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  • 99
    Thermo Fisher high capacity rna to cdna kit
    Relative expression of TREM-1 and DAP12 genes. THP-1 cells were exposed to bacteria at multiplicity of infection (MOI) of 10 or 100. After 4 and 24 h post exposure <t>RNA</t> was extracted, <t>cDNA</t> synthesized and the relative gene expression was determined by quantitative polymerase chain reaction, as described. (A) TREM-1 at 4 h; (B) TREM-1 at 24 h, (C) DAP12 at 4 h, (D) DAP12 at 24 h. Experiments were repeated three times. Data shown are mean ± SD of triplicates from a representative experiment. * P ≤ 0.05 compared with uninfected control.
    High Capacity Rna To Cdna Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19456 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher high capacity cdna reverse transcription kit
    α 2 M expression in primary villous cytotrophoblastic cells and BeWo cells. vCTB were purified from early (8 weeks of gestation) and late first-trimester (11 weeks of gestation) trophoblast and term placenta and seeded for 24, 48, 72 and 96 h. BeWo cells were seeded and treated or not with 20 µM Forskolin (FSK) for 48 h. <t>RNA</t> was retrotranscribed, and 50 ng of <t>cDNA</t> was used to perform α 2 M and GAPDH PCR. n = 3.
    High Capacity Cdna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 119681 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher high capacity cdna archive kit
    Genomic structures of novel TUs . A. TU4. B. TU7. C. TU11. D. TU52. E. TU54. Initial paired-end ditag: dark blue. Exons of validated full-length <t>cDNA</t> sequences: orange. Exons of previously known transcripts at each locus: teal (protein-coding sequence: shaded). 3' terminal exon arrows: direction of transcription. Quantitative real-time <t>PCR</t> primer locations: red arrows. All validated cDNA sequences can be found in Additional file 2 .
    High Capacity Cdna Archive Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19389 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher high capacity reverse transcription kit
    Genomic structures of novel TUs . A. TU4. B. TU7. C. TU11. D. TU52. E. TU54. Initial paired-end ditag: dark blue. Exons of validated full-length <t>cDNA</t> sequences: orange. Exons of previously known transcripts at each locus: teal (protein-coding sequence: shaded). 3' terminal exon arrows: direction of transcription. Quantitative real-time <t>PCR</t> primer locations: red arrows. All validated cDNA sequences can be found in Additional file 2 .
    High Capacity Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 5378 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pierce streptavidin coated high capacity plates
    Glycosylation in intestinal organoids transfected with mut-AGX1 and BH-GalNAc-T2 or non-transfected, control samples. Organoids were fed with DMSO (top) or 50 µM compound 11 (bottom), fixed and treated with biotin alkyne under CuAAC conditions followed by <t>Streptavidin</t> Alexa Fluor 647 staining. Data are from one representative out of two independent experiments and shown as grayscale images for each channel and a color merge image of all three channels. Scale bar, 100 µm.
    Pierce Streptavidin Coated High Capacity Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher high capacity reverse transcriptase kit
    Glycosylation in intestinal organoids transfected with mut-AGX1 and BH-GalNAc-T2 or non-transfected, control samples. Organoids were fed with DMSO (top) or 50 µM compound 11 (bottom), fixed and treated with biotin alkyne under CuAAC conditions followed by <t>Streptavidin</t> Alexa Fluor 647 staining. Data are from one representative out of two independent experiments and shown as grayscale images for each channel and a color merge image of all three channels. Scale bar, 100 µm.
    High Capacity Reverse Transcriptase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 759 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher savant speedvac concentrator
    Glycosylation in intestinal organoids transfected with mut-AGX1 and BH-GalNAc-T2 or non-transfected, control samples. Organoids were fed with DMSO (top) or 50 µM compound 11 (bottom), fixed and treated with biotin alkyne under CuAAC conditions followed by <t>Streptavidin</t> Alexa Fluor 647 staining. Data are from one representative out of two independent experiments and shown as grayscale images for each channel and a color merge image of all three channels. Scale bar, 100 µm.
    Savant Speedvac Concentrator, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 496 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher high capacity streptavidin agarose beads
    ADAM17 cleaves the CA IX ectodomain and deletion of amino acids 393–402 in the CA IX stalk region prevents the ECD shedding. a Verification of the cleavage activity of the recombinant human TACE/ADAM17 (rhADAM17) towards the fluorogenic peptide Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Ser-Arg-NH2. The peptide was used at the final concentration of 10 μM in a total of 100 μL reaction mixture with 10 ng of the rhADAM17. Time-related increase of the fluorescence emitted from the pro-TNF-α-derived peptide proves that rhADAM17 was active. b Recombinant CA IX-SBP fusion protein attached to a <t>streptavidin-sepharose</t> matrix was cleaved by rhADAM17, eluted and detected by ELISA using CA IX-specific monoclonal antibodies. c Cells transiently expressing FL-CA IX were treated with ADAM17/10 inhibitor for 24 h and CA IX ECD was detected in culture media by ELISA. Results in b and c support the role of ADAM17 in cleavage of CA IX. d Schematic illustration of the CA IX domain structure and positions of deletions in the mutants. Scissors indicates extracellular domain cleavage region. Missing amino-acid residues of individual deletion variants are represented by numbers on the right side. SP signal peptide, PG proteoglycan-like domain, CA catalytic domain, TM transmembrane region, IC intracellular tail. e Immunofluorescence analysis of CHO-wt, CHO-M2 and CHO-M2-TACE cells transiently expressing the FL-CA IX and two stalk deletion variants. The cells were fixed with methanol, incubated with M75 antibody followed by ALEXA Fluor488-secondary antibody and nuclei were stained with DAPI. Deletion of the cleavage site did not affect the CA IX localisation. f , g ELISA analysis of the CA IX deletion variants for the ECD shedding. The plasmids encoding FL-CA IX and its two deletion mutants were transiently transfected to CHO-wt, CHO-M2 (ADAM17-defective) and CHO-M2-TACE (human ADAM17 expressing) cell lines. 48 h after transfection, the cells were cultivated for 3 h in equal medium volumes in the presence or absence of PMA. Undiluted conditioned media ( f ) and cell lysates diluted 1:10 ( g ) were collected and examined by ELISA using V/10 antibody as a capture and mixture of biotinylated MAbs M75 and IV/18 as a detector. h Biochemical evidence that ADAM17 can cleave FL-CA IX, but not the NS mutant was obtained by treatment of CHO cell variants expressing FL and NS, respectively, with recombinant rhADAM17 added to medium at a concentration of 50 μg/ml for 24 h. Collected media were analysed by ELISA. i ADAM17 suppression resulting from infection by lentiviruses expressing ADAM17-specific shRNA led to a decreased CA IX shedding from C33a-FL cells, but not from C33a-NS cells proving the involvement of ADAM17 in the CA IX ECD cleavage. The results (mean ± SD) represent the mean of two measurements in triplicates. (* P
    High Capacity Streptavidin Agarose Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Relative expression of TREM-1 and DAP12 genes. THP-1 cells were exposed to bacteria at multiplicity of infection (MOI) of 10 or 100. After 4 and 24 h post exposure RNA was extracted, cDNA synthesized and the relative gene expression was determined by quantitative polymerase chain reaction, as described. (A) TREM-1 at 4 h; (B) TREM-1 at 24 h, (C) DAP12 at 4 h, (D) DAP12 at 24 h. Experiments were repeated three times. Data shown are mean ± SD of triplicates from a representative experiment. * P ≤ 0.05 compared with uninfected control.

