a treatment of either PBS or CDDP (10 mg/kg body weight). The mice were sacrificed after 3 weeks of CDDP treatment, and the tumor weight was measured. (G) Overexpression of miR-491 inhibits cell proliferation in MG63 xenograft models. Tumor tissues from MG63 xenograft model were stained with Ki-67. (H) Apoptotic cells were detected using a TUNEL assay in the indicated xenograft tumor samples. *p

    Techniques Used: Over Expression, In Vitro, In Vivo, Transfection, Negative Control, Invasion Assay, Expressing, Western Blot, Inhibition, Incubation, MTT Assay, Flow Cytometry, Cytometry, Plasmid Preparation, Injection, Mouse Assay, Microscopy, Stable Transfection, Staining, TUNEL Assay

    23) Product Images from "A small amount of cyclooxygenase 2 (COX2) is constitutively expressed in platelets"

    Article Title: A small amount of cyclooxygenase 2 (COX2) is constitutively expressed in platelets

    Journal: Platelets

    doi: 10.1080/09537104.2016.1203406

    COX2 mRNA is detected in human platelets (A–B) Establishment of linear regressional equations for output variable Ct of RT-PCR and input variable logged cDNA copy number of COX2 and COX1, respectively. (C) Estimate of the COX2 mRNA level relative to that of COX1 in platelets and ovarian cell lines by RT-PCR. Total RNA samples from platelets and ovarian cell lines were assayed by RT-PCR to obtain corresponding Ct values. COX2 and COX1 mRNA copy numbers were calculated by using the above mentioned linear regressional equations and subsequent exponential transformation.
    Figure Legend Snippet: COX2 mRNA is detected in human platelets (A–B) Establishment of linear regressional equations for output variable Ct of RT-PCR and input variable logged cDNA copy number of COX2 and COX1, respectively. (C) Estimate of the COX2 mRNA level relative to that of COX1 in platelets and ovarian cell lines by RT-PCR. Total RNA samples from platelets and ovarian cell lines were assayed by RT-PCR to obtain corresponding Ct values. COX2 and COX1 mRNA copy numbers were calculated by using the above mentioned linear regressional equations and subsequent exponential transformation.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transformation Assay

    24) Product Images from "Role of phospholipase A2 (PLA2) inhibitors in attenuating apoptosis of the corneal epithelial cells and mitigation of Acanthamoeba keratitis"

    Article Title: Role of phospholipase A2 (PLA2) inhibitors in attenuating apoptosis of the corneal epithelial cells and mitigation of Acanthamoeba keratitis

    Journal: Experimental eye research

    doi: 10.1016/j.exer.2013.05.021

    MIP-133 induces cytosolic phospholipase A 2α (cPLA 2α ) up-regulation in HCORN cells. HCORN cells were treated with MIP-133 at doses 7.5, 15, and 50 μg/ml for 6, 12, and 24 hrs following which cells were processed for total RNA isolation and RT-PCR. The amount of mRNA expression was quantified by densitometry of bands in comparison to the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1A ). HCORN cells were homogenized and then cell lysates were used to measure cPLA 2α enzyme analysis (1B) . The data are mean ± SEM of three independent experiments. Asterisk indicates P values
    Figure Legend Snippet: MIP-133 induces cytosolic phospholipase A 2α (cPLA 2α ) up-regulation in HCORN cells. HCORN cells were treated with MIP-133 at doses 7.5, 15, and 50 μg/ml for 6, 12, and 24 hrs following which cells were processed for total RNA isolation and RT-PCR. The amount of mRNA expression was quantified by densitometry of bands in comparison to the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1A ). HCORN cells were homogenized and then cell lysates were used to measure cPLA 2α enzyme analysis (1B) . The data are mean ± SEM of three independent experiments. Asterisk indicates P values

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

    MIP-133 induces up-regulation of CXCL2 in HCORN cells. HCORN cells were exposed to MIP-133 at indicated doses and times, and were then processed for total RNA isolation and RT-PCR analysis. The amount of mRNA expression was quantified by densitometry of bands in comparison to GAPDH (5A) . Supernatants were collected from harvested cells and then processed for protein estimation by ELISA (5B) . The data is mean ± SEM of three independent experiments. Asterisk indicates P values
    Figure Legend Snippet: MIP-133 induces up-regulation of CXCL2 in HCORN cells. HCORN cells were exposed to MIP-133 at indicated doses and times, and were then processed for total RNA isolation and RT-PCR analysis. The amount of mRNA expression was quantified by densitometry of bands in comparison to GAPDH (5A) . Supernatants were collected from harvested cells and then processed for protein estimation by ELISA (5B) . The data is mean ± SEM of three independent experiments. Asterisk indicates P values

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

    25) Product Images from "Assessment of immunosuppressive activity of human mesenchymal stem cells using murine antigen specific CD4 and CD8 T cells in vitro"

    Article Title: Assessment of immunosuppressive activity of human mesenchymal stem cells using murine antigen specific CD4 and CD8 T cells in vitro

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/scrt339

    hMSCs inhibit expression of T-bet and GATA-3 in murine CD4 + and CD8 + T cells. Total RNA was extracted from murine T cells 48 hrs after stimulation with specific peptides and co-culturing with human bone marrow-derived mesenchymal stem cells (hMSCs). The RNA was reverse transcribed into cDNA and specifically amplified using probes for murine T-bet and GATA-3. Bars in each panel represent fold change of mRNA expression as compared with un-stimulated T cells. Results for CD4 + T cells are shown in panel A and for CD8 + T cells in panel B . Every assay was done in triplicate, values are expressed as the mean and SD of triplicates. Statistical analysis was carried out by applying the Student’s t- test. *indicates P
    Figure Legend Snippet: hMSCs inhibit expression of T-bet and GATA-3 in murine CD4 + and CD8 + T cells. Total RNA was extracted from murine T cells 48 hrs after stimulation with specific peptides and co-culturing with human bone marrow-derived mesenchymal stem cells (hMSCs). The RNA was reverse transcribed into cDNA and specifically amplified using probes for murine T-bet and GATA-3. Bars in each panel represent fold change of mRNA expression as compared with un-stimulated T cells. Results for CD4 + T cells are shown in panel A and for CD8 + T cells in panel B . Every assay was done in triplicate, values are expressed as the mean and SD of triplicates. Statistical analysis was carried out by applying the Student’s t- test. *indicates P

    Techniques Used: Expressing, Derivative Assay, Amplification

    26) Product Images from "Pan-genomic analysis of bovine monocyte-derived macrophage gene expression in response to in vitro infection with Mycobacterium avium subspecies paratuberculosis"

    Article Title: Pan-genomic analysis of bovine monocyte-derived macrophage gene expression in response to in vitro infection with Mycobacterium avium subspecies paratuberculosis

    Journal: Veterinary Research

    doi: 10.1186/1297-9716-43-25

    Real time qRT-PCR analysis of the CD40, IL1B, IL6 and TNF genes following infection with M. avium subsp. paratuberculosis . Log 2 fold-changes in expression in the M. avium subsp. paratuberculosis -infected MDM (MPTb) relative to the non-infected control MDM at all three time points are shown. All data presented is based on the conventionally-prepared cDNA template. For comparison, the expression profiles for these genes as per the microarray data are also shown. The significance of the fold-changes in expression for each gene based on the real time qRT-PCR analysis only are denoted by asterisks in the figure (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). In addition, the log 2 fold-change in expression for the non-infected control MDM at each time point relative to the 0 hour non-infected control MDM are also shown for both the microarray and real time qRT-PCR data; no significant differences in gene expression between the non-infected control MDM relative to the 0 h non-infected control was observed at each time point ( P ≥ 0.05).
    Figure Legend Snippet: Real time qRT-PCR analysis of the CD40, IL1B, IL6 and TNF genes following infection with M. avium subsp. paratuberculosis . Log 2 fold-changes in expression in the M. avium subsp. paratuberculosis -infected MDM (MPTb) relative to the non-infected control MDM at all three time points are shown. All data presented is based on the conventionally-prepared cDNA template. For comparison, the expression profiles for these genes as per the microarray data are also shown. The significance of the fold-changes in expression for each gene based on the real time qRT-PCR analysis only are denoted by asterisks in the figure (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). In addition, the log 2 fold-change in expression for the non-infected control MDM at each time point relative to the 0 hour non-infected control MDM are also shown for both the microarray and real time qRT-PCR data; no significant differences in gene expression between the non-infected control MDM relative to the 0 h non-infected control was observed at each time point ( P ≥ 0.05).

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

    27) Product Images from "Respiratory Syncytial Virus Regulates Human MicroRNAs by Using Mechanisms Involving Beta Interferon and NF-?B"

    Article Title: Respiratory Syncytial Virus Regulates Human MicroRNAs by Using Mechanisms Involving Beta Interferon and NF-?B

    Journal: mBio

    doi: 10.1128/mBio.00220-12

    let-7b is increased in MDDCs and let-7c, let-7i, and miR-30b are increased in NHBE cells after RSV inoculation. Cells were inoculated with RSV and harvested 48 h later. The microRNAs let-7b, let-7c, let-7i, miR-30b, and miR-221 were assayed in MDDCs (A) or NHBE cells (B) by real-time PCR. The relative fold change of the miRNAs was calculated by normalizing values against the GAPDH control in comparison to mock-infected cells. Data are plotted as means; error bars represent the standard errors of the means. Each point represents a biological replicate, and graphs are representative of three technical replicates. The RSV G RNA level also was assayed by real-time PCR in inoculated MDDCs and plotted as log 10 -transformed relative RNA level in comparison to mock-infected cells. The let-7b miRNA level was plotted against the RSV G RNA level (C).
    Figure Legend Snippet: let-7b is increased in MDDCs and let-7c, let-7i, and miR-30b are increased in NHBE cells after RSV inoculation. Cells were inoculated with RSV and harvested 48 h later. The microRNAs let-7b, let-7c, let-7i, miR-30b, and miR-221 were assayed in MDDCs (A) or NHBE cells (B) by real-time PCR. The relative fold change of the miRNAs was calculated by normalizing values against the GAPDH control in comparison to mock-infected cells. Data are plotted as means; error bars represent the standard errors of the means. Each point represents a biological replicate, and graphs are representative of three technical replicates. The RSV G RNA level also was assayed by real-time PCR in inoculated MDDCs and plotted as log 10 -transformed relative RNA level in comparison to mock-infected cells. The let-7b miRNA level was plotted against the RSV G RNA level (C).