    Journal: Molecular oral microbiology

    Article Title: Activation of the TREM-1 pathway in human monocytes by periodontal pathogens and oral commensal bacteria

    doi: 10.1111/omi.12169

    Figure Lengend Snippet: Relative expression of TREM-1 and DAP12 genes. THP-1 cells were exposed to bacteria at multiplicity of infection (MOI) of 10 or 100. After 4 and 24 h post exposure RNA was extracted, cDNA synthesized and the relative gene expression was determined by quantitative polymerase chain reaction, as described. (A) TREM-1 at 4 h; (B) TREM-1 at 24 h, (C) DAP12 at 4 h, (D) DAP12 at 24 h. Experiments were repeated three times. Data shown are mean ± SD of triplicates from a representative experiment. * P ≤ 0.05 compared with uninfected control.

    Article Snippet: Reverse transcription was performed using the High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA) according to manufacturer’s instructions.

    Techniques: Expressing, Infection, Synthesized, Real-time Polymerase Chain Reaction

    Collagen homeostasis is disrupted in the myocardium of Col14a1 −/− mice Taqman Low Density Array was used to examine changes in mRNA levels of fibril-forming (A), network and beaded-forming (B) and FACIT-forming (C) collagens in cDNA generated from E11.5, PND1 and 3 months Col14a1 −/− and Col14a1 +/+ ( wild type ) ventricles. *, indicate p

    Journal: Journal of molecular and cellular cardiology

    Article Title: Collagen XIV is important for growth and structural integrity of the myocardium

    doi: 10.1016/j.yjmcc.2012.08.002

    Figure Lengend Snippet: Collagen homeostasis is disrupted in the myocardium of Col14a1 −/− mice Taqman Low Density Array was used to examine changes in mRNA levels of fibril-forming (A), network and beaded-forming (B) and FACIT-forming (C) collagens in cDNA generated from E11.5, PND1 and 3 months Col14a1 −/− and Col14a1 +/+ ( wild type ) ventricles. *, indicate p

    Article Snippet: 200~400 ng mRNA was used to generate cDNA using the high-capacity cDNA kit (Applied Biosystems) [ ].

    Techniques: Mouse Assay, TLDA Assay, Generated

    Clone 13 efficiently infects CD8α neg CD11c pos DC. Adult C57BL/6 were infected with LCMV Arm, or Cl13 and spleens harvested 7 days post infection. CD11c pos cells were positively selected by MACS bead isolation following CD19/90 depletion. (A) Isolated cells were stained for CD11c, CD90, CD19, CD8α and cell surface (top two rows) LCMV NP or intracellular (bottom row) LCMV NP. Gating controls are as follows: Naïve DC (dashed line), DCs from Cl13 infected mice incubated with isotype control antibody for LCMV NP Ab (filled grey histogram). (B C) CD8α neg DCs were sorted based on cell surface NP expression. (B) RNA was extracted from sorted DCs; reverse transcription was performed and generated cDNA used in Real Time PCR reaction to determine the copy number of LCMV GP, relative to known concentration of control plasmid. ND indicates below detection limits (C) 4×10 4 sorted DCs were cultured for 2 days in cRPMI. IL-10 levels were measured in culture supernatants of Cl13 infected (NP pos ), exposed (NP neg ) DCs and Naïve CD8α neg DCs. n = 4–5 mice per treatment per experiment. RealTime and ELISA data from one representative of three independent experiments is shown. Standard deviation is shown.

    Journal: PLoS ONE

    Article Title: Direct Infection of Dendritic Cells during Chronic Viral Infection Suppresses Antiviral T Cell Proliferation and Induces IL-10 Expression in CD4 T Cells

    doi: 10.1371/journal.pone.0090855

    Figure Lengend Snippet: Clone 13 efficiently infects CD8α neg CD11c pos DC. Adult C57BL/6 were infected with LCMV Arm, or Cl13 and spleens harvested 7 days post infection. CD11c pos cells were positively selected by MACS bead isolation following CD19/90 depletion. (A) Isolated cells were stained for CD11c, CD90, CD19, CD8α and cell surface (top two rows) LCMV NP or intracellular (bottom row) LCMV NP. Gating controls are as follows: Naïve DC (dashed line), DCs from Cl13 infected mice incubated with isotype control antibody for LCMV NP Ab (filled grey histogram). (B C) CD8α neg DCs were sorted based on cell surface NP expression. (B) RNA was extracted from sorted DCs; reverse transcription was performed and generated cDNA used in Real Time PCR reaction to determine the copy number of LCMV GP, relative to known concentration of control plasmid. ND indicates below detection limits (C) 4×10 4 sorted DCs were cultured for 2 days in cRPMI. IL-10 levels were measured in culture supernatants of Cl13 infected (NP pos ), exposed (NP neg ) DCs and Naïve CD8α neg DCs. n = 4–5 mice per treatment per experiment. RealTime and ELISA data from one representative of three independent experiments is shown. Standard deviation is shown.

    Article Snippet: Equal amounts of total RNA were used to generate cDNA via reverse transcription with the High Capacity RNA to DNA kit (Applied Biosystems).

    Techniques: Infection, Magnetic Cell Separation, Isolation, Staining, Mouse Assay, Incubation, Expressing, Generated, Real-time Polymerase Chain Reaction, Concentration Assay, Plasmid Preparation, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Effects of the treatment with lipid nanoemulsion-methotrexate (LDE-MTX) on gene expression of inflammation markers and lipoprotein receptors in grafted hearts. Gene expression of TNF-α (A), MCP-1 (B), IL-18 (C), VCAM-1 (D), IL-10 (E), IL-1β (F), MMP-9 (G), MMP-12 (H), LDLR (I), LRP-1 (J), CD-36 (K), in control native and grafted hearts, and LDE-MTX grafted hearts of the rabbits. Total RNA was isolated from cardiac tissue and pooled samples of each group were reverse transcribed into cDNA. Quantitative real time PCR was used to calculate relative gene expression of target genes comparing the mean of the pool of grafted hearts of control and LDE-MTX groups versus the mean of the pool of native hearts of control group.

    Journal: Brazilian Journal of Medical and Biological Research

    Article Title: Methotrexate associated to lipid core nanoparticles improves cardiac allograft vasculopathy and the inflammatory profile in a rabbit heart graft model

    doi: 10.1590/1414-431X20176225

    Figure Lengend Snippet: Effects of the treatment with lipid nanoemulsion-methotrexate (LDE-MTX) on gene expression of inflammation markers and lipoprotein receptors in grafted hearts. Gene expression of TNF-α (A), MCP-1 (B), IL-18 (C), VCAM-1 (D), IL-10 (E), IL-1β (F), MMP-9 (G), MMP-12 (H), LDLR (I), LRP-1 (J), CD-36 (K), in control native and grafted hearts, and LDE-MTX grafted hearts of the rabbits. Total RNA was isolated from cardiac tissue and pooled samples of each group were reverse transcribed into cDNA. Quantitative real time PCR was used to calculate relative gene expression of target genes comparing the mean of the pool of grafted hearts of control and LDE-MTX groups versus the mean of the pool of native hearts of control group.

    Article Snippet: After RNA quantification in Nanodrop 2000 Spectrophotometer instrument (Thermo Fisher Scientific Inc., USA), one microgram of total RNA of each pool was reverse-transcribed using the High Capacity RNA-to-cDNA Master Mix (Applied Biosystems Life Technology, USA) according to the manufacturer’s specifications.

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

    Differentiated adipocytes and fat depots express CXCR2 . (A) 3T3‐L1 cells were differentiated into adipocytes and cells stained with oil red‐O (scale bars: 50 μm). (B) mRNA was extracted before and after differentiation, reverse transcribed to cDNA and analyzed for CXCR2 expression relative to the house‐keeping gene 18s. (C) Oil red‐O staining was quantified in undifferentiated and differentiated adipocytes in the absence or presence of two different CXCR2 inhibitors (expressed relative to differentiated cells). (D) Adipocytes differentiated in the absence and presence of a CXCR2 inhibitor 1 were analyzed for PPARγ protein expression relative to GAPDH levels (left panel) and quantified relative to GAPDH using densitometry. Data are plotted as mean (± sem ), where each symbol represents an experimental replicate. Analyzed using an unpaired t test with Welch's correction (B and D) or one‐way ANOVA with TUKEY's multiple comparison test (C). Data are representative of two separate experiments

    Journal: Journal of Leukocyte Biology

    Article Title: The chemokine receptor CXCR2 contributes to murine adipocyte development. The chemokine receptor CXCR2 contributes to murine adipocyte development

    doi: 10.1002/JLB.1A0618-216RR

    Figure Lengend Snippet: Differentiated adipocytes and fat depots express CXCR2 . (A) 3T3‐L1 cells were differentiated into adipocytes and cells stained with oil red‐O (scale bars: 50 μm). (B) mRNA was extracted before and after differentiation, reverse transcribed to cDNA and analyzed for CXCR2 expression relative to the house‐keeping gene 18s. (C) Oil red‐O staining was quantified in undifferentiated and differentiated adipocytes in the absence or presence of two different CXCR2 inhibitors (expressed relative to differentiated cells). (D) Adipocytes differentiated in the absence and presence of a CXCR2 inhibitor 1 were analyzed for PPARγ protein expression relative to GAPDH levels (left panel) and quantified relative to GAPDH using densitometry. Data are plotted as mean (± sem ), where each symbol represents an experimental replicate. Analyzed using an unpaired t test with Welch's correction (B and D) or one‐way ANOVA with TUKEY's multiple comparison test (C). Data are representative of two separate experiments

    Article Snippet: Purified RNA (500 ng) was converted to cDNA using the high capacity RNA to cDNA kit (Thermo Scientific). cDNA was analyzed for CXCR2 expression using the indicated primers (Table ), Q5 High‐Fidelity DNA polymerase and DNTP mix (NewEngland Biolabs, Ipswich, MA, USA) using a standard thermocycler programme.

    Techniques: Staining, Expressing

    Analysis of mRNA levels for K3, ORF49, and ORF64 during KSHV primary infection at 1, 2, 4, and 7 days p.i. Human PBMCs were infected by wt KSHV, RTA1 st , RTA all , and LANAp, and cells were harvested at 1, 2, 4, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of K3 (A), ORF49 (B), and ORF64 (C) were quantified by qRT-PCR on a StepOnePlus real-time PCR system. (D) The mRNA levels of K3, ORF49, and ORF64 in BJAB, BCBL1, and BC3 cells were analyzed by qRT-PCR. dpi, days postinfection; RQ, relative quantity.

    Journal: mBio

    Article Title: Kaposi’s Sarcoma-Associated Herpesvirus Genome Programming during the Early Stages of Primary Infection of Peripheral Blood Mononuclear Cells

    doi: 10.1128/mBio.02261-14

    Figure Lengend Snippet: Analysis of mRNA levels for K3, ORF49, and ORF64 during KSHV primary infection at 1, 2, 4, and 7 days p.i. Human PBMCs were infected by wt KSHV, RTA1 st , RTA all , and LANAp, and cells were harvested at 1, 2, 4, and 7 days p.i. Total RNAs were extracted by using TRIzol (Invitrogen), and cDNAs were synthesized using a high capacity RNA-to-cDNA kit. The mRNA levels of K3 (A), ORF49 (B), and ORF64 (C) were quantified by qRT-PCR on a StepOnePlus real-time PCR system. (D) The mRNA levels of K3, ORF49, and ORF64 in BJAB, BCBL1, and BC3 cells were analyzed by qRT-PCR. dpi, days postinfection; RQ, relative quantity.

    Article Snippet: Total RNAs from infected PBMCs were extracted by using TRIzol (Invitrogen, Inc., Carlsbad, CA), and 1.0 µg DNase-treated total RNA was used to generate cDNA using the high capacity RNA-to-cDNA kit (Applied Biosystems Inc., Foster City, CA) according to the manufacturer’s instructions.

    Techniques: Infection, Synthesized, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    α 2 M expression in primary villous cytotrophoblastic cells and BeWo cells. vCTB were purified from early (8 weeks of gestation) and late first-trimester (11 weeks of gestation) trophoblast and term placenta and seeded for 24, 48, 72 and 96 h. BeWo cells were seeded and treated or not with 20 µM Forskolin (FSK) for 48 h. RNA was retrotranscribed, and 50 ng of cDNA was used to perform α 2 M and GAPDH PCR. n = 3.

    Journal: Scientific Reports

    Article Title: Activated α2-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion

    doi: 10.1038/s41598-020-66554-0

    Figure Lengend Snippet: α 2 M expression in primary villous cytotrophoblastic cells and BeWo cells. vCTB were purified from early (8 weeks of gestation) and late first-trimester (11 weeks of gestation) trophoblast and term placenta and seeded for 24, 48, 72 and 96 h. BeWo cells were seeded and treated or not with 20 µM Forskolin (FSK) for 48 h. RNA was retrotranscribed, and 50 ng of cDNA was used to perform α 2 M and GAPDH PCR. n = 3.

    Article Snippet: Reverse transcription was performed with 1 µg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies).

    Techniques: Expressing, Purification, Polymerase Chain Reaction

    α 2 M* induced BeWo cell fusion through p-CREB, p-ERK1/2 and p-JNK activation, without affecting syncytin expression. ( A,B ) BeWo cells were seeded for 24 h prior to 24 h of starvation. Subsequently, cells were treated with 5 μM KT5720, 10 μM SP600125 or 10 μM UO126 for 1 h, and 100 pM of α 2 M* was added or not for 30 min. ( A ) Western blotting was performed. p-CREB, CREB, p-ERK1/2, ERK1/2, p-JNK and JNK levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation. ( B ) Nuclei and syncytia were counted, and a fusion index was calculated. n = 3. Data represented as mean±SEM. ns (not significant), *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.005; ANOVA comparison test. ( C , D ) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M*. ( C ) RNA was retrotranscribed, and 10 ng of cDNA was used to perform qPCR using syncytin-1 and syncytin-2 primers. n = 3. Data represented as mean±SEM. **P ≤ 0.01; t-test comparison test. ( D ) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M* for 48 h. Western blotting was performed. Syncytin-1 and GAPDH levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation.