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

    28) Product Images from "Down-Regulated CK8 Expression in Human Intervertebral Disc Degeneration"

    Article Title: Down-Regulated CK8 Expression in Human Intervertebral Disc Degeneration

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.5642

    qRT-PCR showed significant decreased expression of CK8 in IDD at the mRNA level. Error bars represent SEM. ∗ p
    Figure Legend Snippet: qRT-PCR showed significant decreased expression of CK8 in IDD at the mRNA level. Error bars represent SEM. ∗ p

    Techniques Used: Quantitative RT-PCR, Expressing

    29) Product Images from "Preferential Activation of the Hedgehog Pathway by Epigenetic Modulations in HPV Negative HNSCC Identified with Meta-Pathway Analysis"

    Article Title: Preferential Activation of the Hedgehog Pathway by Epigenetic Modulations in HPV Negative HNSCC Identified with Meta-Pathway Analysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078127

    Meta-pathway activity identified in 44 HNSCC and 25 UPPP samples. Relative activity of meta-pathways associated with (a) UPPP, (b) HNSCC, (c) HPV-positive, and (d) HPV-negative samples.
    Figure Legend Snippet: Meta-pathway activity identified in 44 HNSCC and 25 UPPP samples. Relative activity of meta-pathways associated with (a) UPPP, (b) HNSCC, (c) HPV-positive, and (d) HPV-negative samples.

    Techniques Used: Activity Assay

    Clustering combined DNA methylation and gene expression data from 44 HNSCC samples and 25 UPPP samples. Patterns identified with hierarchical clustering for sample-by-sample Pearson correlations for combined gene expression and log transformed DNA methylation data, colored by whether samples are normal (black, labeled “N”), HPV-positive (blue, labeled “+”) and HPV-negative (red, labeled “−”) samples.
    Figure Legend Snippet: Clustering combined DNA methylation and gene expression data from 44 HNSCC samples and 25 UPPP samples. Patterns identified with hierarchical clustering for sample-by-sample Pearson correlations for combined gene expression and log transformed DNA methylation data, colored by whether samples are normal (black, labeled “N”), HPV-positive (blue, labeled “+”) and HPV-negative (red, labeled “−”) samples.

    Techniques Used: DNA Methylation Assay, Expressing, Transformation Assay, Labeling

    30) Product Images from "Genome-wide identification and functional analyses of microRNA signatures associated with cancer pain"

    Article Title: Genome-wide identification and functional analyses of microRNA signatures associated with cancer pain

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201302797

    Manipulation of miRNA expression in lumbar DRGs in mice in vivo via intrathecal application of miRNA mimics and inhibitors Experimental scheme established in this study which enables effective knockdown/induction of miRNA expression in DRGs in vivo and analysis of tumour pain-associated behaviours. Microscopic analyses of whole-mount DRGs or cryosections showing uptake of FAM-conjugated miRNA inhibitors. Scale bar is 50 µm in all panels. Typical examples of qRT-PCR verification of efficacy of miRNA inhibitors in reversing tumour-induced upregulation of miRNAs in ipsilateral DRGs in vivo . Typical examples of qRT-PCR verification of efficacy of miRNA mimics in reversing tumour-induced downregulation of miRNAs and inducing overexpression of miRNAs in ipsilateral DRGs in vivo . In panel (C), * denotes p = 0.02 for miR-1a-3p, 0.04 for miR-34c-3p as compared to corresponding mismatch inhibitor and in panel (D), * denotes p = 0.001 for miR-370-3p and
    Figure Legend Snippet: Manipulation of miRNA expression in lumbar DRGs in mice in vivo via intrathecal application of miRNA mimics and inhibitors Experimental scheme established in this study which enables effective knockdown/induction of miRNA expression in DRGs in vivo and analysis of tumour pain-associated behaviours. Microscopic analyses of whole-mount DRGs or cryosections showing uptake of FAM-conjugated miRNA inhibitors. Scale bar is 50 µm in all panels. Typical examples of qRT-PCR verification of efficacy of miRNA inhibitors in reversing tumour-induced upregulation of miRNAs in ipsilateral DRGs in vivo . Typical examples of qRT-PCR verification of efficacy of miRNA mimics in reversing tumour-induced downregulation of miRNAs and inducing overexpression of miRNAs in ipsilateral DRGs in vivo . In panel (C), * denotes p = 0.02 for miR-1a-3p, 0.04 for miR-34c-3p as compared to corresponding mismatch inhibitor and in panel (D), * denotes p = 0.001 for miR-370-3p and

    Techniques Used: Expressing, Mouse Assay, In Vivo, Quantitative RT-PCR, Over Expression

    31) Product Images from "Skeletal Muscle Cells Express ICAM-1 after Muscle Overload and ICAM-1 Contributes to the Ensuing Hypertrophic Response"

    Article Title: Skeletal Muscle Cells Express ICAM-1 after Muscle Overload and ICAM-1 Contributes to the Ensuing Hypertrophic Response

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0058486

    Protein synthesis, measured using the nonradioactive SUnSET technique, in plantaris muscles. A) Representative western blot (50 ug of protein/lane) of puromycin in control (CT) and 7 d overloaded muscles of wild type (WT) and ICAM-1-/- (IC) mice. B) Coomassie blue stained 10% SDS PAGE gel containing the same samples shown in panel A. C) Quantitative analysis of the relative abundance of puromycin incorporation into proteins (n = 7-8/group). #, significant interaction at 7 d of overload. *, significantly elevated at 7 d of overload compared to controls for wild type mice.
    Figure Legend Snippet: Protein synthesis, measured using the nonradioactive SUnSET technique, in plantaris muscles. A) Representative western blot (50 ug of protein/lane) of puromycin in control (CT) and 7 d overloaded muscles of wild type (WT) and ICAM-1-/- (IC) mice. B) Coomassie blue stained 10% SDS PAGE gel containing the same samples shown in panel A. C) Quantitative analysis of the relative abundance of puromycin incorporation into proteins (n = 7-8/group). #, significant interaction at 7 d of overload. *, significantly elevated at 7 d of overload compared to controls for wild type mice.

    Techniques Used: Western Blot, Mouse Assay, Staining, SDS Page

    Measures of skeletal muscle hypertrophy. A) Wet plantaris mass in control and overloaded mice (n = 16-20/group). B) Total protein in muscle homogenates of control (n = 6/group) and overloaded mice (n = 8/group). C) Mean cross-sectional area of normal myofibers in control (n = 1813 and 1697 myofibers for wild type and ICAM-1-/- mice, respectively)/strain) and 14 d overloaded (n = 2617 and 1096 myofibers for wild type and ICAM-1-/- mice, respectively) muscles. D) Frequency distribution of the size of normal myofibers at 14 d of overload #, significant interaction at 14 and 21 d of overload. *, significantly higher at 14 and/or 21 d of overload relative to respective controls.
    Figure Legend Snippet: Measures of skeletal muscle hypertrophy. A) Wet plantaris mass in control and overloaded mice (n = 16-20/group). B) Total protein in muscle homogenates of control (n = 6/group) and overloaded mice (n = 8/group). C) Mean cross-sectional area of normal myofibers in control (n = 1813 and 1697 myofibers for wild type and ICAM-1-/- mice, respectively)/strain) and 14 d overloaded (n = 2617 and 1096 myofibers for wild type and ICAM-1-/- mice, respectively) muscles. D) Frequency distribution of the size of normal myofibers at 14 d of overload #, significant interaction at 14 and 21 d of overload. *, significantly higher at 14 and/or 21 d of overload relative to respective controls.

    Techniques Used: Mouse Assay

    Satellite cells/myoblasts and regenerating myofibers after muscle overload. A) Representative western blot (30 ug of protein/lane) of Pax7 (60 kDa) in control (CT) and overloaded (3, 7 and 14 d) muscles of wild type (WT) and ICAM-1-/- (IC) mice. B) Quantitative analysis of the relative abundance of Pax7 protein (n = 6–8/group). #, significant interaction at 3 d of overload. *, significantly elevated at each overload time point compared to control levels for both wild type and ICAM-1-/- mice. C) Regenerating myofibers expressed as a percentage of the total number of myofibers. #, significant interaction at 7 d of overload.
    Figure Legend Snippet: Satellite cells/myoblasts and regenerating myofibers after muscle overload. A) Representative western blot (30 ug of protein/lane) of Pax7 (60 kDa) in control (CT) and overloaded (3, 7 and 14 d) muscles of wild type (WT) and ICAM-1-/- (IC) mice. B) Quantitative analysis of the relative abundance of Pax7 protein (n = 6–8/group). #, significant interaction at 3 d of overload. *, significantly elevated at each overload time point compared to control levels for both wild type and ICAM-1-/- mice. C) Regenerating myofibers expressed as a percentage of the total number of myofibers. #, significant interaction at 7 d of overload.

    Techniques Used: Western Blot, Mouse Assay

    ICAM-1 expression by cultured skeletal muscle cells. A) Representative flow cytometric quadrant plots of control and TNF-α treated (10 ng/ml for 24 h) cultures of myoblasts. ICAM-1 was detected using a phycoerythrin (PE) conjugated antibody; whereas, myoblasts were identified using an AlexaFlour® 649 (AF649) α7 antibody. ICAM-1 was not expressed by C2C12 and primary myoblasts in control cultures; whereas, TNF-α treatment caused ICAM-1 to be expressed by 30% and 15% of C2C12 and primary myoblasts, respectively. B) Representative western blots for ICAM-1 expression in C212 and primary cells treated with differentiation medium for up to 6 d (20 ug of protein/lane). A cell lysate of a myeloid cell line (RAW 264.7 cells; 5 ug of protein) was used a positive control (+CT). Membranes were probed for α-tubulin (50 kDa) to serve as a control for sample loading. ICAM-1 was not detected in differentiating or differentiated cultures. C) Representative western blot for ICAM-1 after treating proliferating myoblasts or differentiated myotubes (4 d in differentiation medium) with TNF-α (10 ng/ml for 24 h) (20 ug of protein/lane). TNF-α treatment resulted in a ICAM-1 band that was of the same molecule weight (110 kDa) as those found in plantaris muscles from control (CT) and 7 d overloaded (7 d) wild type mice (15 ug of protein/lane).
    Figure Legend Snippet: ICAM-1 expression by cultured skeletal muscle cells. A) Representative flow cytometric quadrant plots of control and TNF-α treated (10 ng/ml for 24 h) cultures of myoblasts. ICAM-1 was detected using a phycoerythrin (PE) conjugated antibody; whereas, myoblasts were identified using an AlexaFlour® 649 (AF649) α7 antibody. ICAM-1 was not expressed by C2C12 and primary myoblasts in control cultures; whereas, TNF-α treatment caused ICAM-1 to be expressed by 30% and 15% of C2C12 and primary myoblasts, respectively. B) Representative western blots for ICAM-1 expression in C212 and primary cells treated with differentiation medium for up to 6 d (20 ug of protein/lane). A cell lysate of a myeloid cell line (RAW 264.7 cells; 5 ug of protein) was used a positive control (+CT). Membranes were probed for α-tubulin (50 kDa) to serve as a control for sample loading. ICAM-1 was not detected in differentiating or differentiated cultures. C) Representative western blot for ICAM-1 after treating proliferating myoblasts or differentiated myotubes (4 d in differentiation medium) with TNF-α (10 ng/ml for 24 h) (20 ug of protein/lane). TNF-α treatment resulted in a ICAM-1 band that was of the same molecule weight (110 kDa) as those found in plantaris muscles from control (CT) and 7 d overloaded (7 d) wild type mice (15 ug of protein/lane).