    Journal: Scientific Reports

    Article Title: Activated α2-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion

    doi: 10.1038/s41598-020-66554-0

    Figure Lengend Snippet: α 2 M* induced BeWo cell fusion through p-CREB, p-ERK1/2 and p-JNK activation, without affecting syncytin expression. ( A,B ) BeWo cells were seeded for 24 h prior to 24 h of starvation. Subsequently, cells were treated with 5 μM KT5720, 10 μM SP600125 or 10 μM UO126 for 1 h, and 100 pM of α 2 M* was added or not for 30 min. ( A ) Western blotting was performed. p-CREB, CREB, p-ERK1/2, ERK1/2, p-JNK and JNK levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation. ( B ) Nuclei and syncytia were counted, and a fusion index was calculated. n = 3. Data represented as mean±SEM. ns (not significant), *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.005; ANOVA comparison test. ( C , D ) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M*. ( C ) RNA was retrotranscribed, and 10 ng of cDNA was used to perform qPCR using syncytin-1 and syncytin-2 primers. n = 3. Data represented as mean±SEM. **P ≤ 0.01; t-test comparison test. ( D ) BeWo cells were seeded for 24 h prior to treatment with or without 100 pM of α 2 M* for 48 h. Western blotting was performed. Syncytin-1 and GAPDH levels were quantified using the ImageJ software, and data are expressed as the fold change relative to the control. n = 3. The images of bands for the target proteins were taken from the same gel, and each image was cropped, as delineated by black dividing lines, as well as adjusted for image intensity for optimal visualisation.

    Article Snippet: Reverse transcription was performed with 1 µg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies).

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

    Plk1 mRNA and protein expression in BT-474 cells after incubating with nanoparticles. (A) An RT-PCR analysis was performed 48 hours after incubating with nanoparticle formulations using primers specific for Plk1 and for GAPDH for standardization. Representative gels show Plk1 cDNA (top) and GAPDH cDNA for standardization (bottom). Percentage of Plk1 cDNA expression is given as percentage of GAPDH-standardized Plk1 cDNA expression in control cells ( n = 8, mean ± SD). A quantitative real-time PCR analysis was performed (B) 24 and (C) 48 hours after incubating with the nanoparticle formulations using Plk1- and GAPDH-specific primers. Graphical summary of gene expression values of treated cells standardized to control cells are shown ( n = 3–5, mean ± SD). (D–F) Western blot analyses were performed using anti-Plk1 antibodies 24, 48, and 72 hours after incubating with the nanoparticle formulations. To control for variability of loading, membranes were reprobed with antibodies against p38 (24 and 48 hours) or β-actin (72 hours). Representative Western blots show Plk1 (top) and β-actin or p38 protein (bottom) for standardization. Percentage of Plk1 protein expression is given as percentage of β-actin- or p38-standardized Plk1 levels in control cells ( n = 3, mean ± SD).

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Downregulation of Plk1 Expression By Receptor-Mediated Uptake of Antisense Oligonucleotide-Loaded Nanoparticles 1

    doi:

    Figure Lengend Snippet: Plk1 mRNA and protein expression in BT-474 cells after incubating with nanoparticles. (A) An RT-PCR analysis was performed 48 hours after incubating with nanoparticle formulations using primers specific for Plk1 and for GAPDH for standardization. Representative gels show Plk1 cDNA (top) and GAPDH cDNA for standardization (bottom). Percentage of Plk1 cDNA expression is given as percentage of GAPDH-standardized Plk1 cDNA expression in control cells ( n = 8, mean ± SD). A quantitative real-time PCR analysis was performed (B) 24 and (C) 48 hours after incubating with the nanoparticle formulations using Plk1- and GAPDH-specific primers. Graphical summary of gene expression values of treated cells standardized to control cells are shown ( n = 3–5, mean ± SD). (D–F) Western blot analyses were performed using anti-Plk1 antibodies 24, 48, and 72 hours after incubating with the nanoparticle formulations. To control for variability of loading, membranes were reprobed with antibodies against p38 (24 and 48 hours) or β-actin (72 hours). Representative Western blots show Plk1 (top) and β-actin or p38 protein (bottom) for standardization. Percentage of Plk1 protein expression is given as percentage of β-actin- or p38-standardized Plk1 levels in control cells ( n = 3, mean ± SD).

    Article Snippet: After isolation of total RNA using RNeasy Mini Kits (Qiagen) according to the manufacturer's protocol 24 and 48 hours after incubating with the nanoparticle formulations, the mRNA was transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Darmstadt, Germany).

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

    Increased expression of GFAT in lung cancer cell lines and tissues. ( A ) Expression of GFAT mRNA in HBECs and lung cancer cell lines. Total RNA was extracted from cell lines; cDNA was synthesized by reverse transcription and used for PCR with specific primers for GFAT1, GFAT2, and β-actin as loading control. Products were run in agarose gel with EB. ( B ) GFAT protein and O-GlcNAcylation levels in HBECs and lung cancer cell lines as examined with Western blot in total cell lysates. β-Actin was probed as a loading control. ( C ) GFAT mRNA expression in human lung cancer tissues examined with TaqMan assay. GFAT expression in 12 adenocarcinomas, 12 squamous cell carcinomas, and their corresponding distant normal tissues was normalized to respective β-actin, and then cancer over normal expression was calculated. * P

    Journal: Molecular carcinogenesis

    Article Title: Inhibition of the hexosamine biosynthesis pathway potentiates cisplatin cytotoxicity by decreasing BiP expression in non-small cell lung cancer cells

    doi: 10.1002/mc.22992

    Figure Lengend Snippet: Increased expression of GFAT in lung cancer cell lines and tissues. ( A ) Expression of GFAT mRNA in HBECs and lung cancer cell lines. Total RNA was extracted from cell lines; cDNA was synthesized by reverse transcription and used for PCR with specific primers for GFAT1, GFAT2, and β-actin as loading control. Products were run in agarose gel with EB. ( B ) GFAT protein and O-GlcNAcylation levels in HBECs and lung cancer cell lines as examined with Western blot in total cell lysates. β-Actin was probed as a loading control. ( C ) GFAT mRNA expression in human lung cancer tissues examined with TaqMan assay. GFAT expression in 12 adenocarcinomas, 12 squamous cell carcinomas, and their corresponding distant normal tissues was normalized to respective β-actin, and then cancer over normal expression was calculated. * P

    Article Snippet: Briefly, total RNA extracted with Trizol (Invitrogen) was used for reverse transcription with High Capacity cDNA RT kit (Applied Biosystems, Foster City, CA).