    Techniques Used: Expressing, Cell Culture, Flow Cytometry, Western Blot, Positive Control, Mouse Assay

    ICAM-1 localization in 7 and 14 d overloaded muscles of wild type mice. Representative images from confocal microscopy. A) ICAM-1 (green) was found to be co-localized to CD31+ endothelial cells (cyan) and to be associated with the membrane of myofibers (WGA+; red). Cells (DAPI+; blue) in the interstitium were also found to express ICAM-1 (arrow). Column labeled as “MERGED” represents an overlay of the ICAM-1, CD31, WGA and DAPI images. B) Higher magnification clearly revealed the colocalization of ICAM-1 (green) with the membrane marker WGA (red) in overloaded muscles. Column labeled as “MERGED” include images of ICAM-1, WGA, and DAPI.
    Figure Legend Snippet: ICAM-1 localization in 7 and 14 d overloaded muscles of wild type mice. Representative images from confocal microscopy. A) ICAM-1 (green) was found to be co-localized to CD31+ endothelial cells (cyan) and to be associated with the membrane of myofibers (WGA+; red). Cells (DAPI+; blue) in the interstitium were also found to express ICAM-1 (arrow). Column labeled as “MERGED” represents an overlay of the ICAM-1, CD31, WGA and DAPI images. B) Higher magnification clearly revealed the colocalization of ICAM-1 (green) with the membrane marker WGA (red) in overloaded muscles. Column labeled as “MERGED” include images of ICAM-1, WGA, and DAPI.

    Techniques Used: Mouse Assay, Confocal Microscopy, Whole Genome Amplification, Labeling, Marker

    ICAM-1 and CD11b localization in 7 d overloaded muscle of wild type mice. Representative images from confocal microscopy. ICAM-1 (green) and CD11b (red) co-localized on the membrane of myofibers (arrow) and CD11b+ cells were closely associated with ICAM-1+ myofibers (arrowhead). Numerous CD11b+ cells residing in overloaded muscles expressed ICAM-1.
    Figure Legend Snippet: ICAM-1 and CD11b localization in 7 d overloaded muscle of wild type mice. Representative images from confocal microscopy. ICAM-1 (green) and CD11b (red) co-localized on the membrane of myofibers (arrow) and CD11b+ cells were closely associated with ICAM-1+ myofibers (arrowhead). Numerous CD11b+ cells residing in overloaded muscles expressed ICAM-1.

    Techniques Used: Mouse Assay, Confocal Microscopy

    Gene and protein expression of ICAM-1. A) Fold change in ICAM-1 gene expression (n = 8/group). Gene transcripts tended (p = 0.054) to be lower at 14 d relative to 3 d of overload for both strains of mice. B) Representative western blot of ICAM-1 (110 kDa) in control and overloaded (3, 7, and 14 d) muscles of wild type and CD18-/- mice (15 ug of protein/lane). C) Quantitative analysis of ICAM-1 protein (n = 7/group). *, significantly elevated at each overload time point compared to control levels for both strains of mice.
    Figure Legend Snippet: Gene and protein expression of ICAM-1. A) Fold change in ICAM-1 gene expression (n = 8/group). Gene transcripts tended (p = 0.054) to be lower at 14 d relative to 3 d of overload for both strains of mice. B) Representative western blot of ICAM-1 (110 kDa) in control and overloaded (3, 7, and 14 d) muscles of wild type and CD18-/- mice (15 ug of protein/lane). C) Quantitative analysis of ICAM-1 protein (n = 7/group). *, significantly elevated at each overload time point compared to control levels for both strains of mice.

    Techniques Used: Expressing, Mouse Assay, Western Blot

    ICAM-1 expression by satellite cells/myoblasts. A) Confocal images of wild type muscle overloaded for 7 d. Arrow indicates a presumptive satellite cell that is positive for ICAM-1. B) Representative plots from flow cytometric analysis of cells isolated from control and 7 d overloaded muscles of wild type mice. ICAM-1 was detected using a phycoerythrin (PE) conjugated antibody; whereas, satellite cells/myoblasts were identified using an AlexaFlour® 649 (AF649) α7 antibody. Satellite cells/myoblasts were operational defined as CD45 neg CD31 neg integrin α7+cells. C) Flow cytometric determination of ICAM-1 expression by satellite cells/myoblasts (CD45 neg CD31 neg integrin α7+ICAM-1+) expressed relative to muscle mass (mg). *, significantly higher for 7 d overloaded muscles (n = 6) compared to control muscles (n = 4).
    Figure Legend Snippet: ICAM-1 expression by satellite cells/myoblasts. A) Confocal images of wild type muscle overloaded for 7 d. Arrow indicates a presumptive satellite cell that is positive for ICAM-1. B) Representative plots from flow cytometric analysis of cells isolated from control and 7 d overloaded muscles of wild type mice. ICAM-1 was detected using a phycoerythrin (PE) conjugated antibody; whereas, satellite cells/myoblasts were identified using an AlexaFlour® 649 (AF649) α7 antibody. Satellite cells/myoblasts were operational defined as CD45 neg CD31 neg integrin α7+cells. C) Flow cytometric determination of ICAM-1 expression by satellite cells/myoblasts (CD45 neg CD31 neg integrin α7+ICAM-1+) expressed relative to muscle mass (mg). *, significantly higher for 7 d overloaded muscles (n = 6) compared to control muscles (n = 4).

    Techniques Used: Expressing, Flow Cytometry, Isolation, Mouse Assay

    Myeloid cell accumulation in plantaris muscles. A) Neutrophil concentrations (Ly6G+ cells/mm 3 ; n = 6–8/group). *, elevated relative to respective controls. #, higher for wild type compared to ICAM-1-/- mice at 7 d of overload. B) Macrophage concentrations (F4/80+ cells/mm 3 ; n = 6-8/group). *, elevated relative to controls.
    Figure Legend Snippet: Myeloid cell accumulation in plantaris muscles. A) Neutrophil concentrations (Ly6G+ cells/mm 3 ; n = 6–8/group). *, elevated relative to respective controls. #, higher for wild type compared to ICAM-1-/- mice at 7 d of overload. B) Macrophage concentrations (F4/80+ cells/mm 3 ; n = 6-8/group). *, elevated relative to controls.

    Techniques Used: Mouse Assay

    ICAM-1 localization in control muscle of wild type mice. Representative images from confocal microscopy. A) ICAM-1 (green), B) WGA (membrane marker; red) and ICAM-1 (green), C) CD31 (endothelial cell marker; purple), and D) Merged image of ICAM-1 (panel A), and CD31 (panel C). Co-localization analysis revealed that the majority (90–95%) of the ICAM-1 labeling in control muscles was expressed by CD31+ endothelial cells. ICAM-1 was not found on the membrane of myofibers in control muscles.
    Figure Legend Snippet: ICAM-1 localization in control muscle of wild type mice. Representative images from confocal microscopy. A) ICAM-1 (green), B) WGA (membrane marker; red) and ICAM-1 (green), C) CD31 (endothelial cell marker; purple), and D) Merged image of ICAM-1 (panel A), and CD31 (panel C). Co-localization analysis revealed that the majority (90–95%) of the ICAM-1 labeling in control muscles was expressed by CD31+ endothelial cells. ICAM-1 was not found on the membrane of myofibers in control muscles.

    Techniques Used: Mouse Assay, Confocal Microscopy, Whole Genome Amplification, Marker, Labeling

    32) Product Images from "1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) Signaling Capacity and the Epithelial-Mesenchymal Transition in Non-Small Cell Lung Cancer (NSCLC): Implications for Use of 1,25(OH)2D3 in NSCLC Treatment"

    Article Title: 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) Signaling Capacity and the Epithelial-Mesenchymal Transition in Non-Small Cell Lung Cancer (NSCLC): Implications for Use of 1,25(OH)2D3 in NSCLC Treatment

    Journal: Cancers

    doi: 10.3390/cancers5041504

    NSCLC cells that are VDR high and vitamin D-sensitive preferentially express markers of an epithelial phenotype. The indicated NSCLC cell lines were grown under basal growth conditions until they achieved 50%–70% confluence. ( A ) RNA was extracted and used to prepare cDNA. One microliter of each cDNA was used in a quantitative PCR assay to measure expression of representative epithelial markers ( CDH1 , SCNN1A , and EPCAM ) and mesencyhmal markers ( ZEB1 , VIM , and LIX1L ). Data are the mean ± SD for triplicate determinations within a single experiment. Data were normalized to that obtained for H3122 cells. H3122 gene expression was arbitrarily assigned a value of 1.0. VDR high cells are indicated with white bars. VDR low cells are indicated with black bars; ( B ) Protein was extracted 24 h post-seeding of 5 × 10 6 cells and analyzed by immunoblot for VDR, E-cadherin and VIM. Thirty micrograms of total protein was analyzed per sample.
    Figure Legend Snippet: NSCLC cells that are VDR high and vitamin D-sensitive preferentially express markers of an epithelial phenotype. The indicated NSCLC cell lines were grown under basal growth conditions until they achieved 50%–70% confluence. ( A ) RNA was extracted and used to prepare cDNA. One microliter of each cDNA was used in a quantitative PCR assay to measure expression of representative epithelial markers ( CDH1 , SCNN1A , and EPCAM ) and mesencyhmal markers ( ZEB1 , VIM , and LIX1L ). Data are the mean ± SD for triplicate determinations within a single experiment. Data were normalized to that obtained for H3122 cells. H3122 gene expression was arbitrarily assigned a value of 1.0. VDR high cells are indicated with white bars. VDR low cells are indicated with black bars; ( B ) Protein was extracted 24 h post-seeding of 5 × 10 6 cells and analyzed by immunoblot for VDR, E-cadherin and VIM. Thirty micrograms of total protein was analyzed per sample.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    33) Product Images from "Effects of Direct Renin Inhibition on Myocardial Fibrosis and Cardiac Fibroblast Function"