    Techniques: Expressing, Synthesized, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, TaqMan Assay

    Analysis of mRNA expression levels of galectins in normal and infected corneas by qRT-PCR. Complementary DNA was synthesized from 100 ng each of total RNA preparations of normal and infected corneas using the High-Capacity cDNA Reverse Transcriptase Kit, and PCR amplification was performed in triplicate using gene-specific primers for β-actin, Gal-1, -3, -7, -8, and -9 and a Taqman master mix according to the manufacturer's instructions. A threshold cycle value (C t ) was calculated from each amplification plot. Quantification data of each gene were normalized to the expression of β-actin, a value of 1.0 was given to the expression of each gene in the control cornea and the expression values for galectins in infected corneas were calculated as a change in expression level with respect to the control cornea. At least four corneas were pooled and considered one biological replica. N = 4 for all galectins. Data are plotted as mean ± SEM and analyzed using one-way ANOVA. * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Fingerprinting of Galectins in Normal, P. aeruginosa–Infected, and Chemically Burned Mouse Corneas

    doi: 10.1167/iovs.14-15338

    Figure Lengend Snippet: Analysis of mRNA expression levels of galectins in normal and infected corneas by qRT-PCR. Complementary DNA was synthesized from 100 ng each of total RNA preparations of normal and infected corneas using the High-Capacity cDNA Reverse Transcriptase Kit, and PCR amplification was performed in triplicate using gene-specific primers for β-actin, Gal-1, -3, -7, -8, and -9 and a Taqman master mix according to the manufacturer's instructions. A threshold cycle value (C t ) was calculated from each amplification plot. Quantification data of each gene were normalized to the expression of β-actin, a value of 1.0 was given to the expression of each gene in the control cornea and the expression values for galectins in infected corneas were calculated as a change in expression level with respect to the control cornea. At least four corneas were pooled and considered one biological replica. N = 4 for all galectins. Data are plotted as mean ± SEM and analyzed using one-way ANOVA. * P

    Article Snippet: Complementary DNA was synthesized from 100 ng total RNA using the High-Capacity cDNA Reverse Transcriptase Kit (Invitrogen).

    Techniques: Expressing, Infection, Quantitative RT-PCR, Synthesized, Polymerase Chain Reaction, Amplification

    Interaction with K562 CML cells and its paracrine factors modified cell surface antigen expression in MSC. a Cell surface expression of CD13, CD73, CD90, CD95 and CD105 in control-MSC (CON-MSC) and CML-MSC was analyzed by flow cytometry. Mean (geometric) fluorescent intensity (MFI) was calculated for each marker against its isotype control. b , c Control-MSC were cultured in conditioned media derived from K562 cells for one week and their cell surface gene expression in control-MSC (CON) and conditioned media treated MSC (MSC + CM) was analyzed by flow cytometry. MFI of analyzed markers was normalized to control-MSC. c Representative flow cytometry histogram showing cell surface antigen expression levels in CON and MSC + CM conditions. Grey line represents the isotype control, blue and red line represents the stained cells. d , e Control-MSC were co-cultured without (CON) or with K562 cells (MSC + K) for one week and their cell surface gene expression profile was determined by flow cytometry. MFI was calculated for each antigen and normalized to control-MSC. e Representative flow cytometry histograms showing the cell surface antigen expression in CON (blue line), MSC + K (red line) and isotype control (grey line). f , g RNA was extracted from control-MSC (CON) and K562 co-cultured MSC (MSC + K) and reverse transcribed into cDNA. mRNA expression levels of ( f ) CD90, ( g ) CAT and MnSOD was analyzed by real-time PCR. (h) Mitochondrial ROS levels in CON and MSC + K were analyzed by staining with mitosox red and MFI of mitosox was normalized to unstained cells. Values are mean ± SE, *p

    Journal: Journal of Cell Communication and Signaling

    Article Title: K562 chronic myeloid leukemia cells modify osteogenic differentiation and gene expression of bone marrow stromal cells

    doi: 10.1007/s12079-017-0412-8

    Figure Lengend Snippet: Interaction with K562 CML cells and its paracrine factors modified cell surface antigen expression in MSC. a Cell surface expression of CD13, CD73, CD90, CD95 and CD105 in control-MSC (CON-MSC) and CML-MSC was analyzed by flow cytometry. Mean (geometric) fluorescent intensity (MFI) was calculated for each marker against its isotype control. b , c Control-MSC were cultured in conditioned media derived from K562 cells for one week and their cell surface gene expression in control-MSC (CON) and conditioned media treated MSC (MSC + CM) was analyzed by flow cytometry. MFI of analyzed markers was normalized to control-MSC. c Representative flow cytometry histogram showing cell surface antigen expression levels in CON and MSC + CM conditions. Grey line represents the isotype control, blue and red line represents the stained cells. d , e Control-MSC were co-cultured without (CON) or with K562 cells (MSC + K) for one week and their cell surface gene expression profile was determined by flow cytometry. MFI was calculated for each antigen and normalized to control-MSC. e Representative flow cytometry histograms showing the cell surface antigen expression in CON (blue line), MSC + K (red line) and isotype control (grey line). f , g RNA was extracted from control-MSC (CON) and K562 co-cultured MSC (MSC + K) and reverse transcribed into cDNA. mRNA expression levels of ( f ) CD90, ( g ) CAT and MnSOD was analyzed by real-time PCR. (h) Mitochondrial ROS levels in CON and MSC + K were analyzed by staining with mitosox red and MFI of mitosox was normalized to unstained cells. Values are mean ± SE, *p

    Article Snippet: The resulting RNA was reverse transcribed using high capacity cDNA synthesis kit and oligodT primers at 37°C for 120 min. Real-time PCR was performed using Power SyBr Green reagents in an ABI 7500 system (Thermo Fisher Scientific).

    Techniques: Modification, Expressing, Flow Cytometry, Cytometry, Marker, Cell Culture, Derivative Assay, Staining, Real-time Polymerase Chain Reaction

    Male Balb/c mice were injected with either control buffer (Con) or tunicamycin (Tun). After 24 hrs, the epididymal fat pads were dissected and ( A ) total RNA was extracted and transcribed to cDNA. Relative mRNA levels of the indicated genes were quantified using real-time PCR and normalized to 18S rRNA. The value obtained for the control for each gene was assigned a value of 1. The value obtained following injection with tunicamycin was expressed relative to the control value. Results represent the mean ± SD of n = 3–8 experiments. ▪ Control, □ Tunicamycin. ( B ) Equal amounts of protein were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing the indicated proteins. For IRE1α, GRP78 and GAPDH intervening lanes have been removed. ( C – E ) The intensities of the bands in ( B ) were quantified from n = 3–6 experiments. The control values were set to 1. The values obtained from tunicamycin treated mice were expressed relative to the control. * Indicates P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Endoplasmic reticulum stress in adipose tissue augments lipolysis

    doi: 10.1111/jcmm.12384

    Figure Lengend Snippet: Male Balb/c mice were injected with either control buffer (Con) or tunicamycin (Tun). After 24 hrs, the epididymal fat pads were dissected and ( A ) total RNA was extracted and transcribed to cDNA. Relative mRNA levels of the indicated genes were quantified using real-time PCR and normalized to 18S rRNA. The value obtained for the control for each gene was assigned a value of 1. The value obtained following injection with tunicamycin was expressed relative to the control value. Results represent the mean ± SD of n = 3–8 experiments. ▪ Control, □ Tunicamycin. ( B ) Equal amounts of protein were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing the indicated proteins. For IRE1α, GRP78 and GAPDH intervening lanes have been removed. ( C – E ) The intensities of the bands in ( B ) were quantified from n = 3–6 experiments. The control values were set to 1. The values obtained from tunicamycin treated mice were expressed relative to the control. * Indicates P

    Article Snippet: RNA (2 μg) was transcribed to cDNA using the high capacity cDNA reverse transcription kit (Applied Biosystems, Burlington, ON, Canada).