    Article Title: Effects of Direct Renin Inhibition on Myocardial Fibrosis and Cardiac Fibroblast Function

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081612

    Effects of aliskiren on myocardial matrix gene expression. Real time PCR was performed with ventricular tissue obtained from mice after 12-inducing diet and different doses of aliskiren. Figure 3A demonstrates changes in expression of COL1A1(alpha 1 chain of type I collagen) and 3B shows changes in COL1A2(alpha2 chain of type I collagen) genes. Lower right panels (3C and 3D) demonstrate the results of western blotting for collagen type I in various groups. Hhe – Hhe-inducing diet; AL 0.5– aliskiren 0.5 mg/kg/day; AL 5– aliskiren 5 mg/kg/day; and AL 50– aliskiren 50 mg/kg/day. * p
    Figure Legend Snippet: Effects of aliskiren on myocardial matrix gene expression. Real time PCR was performed with ventricular tissue obtained from mice after 12-inducing diet and different doses of aliskiren. Figure 3A demonstrates changes in expression of COL1A1(alpha 1 chain of type I collagen) and 3B shows changes in COL1A2(alpha2 chain of type I collagen) genes. Lower right panels (3C and 3D) demonstrate the results of western blotting for collagen type I in various groups. Hhe – Hhe-inducing diet; AL 0.5– aliskiren 0.5 mg/kg/day; AL 5– aliskiren 5 mg/kg/day; and AL 50– aliskiren 50 mg/kg/day. * p

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

    Effects of aliskiren on homocysteine-induced matrix expression in cultured cardiac fibroblasts. Fibroblasts were cultured in the presence or absence of D,L, homocysteine 200 µmol/L for 24 hours after pretreatment with varying concentrations of aliskiren (0.1–10 µmol/L). Real time PCR was done to measure relative expression of COLIA2 (alpha 2 chain of type I collagen; A) and COL3A1 (alpha 1 chain of type III collagen; B) genes. Figure 5C demonstrates the results of real time PCR to compare the effects of losartan and captopril with aliskiren on homocysteine induced expression of COL1A2 gene. Western blotting was conducted to measure the expression of collagen type I protein (D and E) Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as internal control. Hcy-D,L, homocysteine; AL-aliskiren; Los – losartan; and Cap – captopril. *p
    Figure Legend Snippet: Effects of aliskiren on homocysteine-induced matrix expression in cultured cardiac fibroblasts. Fibroblasts were cultured in the presence or absence of D,L, homocysteine 200 µmol/L for 24 hours after pretreatment with varying concentrations of aliskiren (0.1–10 µmol/L). Real time PCR was done to measure relative expression of COLIA2 (alpha 2 chain of type I collagen; A) and COL3A1 (alpha 1 chain of type III collagen; B) genes. Figure 5C demonstrates the results of real time PCR to compare the effects of losartan and captopril with aliskiren on homocysteine induced expression of COL1A2 gene. Western blotting was conducted to measure the expression of collagen type I protein (D and E) Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was used as internal control. Hcy-D,L, homocysteine; AL-aliskiren; Los – losartan; and Cap – captopril. *p

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

    34) Product Images from "Targeting Gastrin-Releasing Peptide Suppresses Neuroblastoma Progression via Upregulation of PTEN Signaling"

    Article Title: Targeting Gastrin-Releasing Peptide Suppresses Neuroblastoma Progression via Upregulation of PTEN Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072570

    GRP silencing on PTEN/AKT/mTOR signaling and MYCN, TWIST, FAK. (A) BE(2)-C/Tet/shGRP (+DOX) cells and SH-SY5Y/Tet/shGRP (+DOX) cells had an increase in PTEN expression along with correlative decreases in pAKT and pmTOR expression when compared to control cells (without doxycycline; -DOX). (B) Doxycycline-induced GRP silencing in BE (2)-C/Tet/shGRP cells decreased the mRNA levels of MYCN , TWIST and FAK by ∼50–60% as assessed by QRT-PCR (mean ± SEM; * = p
    Figure Legend Snippet: GRP silencing on PTEN/AKT/mTOR signaling and MYCN, TWIST, FAK. (A) BE(2)-C/Tet/shGRP (+DOX) cells and SH-SY5Y/Tet/shGRP (+DOX) cells had an increase in PTEN expression along with correlative decreases in pAKT and pmTOR expression when compared to control cells (without doxycycline; -DOX). (B) Doxycycline-induced GRP silencing in BE (2)-C/Tet/shGRP cells decreased the mRNA levels of MYCN , TWIST and FAK by ∼50–60% as assessed by QRT-PCR (mean ± SEM; * = p

    Techniques Used: Expressing, Quantitative RT-PCR

    35) Product Images from "Upregulation of Voltage-Gated Calcium Channel Cav1.3 in Bovine Somatotropes Treated with Ghrelin"

    Article Title: Upregulation of Voltage-Gated Calcium Channel Cav1.3 in Bovine Somatotropes Treated with Ghrelin

    Journal: Journal of Signal Transduction

    doi: 10.1155/2013/527253

    Expression of L-type Ca 2+ channel α 1 subunits mRNA in bovine somatotropes. (a) Electrophoresis of L-type Ca 2+ channel a 1.2 and 1.3 subunits mRNA, bGH, and β -actin (B-Ac). (b) Summary of three independent experiments; the principal subunit expressed is 1.3. (c) Electrophoresis of somatotropes cells treated by GHRP-6 100 nM for 48 h. (d) Summary of three independent experiments; GHRP-6 increases the mRNA for HVA calcium channels L-type 1.3 and the mRNA for bGH.
    Figure Legend Snippet: Expression of L-type Ca 2+ channel α 1 subunits mRNA in bovine somatotropes. (a) Electrophoresis of L-type Ca 2+ channel a 1.2 and 1.3 subunits mRNA, bGH, and β -actin (B-Ac). (b) Summary of three independent experiments; the principal subunit expressed is 1.3. (c) Electrophoresis of somatotropes cells treated by GHRP-6 100 nM for 48 h. (d) Summary of three independent experiments; GHRP-6 increases the mRNA for HVA calcium channels L-type 1.3 and the mRNA for bGH.

    Techniques Used: Expressing, Electrophoresis

    36) Product Images from "The Role of the Staphylococcal VraTSR Regulatory System on Vancomycin Resistance and vanA Operon Expression in Vancomycin-Resistant Staphylococcus aureus"

    Article Title: The Role of the Staphylococcal VraTSR Regulatory System on Vancomycin Resistance and vanA Operon Expression in Vancomycin-Resistant Staphylococcus aureus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085873

    Effect of deletion of vraTSR on vanA and vanR gene induction by vancomycin. Expression of vanA operon genes in wildtype VRSA clinical strain (VRS1), the vraTSR deletion mutant (VRS1Δvra) and the vraTSR -complemented mutant (VRS1cΔvra), as measured by qRT-PCR. Vancomycin was added to early log cultures (when cultures reached an OD 600 of 0.2) to induce vanA expression and RNA was isolated 1 hour later. Expression of each gene target was evaluated using relative quantification (RQ) with the comparative ΔΔCt method using strain VRS1 treated with vancomycin as the reference (after each probe was normalized to an endogenous control). Error bars reflect the range of RQ values from 3 experimental triplicates. The vanA gene probe was labeled with FAM and used in duplex reactions with the 16S rRNA gene probe labeled with fluorophore Cy5 as the endogenous control. A FAM-labled vanR probe was used in a duplex reaction with a gyrB probe labeled with fluorophore Cy5 as the endogenous control. The choice of the endogenous controls were based on compatibility with the target in the duplex reaction.
    Figure Legend Snippet: Effect of deletion of vraTSR on vanA and vanR gene induction by vancomycin. Expression of vanA operon genes in wildtype VRSA clinical strain (VRS1), the vraTSR deletion mutant (VRS1Δvra) and the vraTSR -complemented mutant (VRS1cΔvra), as measured by qRT-PCR. Vancomycin was added to early log cultures (when cultures reached an OD 600 of 0.2) to induce vanA expression and RNA was isolated 1 hour later. Expression of each gene target was evaluated using relative quantification (RQ) with the comparative ΔΔCt method using strain VRS1 treated with vancomycin as the reference (after each probe was normalized to an endogenous control). Error bars reflect the range of RQ values from 3 experimental triplicates. The vanA gene probe was labeled with FAM and used in duplex reactions with the 16S rRNA gene probe labeled with fluorophore Cy5 as the endogenous control. A FAM-labled vanR probe was used in a duplex reaction with a gyrB probe labeled with fluorophore Cy5 as the endogenous control. The choice of the endogenous controls were based on compatibility with the target in the duplex reaction.

    Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR, Isolation, Labeling

    37) Product Images from "Comparison of Epithelial Differentiation and Immune Regulatory Properties of Mesenchymal Stromal Cells Derived from Human Lung and Bone Marrow"

    Article Title: Comparison of Epithelial Differentiation and Immune Regulatory Properties of Mesenchymal Stromal Cells Derived from Human Lung and Bone Marrow

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035639

    Epithelial differentiation. ( A ) Morphology changes after epithelial differentiation. Inverted phase-contrast microscopy (Zeiss Observer.Z1; 100x) of BM-hMSCs and lung-hMSCs without and with RA treatment ( Ctrl; +RA ). ( B ) Evaluation of epithelial differentiation by qRT-PCR on BM-hMSCs (light grey) and lung-hMSCs (dark grey), before (basal) and after RA treatment for the induction of epithelial differentiation. Panel shows the expression of the epithelial genes cytokeratin 18 (krt18), tight junction protein (tjp1), also named zona occludens 1, occludin (ocln) and mesenchymal genes vimentin (vim) and e-cadherin repressor snai1. Data are normalized on actb (beta actin) expression and related to basal BM-hMSCs. The error bars represent standard deviation; * = p
    Figure Legend Snippet: Epithelial differentiation. ( A ) Morphology changes after epithelial differentiation. Inverted phase-contrast microscopy (Zeiss Observer.Z1; 100x) of BM-hMSCs and lung-hMSCs without and with RA treatment ( Ctrl; +RA ). ( B ) Evaluation of epithelial differentiation by qRT-PCR on BM-hMSCs (light grey) and lung-hMSCs (dark grey), before (basal) and after RA treatment for the induction of epithelial differentiation. Panel shows the expression of the epithelial genes cytokeratin 18 (krt18), tight junction protein (tjp1), also named zona occludens 1, occludin (ocln) and mesenchymal genes vimentin (vim) and e-cadherin repressor snai1. Data are normalized on actb (beta actin) expression and related to basal BM-hMSCs. The error bars represent standard deviation; * = p

    Techniques Used: Microscopy, Quantitative RT-PCR, Expressing, Standard Deviation

    Characterization of lung-hMSCs. ( A ) Morphology of lung-hMSCs. Inverted phase-contrast microscopy of primary colonies of lung-hMSCs ( left panel ) and expanded colonies at confluence ( right panel ). ( B ) ( left panel ) Characterization by qRT-PCR of stemness profile of cells extracted from lung tissues (dark grey columns) in comparison with BM-hMSCs (ligh grey columns). Relative gene expression fold induction of nestin (nes), smad4, nanog, pou5f1(or Oct4), sox2 and tdgf1 (or Cripto). Gene expression is normalized on actb gene (beta actin) and related to BM-hMSCs as basal expression. Mean ± SD; * = p
    Figure Legend Snippet: Characterization of lung-hMSCs. ( A ) Morphology of lung-hMSCs. Inverted phase-contrast microscopy of primary colonies of lung-hMSCs ( left panel ) and expanded colonies at confluence ( right panel ). ( B ) ( left panel ) Characterization by qRT-PCR of stemness profile of cells extracted from lung tissues (dark grey columns) in comparison with BM-hMSCs (ligh grey columns). Relative gene expression fold induction of nestin (nes), smad4, nanog, pou5f1(or Oct4), sox2 and tdgf1 (or Cripto). Gene expression is normalized on actb gene (beta actin) and related to BM-hMSCs as basal expression. Mean ± SD; * = p

    Techniques Used: Microscopy, Quantitative RT-PCR, Expressing

    38) Product Images from "Dose-Dependent Activation of Putative Oncogene SBSN by BORIS"

    Article Title: Dose-Dependent Activation of Putative Oncogene SBSN by BORIS

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0040389

    Changes in histone modifications around SBSN TSS upon BORIS induction. (A) Enrichment of active chromatin modification, H3K4me3, near the SBSN TSS was measured by ChIP experiment with H3K4me3 antibodies for H358 transfected with BORIS and induced by indicated doxycycline concentrations. The region from +202 bp to +374 bp was analyzed. Note that cell lysates from the same experiments was used for all ChIP experiments ( Figure 2C ). Enrichment level was measured relative to IgG in qRT-PCR as described in the Methods. (B) Enrichment of active chromatin modification – H3Ac. (C) Enrichment of repressive chromatin modification, H3K9me3. (*, p-value
    Figure Legend Snippet: Changes in histone modifications around SBSN TSS upon BORIS induction. (A) Enrichment of active chromatin modification, H3K4me3, near the SBSN TSS was measured by ChIP experiment with H3K4me3 antibodies for H358 transfected with BORIS and induced by indicated doxycycline concentrations. The region from +202 bp to +374 bp was analyzed. Note that cell lysates from the same experiments was used for all ChIP experiments ( Figure 2C ). Enrichment level was measured relative to IgG in qRT-PCR as described in the Methods. (B) Enrichment of active chromatin modification – H3Ac. (C) Enrichment of repressive chromatin modification, H3K9me3. (*, p-value

    Techniques Used: Modification, Chromatin Immunoprecipitation, Transfection, Quantitative RT-PCR

    SBSN is a target of dose-dependent BORIS transcriptional regulation. (A) Schematic structure of the SBSN gene. The relative positions of exons, CpG islands, and CTCF/BORIS (BORIS) binding sites are shown in black, white and grey, respectively. Transcription start sites (TSS) at the +1 position are indicated by arrows. TSS-relative positions of primers used in qRT-PCR reactions from ChIP-purified DNA are indicated by arrow heads. (B) Relative BORIS mRNA level in the H358 cell line after transient transfection of BORIS. BORIS expression was induced by indicated concentrations of doxycycline (dox) 24 hours after transfection with control empty vector (EV) or with BORIS expressing vector. Expression was quantified relative to GAPDH with the control referred as 1 (*, p-value
    Figure Legend Snippet: SBSN is a target of dose-dependent BORIS transcriptional regulation. (A) Schematic structure of the SBSN gene. The relative positions of exons, CpG islands, and CTCF/BORIS (BORIS) binding sites are shown in black, white and grey, respectively. Transcription start sites (TSS) at the +1 position are indicated by arrows. TSS-relative positions of primers used in qRT-PCR reactions from ChIP-purified DNA are indicated by arrow heads. (B) Relative BORIS mRNA level in the H358 cell line after transient transfection of BORIS. BORIS expression was induced by indicated concentrations of doxycycline (dox) 24 hours after transfection with control empty vector (EV) or with BORIS expressing vector. Expression was quantified relative to GAPDH with the control referred as 1 (*, p-value

    Techniques Used: Binding Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Purification, Transfection, Expressing, Plasmid Preparation

    39) Product Images from "SIV replication is directly downregulated by four antiviral miRNAs"

    Article Title: SIV replication is directly downregulated by four antiviral miRNAs

    Journal: Retrovirology

    doi: 10.1186/1742-4690-10-95

    SIV infection increases levels of mature miR- 29a (A), -29b (B), -9 (C) and -146a (D). Macaque macrophages were infected with SIV (MOI 0.05). Cells were harvested at 2, 4, 8, 12, 24 and 48 hours after infection and RNA was isolated. Taqman miRNA RT-qPCR assays were used to measure levels of mature miRNAs. Results were normalized to U6, then to uninfected samples for the individual time points (ΔΔCq method). Values are expressed as fold induction of miRNAs over uninfected controls using the ΔΔCq method and data shown is an average of at least 3 experiments.
    Figure Legend Snippet: SIV infection increases levels of mature miR- 29a (A), -29b (B), -9 (C) and -146a (D). Macaque macrophages were infected with SIV (MOI 0.05). Cells were harvested at 2, 4, 8, 12, 24 and 48 hours after infection and RNA was isolated. Taqman miRNA RT-qPCR assays were used to measure levels of mature miRNAs. Results were normalized to U6, then to uninfected samples for the individual time points (ΔΔCq method). Values are expressed as fold induction of miRNAs over uninfected controls using the ΔΔCq method and data shown is an average of at least 3 experiments.

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

    TNFα increases levels of mature miRNAs and primary miRNA transcripts in human macrophages. Human macrophages were treated with 20 ng/ml TNFα. Cells were harvested at 2, 4, 8, 12 and 24 hours after treatment and RNA isolated. (A-D) Taqman miRNA RT-qPCR assays were used to measure levels of mature miRNAs. (E-H) Taqman pri-miRNA RT-qPCR assays were used to measure levels of primary miRNA transcripts. Results were normalized to U6. Values are expressed as fold induction of miRNAs over uninfected controls using the ΔΔCq method and data shown is an average of at least 3 experiments.
    Figure Legend Snippet: TNFα increases levels of mature miRNAs and primary miRNA transcripts in human macrophages. Human macrophages were treated with 20 ng/ml TNFα. Cells were harvested at 2, 4, 8, 12 and 24 hours after treatment and RNA isolated. (A-D) Taqman miRNA RT-qPCR assays were used to measure levels of mature miRNAs. (E-H) Taqman pri-miRNA RT-qPCR assays were used to measure levels of primary miRNA transcripts. Results were normalized to U6. Values are expressed as fold induction of miRNAs over uninfected controls using the ΔΔCq method and data shown is an average of at least 3 experiments.

    Techniques Used: Isolation, miRNA RT, Real-time Polymerase Chain Reaction

    Four miRNAs reduce levels of full length and multiply spliced viral RNAs. Primary macaque macrophages were transfected with 100 nM of either miRNA mimics or antagonists, then infected with SIV (MOI 0.05) 24 hours after transfection. Cell lysates were collected at 24, 48 and 72 hours post-infection for miRNA experiments and 48 hours for miRNA antagonist experiments. Levels of full length (A, C) and spliced (B) RNA was measured by RT-qPCR. Values are expressed as fold change over SIV-only controls. Results are reported as percent of SIV-only control using the ΔΔCq method.
    Figure Legend Snippet: Four miRNAs reduce levels of full length and multiply spliced viral RNAs. Primary macaque macrophages were transfected with 100 nM of either miRNA mimics or antagonists, then infected with SIV (MOI 0.05) 24 hours after transfection. Cell lysates were collected at 24, 48 and 72 hours post-infection for miRNA experiments and 48 hours for miRNA antagonist experiments. Levels of full length (A, C) and spliced (B) RNA was measured by RT-qPCR. Values are expressed as fold change over SIV-only controls. Results are reported as percent of SIV-only control using the ΔΔCq method.