    Techniques: Mouse Assay, Injection, Real-time Polymerase Chain Reaction, SDS Page

    Subcutaneous white adipose tissue was obtained during surgery from burned patients (burn) or non-burned patients undergoing elective surgery (non-burn). ( A ) Total RNA was extracted from adipose tissue obtained from non-burned ( n = 3–4) and burned patients ( n = 5–8) and transcribed to cDNA. Real-time quantitative PCR was performed to evaluate the relative mRNA levels of the indicated genes that were normalized to IDH1 mRNA. The value obtained from one non-burn sample was set to 1. The values obtained from all other samples were expressed relative to the non-burn sample. ▪ Non-burn, □ Burn. ( B , Top and Bottom Panels ) Adipose tissue was homogenized and equal amounts of total protein in the tissue homogenates were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing GRP78 and alpha/beta tubulin. ( C ) The intensities of the bands corresponding to GRP78 in ( B ) were quantified. ( D ) Human adipocytes (non-burn) were isolated and cultured either in the presence or absence of tunicamycin (5 μg/ml) at 37°C for the indicated times. The culture media was collected and the concentration of free glycerol determined. To account for the variability between patients in basal lipolysis rates, the amount of glycerol released from control cells from each patient and at each time-point was given a value of 100%. The amount of glycerol released from adipocytes in the presence of tunicamycin is expressed as a percentage of the value obtained from the control cells. Results represent the mean ± SD from n = 3 female patients ages 48 and 61 years, ▪ Control, □ Tunicamycin. * indicates P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Endoplasmic reticulum stress in adipose tissue augments lipolysis

    doi: 10.1111/jcmm.12384

    Figure Lengend Snippet: Subcutaneous white adipose tissue was obtained during surgery from burned patients (burn) or non-burned patients undergoing elective surgery (non-burn). ( A ) Total RNA was extracted from adipose tissue obtained from non-burned ( n = 3–4) and burned patients ( n = 5–8) and transcribed to cDNA. Real-time quantitative PCR was performed to evaluate the relative mRNA levels of the indicated genes that were normalized to IDH1 mRNA. The value obtained from one non-burn sample was set to 1. The values obtained from all other samples were expressed relative to the non-burn sample. ▪ Non-burn, □ Burn. ( B , Top and Bottom Panels ) Adipose tissue was homogenized and equal amounts of total protein in the tissue homogenates were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing GRP78 and alpha/beta tubulin. ( C ) The intensities of the bands corresponding to GRP78 in ( B ) were quantified. ( D ) Human adipocytes (non-burn) were isolated and cultured either in the presence or absence of tunicamycin (5 μg/ml) at 37°C for the indicated times. The culture media was collected and the concentration of free glycerol determined. To account for the variability between patients in basal lipolysis rates, the amount of glycerol released from control cells from each patient and at each time-point was given a value of 100%. The amount of glycerol released from adipocytes in the presence of tunicamycin is expressed as a percentage of the value obtained from the control cells. Results represent the mean ± SD from n = 3 female patients ages 48 and 61 years, ▪ Control, □ Tunicamycin. * indicates P

    Article Snippet: RNA (2 μg) was transcribed to cDNA using the high capacity cDNA reverse transcription kit (Applied Biosystems, Burlington, ON, Canada).

    Techniques: Real-time Polymerase Chain Reaction, SDS Page, Isolation, Cell Culture, Concentration Assay

    RNA and protein content of cellular exosomes from HCC cells (A) RNA was extracted from Hep3B derived exosomes (lane 1) or their corresponding donor cells (lane 2) and analyzed by capillary electrophoresis (Bioanalyzer, Agilent). The RNA content is strikingly different, with the majority of RNA in Hep3B derived exosomes below 2 kb in size and with a very low fraction of 18S ribosomal RNA (rRNA) and 28S rRNA compared to RNA from donor cells. (B) An equivalent amount (600 ng) of RNA from Hep3B or PLC/PRF/5 donor cells, or exosomes obtained from these cells was transcribed to cDNA and the expression of 18S ribosomal RNA (18S rRNA) and small nucleolar RNA U43 (snoRNA U43) examined by real-time PCR. Amplification curves and the mean value ± SEM from four independent samples of threshold cycles for 18S rRNA and snoRNA U43 are shown. Both 18S rRNA and snoRNA U43 show higher CT values in exosomes than in their corresponding donor cells. (C) Protein was isolated from Hep3B derived exosomes or their donor cells and 15 μg of protein was separated on a Bis-Tris gel and stained with SYPRO Ruby (lane 1, donor cells; lane 2, exosomes). The protein content in exosomes was different from that in their corresponding donor cells.

    Journal: Hepatology (Baltimore, Md.)

    Article Title: Inter-cellular nanovesicle mediated microRNA transfer: a mechanism of environmental modulation of hepatocellular cancer cell growth

    doi: 10.1002/hep.24504

    Figure Lengend Snippet: RNA and protein content of cellular exosomes from HCC cells (A) RNA was extracted from Hep3B derived exosomes (lane 1) or their corresponding donor cells (lane 2) and analyzed by capillary electrophoresis (Bioanalyzer, Agilent). The RNA content is strikingly different, with the majority of RNA in Hep3B derived exosomes below 2 kb in size and with a very low fraction of 18S ribosomal RNA (rRNA) and 28S rRNA compared to RNA from donor cells. (B) An equivalent amount (600 ng) of RNA from Hep3B or PLC/PRF/5 donor cells, or exosomes obtained from these cells was transcribed to cDNA and the expression of 18S ribosomal RNA (18S rRNA) and small nucleolar RNA U43 (snoRNA U43) examined by real-time PCR. Amplification curves and the mean value ± SEM from four independent samples of threshold cycles for 18S rRNA and snoRNA U43 are shown. Both 18S rRNA and snoRNA U43 show higher CT values in exosomes than in their corresponding donor cells. (C) Protein was isolated from Hep3B derived exosomes or their donor cells and 15 μg of protein was separated on a Bis-Tris gel and stained with SYPRO Ruby (lane 1, donor cells; lane 2, exosomes). The protein content in exosomes was different from that in their corresponding donor cells.

    Article Snippet: cDNA was transcribed from a total of 600ng of DNase I-treated RNA using the cDNA reverse transcription kit and random primers (Invitrogen, Carlsbad, CA).

    Techniques: Derivative Assay, Electrophoresis, Planar Chromatography, Expressing, Real-time Polymerase Chain Reaction, Amplification, Isolation, Staining

    Cell-to-cell transfer of firefly luciferase by exosomes (A) Real-tine PCR was performed on an cDNA transcribed from an equivalent amount (600 ng) of RNA from PLC-luc derived exosomes or their donor cells (n = 3, each in duplicate). PCR amplification curves for Fluc mRNA and 18S rRNA are shown. (B) PLC/PRF/5 cells were incubated with 15 μg/ml of PLC-luc derived exosomes for 16 hours. RNA was isolated and equivalent amount of RNA (300 ng) was transcribed to cDNA (n = 3). Amplification curves by quantitative real-time PCR for Fluc mRNA and 18S rRNA in PLC/PRF/5 (recipient cells) and PLC-luc (donor cells) are shown. (C) PLC/PRF/5 cells in a 96-well plate were incubated with various concentrations of PLC-luc derived exosomes, and luciferase activity was assessed in these cells after 16 hours. Bars express the mean value of luminescence ± SEM of four separate determinations. *, p

    Journal: Hepatology (Baltimore, Md.)