    Techniques Used: Transfection, Infection, Quantitative RT-PCR

    miRs-29a, -29b, -9 and -146a reduce expression of a luciferase reporter and target predicted binding sites in the 3′ UTR of SIV. (A) SIV nef / U3 - R was cloned into psiCHECK2 plasmid (Promega). 293T cells were co-transfection with 50 nM or 100 nM of each miRNA or scrambled mimic and 100 ng of either psiCHECK+SIV or psiCHECK alone. Control samples were transfected with either psiCHECK or psiCHECK+SIV, and no miRNA mimics. Cells were harvested 24 hours after transfection. Data shown is an average of four experiments and represents miRNA effect on luciferase expression for psiCHECK+SIV over psiCHECK only. (B) WT and mutant biotin-labeled oligos corresponding to SIV RNA sequences containing predicted miRNA binding sites. WT oligo is show on top and mutant on bottom. Boxed bases on WT oligo denote miRNA seed binding site in SIV sequence. Mutant oligo highlighted bases denote the change in seed-binding region. Annotation is based on SIV sequence AY033146. (C) 100 pmol of individual WT or mt oligos were transfected into HeLa cells and lysates collected 24 hours after transfection. Oligos were pulled down using Streptavidin beads and assayed for binding to endogenous cellular miRNAs using Taqman RT-qPCR. Percent input was calculated for each sample (see methods) and data presented as (% input mt )/(% input WT) with % of input of WT set to 100%. In each experiment, the value of the oligo which pulled down the highest percentage of individual endogenous miRNA was set to 100% and binding by other oligos was compared to this value. Data shown is an average of 4 experiments. Statistics represent the comparison of percent of miRNA bound to WT oligo compared to percent of miRNA pulled down by corresponding mt oligo and are reported as a two-tailed t test assuming unequal variance.
    Figure Legend Snippet: miRs-29a, -29b, -9 and -146a reduce expression of a luciferase reporter and target predicted binding sites in the 3′ UTR of SIV. (A) SIV nef / U3 - R was cloned into psiCHECK2 plasmid (Promega). 293T cells were co-transfection with 50 nM or 100 nM of each miRNA or scrambled mimic and 100 ng of either psiCHECK+SIV or psiCHECK alone. Control samples were transfected with either psiCHECK or psiCHECK+SIV, and no miRNA mimics. Cells were harvested 24 hours after transfection. Data shown is an average of four experiments and represents miRNA effect on luciferase expression for psiCHECK+SIV over psiCHECK only. (B) WT and mutant biotin-labeled oligos corresponding to SIV RNA sequences containing predicted miRNA binding sites. WT oligo is show on top and mutant on bottom. Boxed bases on WT oligo denote miRNA seed binding site in SIV sequence. Mutant oligo highlighted bases denote the change in seed-binding region. Annotation is based on SIV sequence AY033146. (C) 100 pmol of individual WT or mt oligos were transfected into HeLa cells and lysates collected 24 hours after transfection. Oligos were pulled down using Streptavidin beads and assayed for binding to endogenous cellular miRNAs using Taqman RT-qPCR. Percent input was calculated for each sample (see methods) and data presented as (% input mt )/(% input WT) with % of input of WT set to 100%. In each experiment, the value of the oligo which pulled down the highest percentage of individual endogenous miRNA was set to 100% and binding by other oligos was compared to this value. Data shown is an average of 4 experiments. Statistics represent the comparison of percent of miRNA bound to WT oligo compared to percent of miRNA pulled down by corresponding mt oligo and are reported as a two-tailed t test assuming unequal variance.

    Techniques Used: Expressing, Luciferase, Binding Assay, Clone Assay, Plasmid Preparation, Cotransfection, Transfection, Mutagenesis, Labeling, Sequencing, Quantitative RT-PCR, Two Tailed Test

    IFNβ increases levels of mature miRNAs and primary miRNA transcripts in human macrophages. Human macrophages were treated with 100 U/ml IFNβ and cells were harvested at 4, 8, 12 and 24 hours after treatment and RNA isolated. (A-D) Taqman miRNA RT-qPCR assays were used to measure levels of mature miRNAs. (E-H) Taqman pri-miRNA RT-qPCR assays were used to measure levels of primary miRNA transcripts. Results were normalized to U6. Values are expressed as fold induction of miRNAs over uninfected controls using the ΔΔCq method and data shown is an average of at least 3 experiments.
    Figure Legend Snippet: IFNβ increases levels of mature miRNAs and primary miRNA transcripts in human macrophages. Human macrophages were treated with 100 U/ml IFNβ and cells were harvested at 4, 8, 12 and 24 hours after treatment and RNA isolated. (A-D) Taqman miRNA RT-qPCR assays were used to measure levels of mature miRNAs. (E-H) Taqman pri-miRNA RT-qPCR assays were used to measure levels of primary miRNA transcripts. Results were normalized to U6. Values are expressed as fold induction of miRNAs over uninfected controls using the ΔΔCq method and data shown is an average of at least 3 experiments.

    Techniques Used: Isolation, miRNA RT, Real-time Polymerase Chain Reaction

    40) Product Images from "Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines"

    Article Title: Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines

    Journal: PeerJ

    doi: 10.7717/peerj.153

    Long-term exposure of BeWo and Jar cells to CBD: changes in BCRP and P-gp mRNA levels. (A), (C) changes in BCRP mRNA in BeWo and Jar cells (respectively). (B), (D) changes in P-gp mRNA in BeWo and Jar cells (respectively). Values are given as fold of change compared to control. Data is displayed as means ± s.d. of at least three ( n = 6) independent experiments for each time point. One-Way ANOVA, followed by Bonferroni’s multiple comparison test. ∗ p
    Figure Legend Snippet: Long-term exposure of BeWo and Jar cells to CBD: changes in BCRP and P-gp mRNA levels. (A), (C) changes in BCRP mRNA in BeWo and Jar cells (respectively). (B), (D) changes in P-gp mRNA in BeWo and Jar cells (respectively). Values are given as fold of change compared to control. Data is displayed as means ± s.d. of at least three ( n = 6) independent experiments for each time point. One-Way ANOVA, followed by Bonferroni’s multiple comparison test. ∗ p

    Techniques Used:

    Related Articles

    Flow Cytometry:

    Article Title: NLRC3 negatively regulates CD4+ T cells and impacts protective immunity during Mycobacterium tuberculosis infection
    Article Snippet: .. Nlrc3 expression Macrophages (Gr-1- CD11b+ ), polymorphonuclear leukocytes (PMNs) (Gr-1+ CD11b+ ), dendritic cells (CD11c+ MHC-IIhi ), CD4+ T cells (CD3+ CD4+ ) and CD8+ T cells (CD3+ CD8+ ) were purified by flow cytometry sorting and total RNA was isolated with Trizol (Invitrogen, USA) according to the manufacturer’s instructions. .. 1 mg of RNA was reverse transcribed to cDNA with random RNAspecific primers using the high-capacity cDNA reverse transcription kit (Applied Biosystems).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway
    Article Snippet: .. For the measurement of Dkk3 mRNA expression, the first-strand cDNA was synthesized from 1 μg of total RNA by a High-Capacity cDNA Archive Kit (Applied Biosystems) and RT-PCR was conducted using a SYBR Premix Ex Taq II Kit (Applied Biosystems). qRT-PCR reaction was carried out using a 7500 Fast Real-Time Sequence detection system (Applied Biosystems). .. The relative gene expression of miR-214 and Dkk3 was calculated using the 2−ΔΔCt method.

    Expressing:

    Article Title: miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway
    Article Snippet: .. For the measurement of Dkk3 mRNA expression, the first-strand cDNA was synthesized from 1 μg of total RNA by a High-Capacity cDNA Archive Kit (Applied Biosystems) and RT-PCR was conducted using a SYBR Premix Ex Taq II Kit (Applied Biosystems). qRT-PCR reaction was carried out using a 7500 Fast Real-Time Sequence detection system (Applied Biosystems). .. The relative gene expression of miR-214 and Dkk3 was calculated using the 2−ΔΔCt method.

    Article Title: NLRC3 negatively regulates CD4+ T cells and impacts protective immunity during Mycobacterium tuberculosis infection
    Article Snippet: .. Nlrc3 expression Macrophages (Gr-1- CD11b+ ), polymorphonuclear leukocytes (PMNs) (Gr-1+ CD11b+ ), dendritic cells (CD11c+ MHC-IIhi ), CD4+ T cells (CD3+ CD4+ ) and CD8+ T cells (CD3+ CD8+ ) were purified by flow cytometry sorting and total RNA was isolated with Trizol (Invitrogen, USA) according to the manufacturer’s instructions. .. 1 mg of RNA was reverse transcribed to cDNA with random RNAspecific primers using the high-capacity cDNA reverse transcription kit (Applied Biosystems).

    Article Title: Direct and indirect pro-inflammatory cytokine response resulting from TC-83 infection of glial cells
    Article Snippet: .. Expression of IL-1β was determined using the ΔΔCτ method with Taqman reagents for IL-1β (Thermo Fisher HS01555410_m1) with 18S as the endogenous control (Thermo Fisher Hs99999901_s1). .. Caspase-3/-7 detection assay Caspase-3/-7 activity was quantitated using the Caspase-Glo 3/7 Assay (Promega G8090) according to the manufacturer’s instructions.

    Stable Transfection:

    Article Title: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer
    Article Snippet: .. To generate A549 cells stably overexpressing circPTK2, we cotransfected the above-mentioned construct or empty vector with packaging plasmids psPAX2 and pMD2.G (Geneseed Biotech) into HEK 293 T cells using Lipofectamine 2000 (Invitrogen). .. After HEK 293 T cells were cultured for 48 h, the packaged lentiviruses were harvested.

    Synthesized:

    Article Title: miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway
    Article Snippet: .. For the measurement of Dkk3 mRNA expression, the first-strand cDNA was synthesized from 1 μg of total RNA by a High-Capacity cDNA Archive Kit (Applied Biosystems) and RT-PCR was conducted using a SYBR Premix Ex Taq II Kit (Applied Biosystems). qRT-PCR reaction was carried out using a 7500 Fast Real-Time Sequence detection system (Applied Biosystems). .. The relative gene expression of miR-214 and Dkk3 was calculated using the 2−ΔΔCt method.

    Mutagenesis:

    Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
    Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

    Isolation:

    Article Title: KDELR2 Competes with Measles Virus Envelope Proteins for Cellular Chaperones Reducing Their Chaperone-Mediated Cell Surface Transport
    Article Snippet: .. Isolated RNA was reverse transcribed in cDNA using the RevertAid first strand cDNA synthesis kit (Fermentas). ..

    Article Title: NLRC3 negatively regulates CD4+ T cells and impacts protective immunity during Mycobacterium tuberculosis infection
    Article Snippet: .. Nlrc3 expression Macrophages (Gr-1- CD11b+ ), polymorphonuclear leukocytes (PMNs) (Gr-1+ CD11b+ ), dendritic cells (CD11c+ MHC-IIhi ), CD4+ T cells (CD3+ CD4+ ) and CD8+ T cells (CD3+ CD8+ ) were purified by flow cytometry sorting and total RNA was isolated with Trizol (Invitrogen, USA) according to the manufacturer’s instructions. .. 1 mg of RNA was reverse transcribed to cDNA with random RNAspecific primers using the high-capacity cDNA reverse transcription kit (Applied Biosystems).

    Transfection:

    Article Title: 25-Hydroxycholesterol and 27-hydroxycholesterol inhibit human rotavirus infection by sequestering viral particles into late endosomes
    Article Snippet: .. Briefly, extraction of total RNA from transfected or untransfected Caco2 cells was performed 72 h after transfection using TRIzol Reagent (Applied Biosystems, Monza, Italy). .. Concentration and purity of the extracted RNA were assessed by spectrophotometry (A260/A280).

    Construct:

    Article Title: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer
    Article Snippet: .. To generate A549 cells stably overexpressing circPTK2, we cotransfected the above-mentioned construct or empty vector with packaging plasmids psPAX2 and pMD2.G (Geneseed Biotech) into HEK 293 T cells using Lipofectamine 2000 (Invitrogen). .. After HEK 293 T cells were cultured for 48 h, the packaged lentiviruses were harvested.