    Article Title: Inter-cellular nanovesicle mediated microRNA transfer: a mechanism of environmental modulation of hepatocellular cancer cell growth

    doi: 10.1002/hep.24504

    Figure Lengend Snippet: Cell-to-cell transfer of firefly luciferase by exosomes (A) Real-tine PCR was performed on an cDNA transcribed from an equivalent amount (600 ng) of RNA from PLC-luc derived exosomes or their donor cells (n = 3, each in duplicate). PCR amplification curves for Fluc mRNA and 18S rRNA are shown. (B) PLC/PRF/5 cells were incubated with 15 μg/ml of PLC-luc derived exosomes for 16 hours. RNA was isolated and equivalent amount of RNA (300 ng) was transcribed to cDNA (n = 3). Amplification curves by quantitative real-time PCR for Fluc mRNA and 18S rRNA in PLC/PRF/5 (recipient cells) and PLC-luc (donor cells) are shown. (C) PLC/PRF/5 cells in a 96-well plate were incubated with various concentrations of PLC-luc derived exosomes, and luciferase activity was assessed in these cells after 16 hours. Bars express the mean value of luminescence ± SEM of four separate determinations. *, p

    Article Snippet: cDNA was transcribed from a total of 600ng of DNase I-treated RNA using the cDNA reverse transcription kit and random primers (Invitrogen, Carlsbad, CA).

    Techniques: Luciferase, Polymerase Chain Reaction, Planar Chromatography, Derivative Assay, Amplification, Incubation, Isolation, Real-time Polymerase Chain Reaction, Activity Assay

    Knockdown of PABPN1 in human cells. HeLa and/or HEK293 cells were grown on 35 mm plates to approximately 30% confluence and transiently transfected with PABPN1-Si or PABPN1-UTR RNAi for indicated length of time. Non transfected (NT) control cells were also maintained in culture for the same duration as transfected cells. (A, B) Following transfection, cells were harvested at 45 h, 66 h and 80 h in Laemmli buffer. Whole cell extracts from Si, UTR, and NT HeLa (A) and HEK293 (B) cells were analyzed for PABPN1 protein by western blotting; GAPDH was used as the loading control. (C) Forty five hours after transfection, HeLa cells grown on coverslip were fixed with Para-formaldehyde and processed for immunostaining with PABPN1 specific antibody and counterstained with Texas-red-conjugated secondary antibody. Processed specimens were then examined with a confocal microscope. Images from two different sections of the slide are shown here. (D) Forty five hours and sixty six hours after transfection, RNA was extracted from HeLa cells using Trizol and reverse transcribed; cDNA thus made were used for PCR with primers specific to β-actin, PABP and PABPN1. Samples from PABPN1-UTR Si and non transfected (NT) cells were collected at 66 h time point. The bands on the gel representing the PCR products were scanned and quantified as described in Materials and Methods . The band intensities are shown in arbitrary unit.

    Journal: PLoS ONE

    Article Title: Depletion of Nuclear Poly(A) Binding Protein PABPN1 Produces a Compensatory Response by Cytoplasmic PABP4 and PABP5 in Cultured Human Cells

    doi: 10.1371/journal.pone.0053036

    Figure Lengend Snippet: Knockdown of PABPN1 in human cells. HeLa and/or HEK293 cells were grown on 35 mm plates to approximately 30% confluence and transiently transfected with PABPN1-Si or PABPN1-UTR RNAi for indicated length of time. Non transfected (NT) control cells were also maintained in culture for the same duration as transfected cells. (A, B) Following transfection, cells were harvested at 45 h, 66 h and 80 h in Laemmli buffer. Whole cell extracts from Si, UTR, and NT HeLa (A) and HEK293 (B) cells were analyzed for PABPN1 protein by western blotting; GAPDH was used as the loading control. (C) Forty five hours after transfection, HeLa cells grown on coverslip were fixed with Para-formaldehyde and processed for immunostaining with PABPN1 specific antibody and counterstained with Texas-red-conjugated secondary antibody. Processed specimens were then examined with a confocal microscope. Images from two different sections of the slide are shown here. (D) Forty five hours and sixty six hours after transfection, RNA was extracted from HeLa cells using Trizol and reverse transcribed; cDNA thus made were used for PCR with primers specific to β-actin, PABP and PABPN1. Samples from PABPN1-UTR Si and non transfected (NT) cells were collected at 66 h time point. The bands on the gel representing the PCR products were scanned and quantified as described in Materials and Methods . The band intensities are shown in arbitrary unit.

    Article Snippet: An aliquot of total RNA (100–500 ng) was reverse transcribed using High Capacity cDNA transcription kit (Applied Biosystems, Life Technologies).

    Techniques: Transfection, Western Blot, Immunostaining, Microscopy, Polymerase Chain Reaction

    Coagulation factor gene expression is elevated in IP mice and malaria-exposed murine trophoblasts. (A) RNA was isolated from conceptuses removed from ED 10 UP (n = 5) and IP (n = 6) mice. Primers specific for the genes indicated were utilized to measure cDNA expression levels in IP relative to UP mice. Data are normalized against murine 18S RNA. Data are expressed as the ratio of fold increase in IP mice to that of UP mice ± SEM. (B) SM9-1 trophoblasts were stimulated with P. chabaudi AS-iRBCs and RNA isolated over the time course indicated. QRT-PCR was conducted as in panel A. Data are expressed as the ratio of fold increase relative to time matched SM9-1 trophoblasts stimulated with uninfected RBC ± SEM and are representative of four separate experiments.

    Journal: PLoS ONE

    Article Title: Maternal Malaria Induces a Procoagulant and Antifibrinolytic State That Is Embryotoxic but Responsive to Anticoagulant Therapy

    doi: 10.1371/journal.pone.0031090

    Figure Lengend Snippet: Coagulation factor gene expression is elevated in IP mice and malaria-exposed murine trophoblasts. (A) RNA was isolated from conceptuses removed from ED 10 UP (n = 5) and IP (n = 6) mice. Primers specific for the genes indicated were utilized to measure cDNA expression levels in IP relative to UP mice. Data are normalized against murine 18S RNA. Data are expressed as the ratio of fold increase in IP mice to that of UP mice ± SEM. (B) SM9-1 trophoblasts were stimulated with P. chabaudi AS-iRBCs and RNA isolated over the time course indicated. QRT-PCR was conducted as in panel A. Data are expressed as the ratio of fold increase relative to time matched SM9-1 trophoblasts stimulated with uninfected RBC ± SEM and are representative of four separate experiments.

    Article Snippet: Briefly, RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription and DNA-free Kits (Applied Biosystems, Carlsbad, CA, USA) or RNeasy® Plus Mini Kit (Qiagen, Valencia, CA, USA) as described by the manufacturers' protocols.

    Techniques: Coagulation, Expressing, Mouse Assay, Isolation, Quantitative RT-PCR

    2.3. Total RNA extraction, cDNA synthesis and quantitative polymerase chain reaction (qPCR)

    Journal: Food & function

    Article Title: Dietary resistant starch type 4-derived butyrate attenuates nuclear factor-kappa-B1 through modulation of lysine 27 trimethylation of histone H3

    doi: 10.1039/c6fo00856a

    Figure Lengend Snippet: 2.3. Total RNA extraction, cDNA synthesis and quantitative polymerase chain reaction (qPCR)

    Article Snippet: The cDNAs were synthesized using 3 µg of RNA for each sample using the High-Capacity cDNA Reverse Transcription (RT) Kit (Invitrogen, Grand Island, NY), following the manufacturers’ protocol.