    Cytometry:

    Article Title: NLRC3 negatively regulates CD4+ T cells and impacts protective immunity during Mycobacterium tuberculosis infection
    Article Snippet: .. Nlrc3 expression Macrophages (Gr-1- CD11b+ ), polymorphonuclear leukocytes (PMNs) (Gr-1+ CD11b+ ), dendritic cells (CD11c+ MHC-IIhi ), CD4+ T cells (CD3+ CD4+ ) and CD8+ T cells (CD3+ CD8+ ) were purified by flow cytometry sorting and total RNA was isolated with Trizol (Invitrogen, USA) according to the manufacturer’s instructions. .. 1 mg of RNA was reverse transcribed to cDNA with random RNAspecific primers using the high-capacity cDNA reverse transcription kit (Applied Biosystems).

    Real-time Polymerase Chain Reaction:

    Article Title: Exosomal miR-21-5p derived from gastric cancer promotes peritoneal metastasis via mesothelial-to-mesenchymal transition
    Article Snippet: .. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from tissues, cultured cells and exosomes using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. .. Then RNA was reverse transcribed into cDNA using PrimeScript RT Reagent (TaKaRa, Japan).

    Sequencing:

    Article Title: miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway
    Article Snippet: .. For the measurement of Dkk3 mRNA expression, the first-strand cDNA was synthesized from 1 μg of total RNA by a High-Capacity cDNA Archive Kit (Applied Biosystems) and RT-PCR was conducted using a SYBR Premix Ex Taq II Kit (Applied Biosystems). qRT-PCR reaction was carried out using a 7500 Fast Real-Time Sequence detection system (Applied Biosystems). .. The relative gene expression of miR-214 and Dkk3 was calculated using the 2−ΔΔCt method.

    Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
    Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

    Cell Culture:

    Article Title: Exosomal miR-21-5p derived from gastric cancer promotes peritoneal metastasis via mesothelial-to-mesenchymal transition
    Article Snippet: .. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from tissues, cultured cells and exosomes using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. .. Then RNA was reverse transcribed into cDNA using PrimeScript RT Reagent (TaKaRa, Japan).

    Quantitative RT-PCR:

    Article Title: miR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/β-catenin signaling pathway
    Article Snippet: .. For the measurement of Dkk3 mRNA expression, the first-strand cDNA was synthesized from 1 μg of total RNA by a High-Capacity cDNA Archive Kit (Applied Biosystems) and RT-PCR was conducted using a SYBR Premix Ex Taq II Kit (Applied Biosystems). qRT-PCR reaction was carried out using a 7500 Fast Real-Time Sequence detection system (Applied Biosystems). .. The relative gene expression of miR-214 and Dkk3 was calculated using the 2−ΔΔCt method.

    Article Title: Exosomal miR-21-5p derived from gastric cancer promotes peritoneal metastasis via mesothelial-to-mesenchymal transition
    Article Snippet: .. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from tissues, cultured cells and exosomes using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. .. Then RNA was reverse transcribed into cDNA using PrimeScript RT Reagent (TaKaRa, Japan).

    High Throughput Screening Assay:

    Article Title: Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV
    Article Snippet: .. High-throughput sequencing of RNAs and sRNAs The total RNA samples were prepared from WT and DCL2b mutant adult leaves using TRIzol reagent (Invitrogen, USA). .. Paired-end mRNA libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations and were sequenced on an Illumina HiSeq 4000 platform; 150 bp reads were generated.

    Purification:

    Article Title: NLRC3 negatively regulates CD4+ T cells and impacts protective immunity during Mycobacterium tuberculosis infection
    Article Snippet: .. Nlrc3 expression Macrophages (Gr-1- CD11b+ ), polymorphonuclear leukocytes (PMNs) (Gr-1+ CD11b+ ), dendritic cells (CD11c+ MHC-IIhi ), CD4+ T cells (CD3+ CD4+ ) and CD8+ T cells (CD3+ CD8+ ) were purified by flow cytometry sorting and total RNA was isolated with Trizol (Invitrogen, USA) according to the manufacturer’s instructions. .. 1 mg of RNA was reverse transcribed to cDNA with random RNAspecific primers using the high-capacity cDNA reverse transcription kit (Applied Biosystems).

    Plasmid Preparation:

    Article Title: Circular RNA hsa_circ_0008305 (circPTK2) inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by controlling TIF1γ in non-small cell lung cancer
    Article Snippet: .. To generate A549 cells stably overexpressing circPTK2, we cotransfected the above-mentioned construct or empty vector with packaging plasmids psPAX2 and pMD2.G (Geneseed Biotech) into HEK 293 T cells using Lipofectamine 2000 (Invitrogen). .. After HEK 293 T cells were cultured for 48 h, the packaged lentiviruses were harvested.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher high capacity cdna reverse transcription kit
    RNF138 reduces Ca V 2.1 protein stability. A , Lack of effect of RNF138/RNF138-H36E overexpression on human Ca V 2.1 mRNA level in HEK293T cells subject to the indicated transfection condition ( p > 0.05; n = 3). To rule out the potential contamination arising from human Ca V 2.1 plasmid in <t>RNA</t> prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of <t>cDNA</t> template (vertical arrows). The signals of Ca V 2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B , RNF138 knock-down does not significantly change rat Ca V 2.1 mRNA level in neurons ( p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized Ca V 2.1 signals were normalized to the shGFP infection control. C , Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human Ca V 2.1 subunit. Ca V 2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D , Quantification of Ca V 2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized Ca V 2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of Ca V 2.1 protein half-life values derived from linear-regression analyses. The estimated Ca V 2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated Ca V 2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E , Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF13 8 on Ca V 2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F , Quantification and comparison of Ca V 2.1 protein half-life values derived from different shRNA infection conditions. The estimated Ca V 2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of Ca V 2.1 in the presence of shGFP is not statistically different ( p > 0.05) from that of Ca V 2.1 with vector in D . Asterisks denote significant difference from the control (* p
    High Capacity Cdna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high capacity cdna reverse transcription kit/product/Thermo Fisher
    Average 99 stars, based on 10121 article reviews
    Price from $9.99 to $1999.99
    high capacity cdna reverse transcription kit - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    91
    Thermo Fisher mrna species
    eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 <t>mRNA</t> (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), <t>RNA-sequencing</t> ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.
    Mrna Species, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrna species/product/Thermo Fisher
    Average 91 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    mrna species - by Bioz Stars, 2020-07
    91/100 stars
      Buy from Supplier

    Image Search Results


    RNF138 reduces Ca V 2.1 protein stability. A , Lack of effect of RNF138/RNF138-H36E overexpression on human Ca V 2.1 mRNA level in HEK293T cells subject to the indicated transfection condition ( p > 0.05; n = 3). To rule out the potential contamination arising from human Ca V 2.1 plasmid in RNA prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of cDNA template (vertical arrows). The signals of Ca V 2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B , RNF138 knock-down does not significantly change rat Ca V 2.1 mRNA level in neurons ( p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized Ca V 2.1 signals were normalized to the shGFP infection control. C , Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human Ca V 2.1 subunit. Ca V 2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D , Quantification of Ca V 2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized Ca V 2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of Ca V 2.1 protein half-life values derived from linear-regression analyses. The estimated Ca V 2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated Ca V 2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E , Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF13 8 on Ca V 2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F , Quantification and comparison of Ca V 2.1 protein half-life values derived from different shRNA infection conditions. The estimated Ca V 2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of Ca V 2.1 in the presence of shGFP is not statistically different ( p > 0.05) from that of Ca V 2.1 with vector in D . Asterisks denote significant difference from the control (* p

    Journal: The Journal of Neuroscience

    Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels

    doi: 10.1523/JNEUROSCI.3070-16.2017

    Figure Lengend Snippet: RNF138 reduces Ca V 2.1 protein stability. A , Lack of effect of RNF138/RNF138-H36E overexpression on human Ca V 2.1 mRNA level in HEK293T cells subject to the indicated transfection condition ( p > 0.05; n = 3). To rule out the potential contamination arising from human Ca V 2.1 plasmid in RNA prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of cDNA template (vertical arrows). The signals of Ca V 2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B , RNF138 knock-down does not significantly change rat Ca V 2.1 mRNA level in neurons ( p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized Ca V 2.1 signals were normalized to the shGFP infection control. C , Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human Ca V 2.1 subunit. Ca V 2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D , Quantification of Ca V 2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized Ca V 2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of Ca V 2.1 protein half-life values derived from linear-regression analyses. The estimated Ca V 2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated Ca V 2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E , Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF13 8 on Ca V 2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F , Quantification and comparison of Ca V 2.1 protein half-life values derived from different shRNA infection conditions. The estimated Ca V 2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of Ca V 2.1 in the presence of shGFP is not statistically different ( p > 0.05) from that of Ca V 2.1 with vector in D . Asterisks denote significant difference from the control (* p

    Article Snippet: RNA was reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Scientific).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Cell Culture, shRNA, Infection, Western Blot, Transformation Assay, Derivative Assay

    Cloning of EXA1 homologs in Nicotiana benthamiana and their functional analysis in potexvirus infection. ( a ) Schematic image of the cDNA structure of NbEXA1a . GYF domain- and eIF4E-binding motif-encoding regions are depicted by stripes. Target regions for virus-induced gene silencing (VIGS) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) are indicated by bars under the image. ( b ) Morphological phenotypes of NbEXA1 -silenced and control plants. Photographs were taken from the top (upper) and side (bottom) of plants at 27 days post-inoculation (dpi). Bars = 5 cm. ( c ) Relative accumulation of NbEXA1 mRNA in NbEXA1 -silenced and control plants. Total RNA was extracted at 27 dpi and analyzed using qRT-PCR. The mean level of NbEXA1 transcript in control plants was used as the standard (1.0), and that in NbEXA1 -silenced plants is shown above the bar. Error bars indicate standard deviation (SD) of 10 samples. ** P

    Journal: Scientific Reports

    Article Title: Functional conservation of EXA1 among diverse plant species for the infection by a family of plant viruses

    doi: 10.1038/s41598-019-42400-w

    Figure Lengend Snippet: Cloning of EXA1 homologs in Nicotiana benthamiana and their functional analysis in potexvirus infection. ( a ) Schematic image of the cDNA structure of NbEXA1a . GYF domain- and eIF4E-binding motif-encoding regions are depicted by stripes. Target regions for virus-induced gene silencing (VIGS) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) are indicated by bars under the image. ( b ) Morphological phenotypes of NbEXA1 -silenced and control plants. Photographs were taken from the top (upper) and side (bottom) of plants at 27 days post-inoculation (dpi). Bars = 5 cm. ( c ) Relative accumulation of NbEXA1 mRNA in NbEXA1 -silenced and control plants. Total RNA was extracted at 27 dpi and analyzed using qRT-PCR. The mean level of NbEXA1 transcript in control plants was used as the standard (1.0), and that in NbEXA1 -silenced plants is shown above the bar. Error bars indicate standard deviation (SD) of 10 samples. ** P

    Article Snippet: RNA isolation, RT-PCR, and qRT-PCR Total RNA was extracted from the plants using ISOGEN (Nippon Gene) and treated with DNase I (Takara Bio, Shiga, Japan), or using the ISOSPIN Plant RNA Kit (Nippongene) following the manufacturer’s instructions. cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Clone Assay, Functional Assay, Infection, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation

    HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 cDNA fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p

    Journal: Molecular Metabolism

    Article Title: Deletion of iRhom2 protects against diet-induced obesity by increasing thermogenesis

    doi: 10.1016/j.molmet.2019.10.006

    Figure Lengend Snippet: HFD-fed iRhom2 KO mice have increased thermogenesis and browning of the white adipose tissue. A-C Thermal images (A), and BAT (B) or body (C) temperature of iRhom2 KO and WT mice fed with SD and HFD for 19 weeks. One experiment with 8 mice per group. D RT-PCR analysis of UCP1, PGC1α, Cidea, PRDM16, and Cox8b expression in SD-fed iRhom2 KO and HFD-fed WT and iRhom2 KO mice BAT samples compared to WT SD-fed control samples. Two experiments with 3–4 replicates in each. E, G Representative photographs of eWAT (E) and sWAT (G) UCP1 immunohistochemistry derived from iRhom2 KO and WT mice fed with HFD for 20 weeks. Scale bar = 100 μm. F, H Graphics showing the UCP1 percentage of area stained in the eWAT (F) and sWAT (H) of the mice described above. One experiment with 8 mice per group (with 2 photographs analyzed per mouse). I RT-PCR analysis of UCP1 in brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT or iRhom2 KO mice. Three independent experiments. J RT-PCR analysis of UCP1 in immortalized WT brown preadipocytes transduced with empty vector or iRhom2-HA, differentiated in vitro and stimulated with norepinephrine for 6 h. Two independent experiments. K HA expression in differentiated immortalized WT brown preadipocytes transduced with retrovirus containing iRhom2 cDNA fused to C-terminal HA tag (iR2-HA). As a negative control, we used the same cells transduced with retrovirus containing the empty vector (EV), and as a loading control we measured p97 protein level. Two independent experiments. L Mitochondrial oxygen consumption rate (OCR) of brown adipocytes differentiated in vitro from the stromal vascular fraction of 4–5 pooled WT and iRhom2 KO mice and stimulated or not with norepinephrine. The results were normalized to the protein content. Two experiments with one or two independent samples per genotype, respectively. M Mitochondrial proton leak of the cells described in L normalized to the protein content. Error bars represent SEM; * represents p

    Article Snippet: First-strand cDNA was synthesized from total RNA using the SuperScript® III First-Strand Synthesis SuperMix or the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific).

    Techniques: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, Derivative Assay, Staining, In Vitro, Transduction, Plasmid Preparation, Negative Control

    eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 mRNA (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), RNA-sequencing ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.

    Journal: PLoS Genetics

    Article Title: A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene

    doi: 10.1371/journal.pgen.1007961

    Figure Lengend Snippet: eQTL analysis of CD58 and microRNA-548ac based on three different data sets. Expression values of CD58 mRNA (labeled in green) and hsa-miR-548ac molecules (labeled in red) measured using microarrays ( A ), RNA-sequencing ( B ), and quantitative real-time PCR ( C ) were plotted for each genotype group. Genotypes 0, 1, and 2 denote the number of MS risk alleles carried, defined either by SNP rs1335532 ( A ) or SNP rs1414273 ( B and C ). The average expression level per group is indicated by a red line. Welch t -test p -values are shown above the brackets for all pairwise genotype comparisons. ( A ) HapMap cohort data (in log2 scale) demonstrated a significant relationship between the MS-associated SNP and CD58 transcript levels in independent populations (n = 82 JPT and n = 82 GIH displayed). ( B ) This could be confirmed by Geuvadis cohort data, presented here for LCLs collected from 282 individuals living in Europe. Interestingly, the eQTL effect is in the opposite direction for hsa-miR-548ac: Significantly higher levels of this miRNA were seen in individuals with increased genetic risk of MS. Numbers below the data points specify the proportion of samples (of n = 276 analyzed LCLs) with zero miRNA read counts. ( C ) Data of the regional MS cohort (n = 32) further substantiated the differences in miRNA levels among the genotypes. A non-significant positive correlation of CD58 mRNA and hsa-miR-548ac expression was found in both the RNA-sequencing data ( B ) and the real-time PCR data ( C ). ( D ) The table gives the F -test p -values calculated for the complete data of the HapMap and Geuvadis cohorts, establishing the cis -mRNA-/miR-eQTL when accounting for population structure in the analysis of covariance (ANCOVA). ANOVA = analysis of variance, eQTL = expression quantitative trait locus, FIN = Finnish in Finland, GBR = British in England and Scotland, GIH = Gujarati Indians in Houston, JPT = Japanese in Tokyo, LCL = lymphoblastoid cell line, MS = multiple sclerosis, PBMC = peripheral blood mononuclear cells, PCR = polymerase chain reaction, SLR = simple linear regression, SNP = single-nucleotide polymorphism, TSI = Toscani in Italia.

    Article Snippet: From each sample, 400 ng of total RNA was reverse transcribed with random primers for mRNA species (High-Capacity cDNA Reverse Transcription Kit), and 10 ng of total RNA was reverse transcribed with specific primers provided with each TaqMan miRNA assay to convert mature miRNAs to cDNA (TaqMan MicroRNA Reverse Transcription Kit, Thermo Fisher Scientific).

    Techniques: Expressing, Labeling, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Biogenesis of microRNA-548ac and genetic variants in the CD58 gene locus. ( A ) Diagram of the processing of an mRNA and an intronic miRNA from the same transcript (adapted from [ 49 ]). Both RNA splicing by the spliceosome and miRNA stem-loop cropping by the Drosha-DGCR8 complex occur cotranscriptionally. Drosha may cleave the miRNA-harboring intron before splicing commitment of the flanking exons. The resulting precursor miRNA is subsequently processed into a mature miRNA, which is loaded into the RNA-induced silencing complex (RISC). ( B ) Annotated secondary structure of hsa-mir-548ac. Highlighted in gray is the 22 nt long sequence of the mature miRNA isoform as assigned by Jima et al . (miRBase accession MIMAT0018938) [ 43 ]. The red circle marks the only common single-nucleotide polymorphism (SNP) within the stem-loop region. The G allele is overrepresented in MS patients. ( C ) Genetic variants in pairwise linkage disequilibrium (LD) with SNP rs1414273. This plot was generated using the LDproxy module of the web-based analysis tool LDlink [ 97 ]. Shown are r 2 LD values of proximal SNPs based on all subpopulations of the 1000 Genomes project (orange dots), recombination rate as estimated from HapMap data (gray line), and the position and exon-intron structure of nearby genes on chromosome 1 (chr1, GRCh37 assembly). The MS-associated SNP rs1335532 is in strong LD with SNP rs1414273 (blue) (correlated forward strand alleles: A = C, G = T). The entire block of LD spans about 50 kb but does not include the promoter region of CD58, which is encoded on the minus strand in the reference genome. ( D ) Worldwide distribution of SNP rs1335532 alleles. Global allele frequencies were visualized as two-color pie charts with the HGDP Selection Browser [ 98 ]. The disease susceptibility variant (A, blue) is the major allele in European populations and the minor allele in East Asian and Southern African populations. cM/Mb = centimorgan per megabase, HGDP = Human Genome Diversity Panel, MS = multiple sclerosis.

    Journal: PLoS Genetics

    Article Title: A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene

    doi: 10.1371/journal.pgen.1007961

    Figure Lengend Snippet: Biogenesis of microRNA-548ac and genetic variants in the CD58 gene locus. ( A ) Diagram of the processing of an mRNA and an intronic miRNA from the same transcript (adapted from [ 49 ]). Both RNA splicing by the spliceosome and miRNA stem-loop cropping by the Drosha-DGCR8 complex occur cotranscriptionally. Drosha may cleave the miRNA-harboring intron before splicing commitment of the flanking exons. The resulting precursor miRNA is subsequently processed into a mature miRNA, which is loaded into the RNA-induced silencing complex (RISC). ( B ) Annotated secondary structure of hsa-mir-548ac. Highlighted in gray is the 22 nt long sequence of the mature miRNA isoform as assigned by Jima et al . (miRBase accession MIMAT0018938) [ 43 ]. The red circle marks the only common single-nucleotide polymorphism (SNP) within the stem-loop region. The G allele is overrepresented in MS patients. ( C ) Genetic variants in pairwise linkage disequilibrium (LD) with SNP rs1414273. This plot was generated using the LDproxy module of the web-based analysis tool LDlink [ 97 ]. Shown are r 2 LD values of proximal SNPs based on all subpopulations of the 1000 Genomes project (orange dots), recombination rate as estimated from HapMap data (gray line), and the position and exon-intron structure of nearby genes on chromosome 1 (chr1, GRCh37 assembly). The MS-associated SNP rs1335532 is in strong LD with SNP rs1414273 (blue) (correlated forward strand alleles: A = C, G = T). The entire block of LD spans about 50 kb but does not include the promoter region of CD58, which is encoded on the minus strand in the reference genome. ( D ) Worldwide distribution of SNP rs1335532 alleles. Global allele frequencies were visualized as two-color pie charts with the HGDP Selection Browser [ 98 ]. The disease susceptibility variant (A, blue) is the major allele in European populations and the minor allele in East Asian and Southern African populations. cM/Mb = centimorgan per megabase, HGDP = Human Genome Diversity Panel, MS = multiple sclerosis.

    Article Snippet: From each sample, 400 ng of total RNA was reverse transcribed with random primers for mRNA species (High-Capacity cDNA Reverse Transcription Kit), and 10 ng of total RNA was reverse transcribed with specific primers provided with each TaqMan miRNA assay to convert mature miRNAs to cDNA (TaqMan MicroRNA Reverse Transcription Kit, Thermo Fisher Scientific).

    Techniques: Sequencing, Generated, Blocking Assay, Selection, Variant Assay