    Techniques: RNA Extraction, Real-time Polymerase Chain Reaction

    Genomic structures of novel TUs . A. TU4. B. TU7. C. TU11. D. TU52. E. TU54. Initial paired-end ditag: dark blue. Exons of validated full-length cDNA sequences: orange. Exons of previously known transcripts at each locus: teal (protein-coding sequence: shaded). 3' terminal exon arrows: direction of transcription. Quantitative real-time PCR primer locations: red arrows. All validated cDNA sequences can be found in Additional file 2 .

    Journal: BMC Genomics

    Article Title: Detailed characterization of the mouse embryonic stem cell transcriptome reveals novel genes and intergenic splicing associated with pluripotency

    doi: 10.1186/1471-2164-9-155

    Figure Lengend Snippet: Genomic structures of novel TUs . A. TU4. B. TU7. C. TU11. D. TU52. E. TU54. Initial paired-end ditag: dark blue. Exons of validated full-length cDNA sequences: orange. Exons of previously known transcripts at each locus: teal (protein-coding sequence: shaded). 3' terminal exon arrows: direction of transcription. Quantitative real-time PCR primer locations: red arrows. All validated cDNA sequences can be found in Additional file 2 .

    Article Snippet: Quantitative Real-Time PCR cDNA synthesis of 2 μg total RNA was performed using High Capacity cDNA Archive Kit (Applied Biosystems) followed by ten-fold dilution of the product.

    Techniques: Sequencing, Real-time Polymerase Chain Reaction

    Glycosylation in intestinal organoids transfected with mut-AGX1 and BH-GalNAc-T2 or non-transfected, control samples. Organoids were fed with DMSO (top) or 50 µM compound 11 (bottom), fixed and treated with biotin alkyne under CuAAC conditions followed by Streptavidin Alexa Fluor 647 staining. Data are from one representative out of two independent experiments and shown as grayscale images for each channel and a color merge image of all three channels. Scale bar, 100 µm.

    Journal: bioRxiv

    Article Title: Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

    doi: 10.1101/2020.04.23.057208

    Figure Lengend Snippet: Glycosylation in intestinal organoids transfected with mut-AGX1 and BH-GalNAc-T2 or non-transfected, control samples. Organoids were fed with DMSO (top) or 50 µM compound 11 (bottom), fixed and treated with biotin alkyne under CuAAC conditions followed by Streptavidin Alexa Fluor 647 staining. Data are from one representative out of two independent experiments and shown as grayscale images for each channel and a color merge image of all three channels. Scale bar, 100 µm.

    Article Snippet: To amplify the amount of viral stock (P1 to P2); 30 mL of Sf21 cells (9×105 cells/mL) were seeded at 6-well plates, incubated overnight and transferred to 30 mL Sf21 cell suspension for incubation at 125 rpm, 27 °C for 3 days.

    Techniques: Transfection, Staining

    UDP-GalNAzMe 5 recognition by GalNAc-Ts and delivery to the living cell. A, in vitro peptide glycosylation by purified GalNAc-Ts. Data are biological duplicates as average of technical duplicates. B , lysate protein glycosylation by GalNAc-T1 and GalNAc-T2. A membrane preparation was used as a lysate protein source, probed with soluble GalNAc-Ts and azide-tagged UDP-sugars, and subjected to CuAAC with clickable biotin. Streptavidin blot was used to visualize glycosylation. Data are from one representative out of three independent experiments. C , Biosynthesis of UDP-GalNAzMe in K-562 cells stably transfected with WT- or mut-AGX1, as assessed by HPAEC-PAD. Standards include UDP-GalNAzMe ( 5 ) and its C4-epimer UDP-GlcNAzMe ( SI-7 ).

    Journal: bioRxiv

    Article Title: Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

    doi: 10.1101/2020.04.23.057208

    Figure Lengend Snippet: UDP-GalNAzMe 5 recognition by GalNAc-Ts and delivery to the living cell. A, in vitro peptide glycosylation by purified GalNAc-Ts. Data are biological duplicates as average of technical duplicates. B , lysate protein glycosylation by GalNAc-T1 and GalNAc-T2. A membrane preparation was used as a lysate protein source, probed with soluble GalNAc-Ts and azide-tagged UDP-sugars, and subjected to CuAAC with clickable biotin. Streptavidin blot was used to visualize glycosylation. Data are from one representative out of three independent experiments. C , Biosynthesis of UDP-GalNAzMe in K-562 cells stably transfected with WT- or mut-AGX1, as assessed by HPAEC-PAD. Standards include UDP-GalNAzMe ( 5 ) and its C4-epimer UDP-GlcNAzMe ( SI-7 ).

    Article Snippet: To amplify the amount of viral stock (P1 to P2); 30 mL of Sf21 cells (9×105 cells/mL) were seeded at 6-well plates, incubated overnight and transferred to 30 mL Sf21 cell suspension for incubation at 125 rpm, 27 °C for 3 days.

    Techniques: In Vitro, Purification, Stable Transfection, Transfection

    An engineered BH-T2 double mutant enhances GalNAzMe labeling. A, in vitro glycosylation using WT- or BH-T2 as enzyme sources. UDP-GalNAz 2 and UDP-GalNAzMe 5 were used as substrates, and UDP-GalNAc 1 was used as a competitor at different concentrations. Azide-labeled glycoproteins were visualized as in Fig. 2B . Data are from one representative out of two independent replicates. B , live cell surface glycosylation by K-562 cells stably transfected with mut-AGX1 and WT- or BH-T2 and fed with DMSO, 50 µM compound 11 , or 3 µM Ac 4 GalNAz. Data are from one representative out of two independent replicates. C , Glycosylation in intestinal organoids transfected with mut-AGX1 and BH-T2. Organoids were fed with 50 µM compound 11 or 1.5 µM Ac 4 GalNAz, fixed and treated with biotin alkyne under CuAAC conditions followed by Streptavidin Alexa Fluor 647 staining.

    Journal: bioRxiv

    Article Title: Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

    doi: 10.1101/2020.04.23.057208

    Figure Lengend Snippet: An engineered BH-T2 double mutant enhances GalNAzMe labeling. A, in vitro glycosylation using WT- or BH-T2 as enzyme sources. UDP-GalNAz 2 and UDP-GalNAzMe 5 were used as substrates, and UDP-GalNAc 1 was used as a competitor at different concentrations. Azide-labeled glycoproteins were visualized as in Fig. 2B . Data are from one representative out of two independent replicates. B , live cell surface glycosylation by K-562 cells stably transfected with mut-AGX1 and WT- or BH-T2 and fed with DMSO, 50 µM compound 11 , or 3 µM Ac 4 GalNAz. Data are from one representative out of two independent replicates. C , Glycosylation in intestinal organoids transfected with mut-AGX1 and BH-T2. Organoids were fed with 50 µM compound 11 or 1.5 µM Ac 4 GalNAz, fixed and treated with biotin alkyne under CuAAC conditions followed by Streptavidin Alexa Fluor 647 staining.

    Article Snippet: To amplify the amount of viral stock (P1 to P2); 30 mL of Sf21 cells (9×105 cells/mL) were seeded at 6-well plates, incubated overnight and transferred to 30 mL Sf21 cell suspension for incubation at 125 rpm, 27 °C for 3 days.

    Techniques: Mutagenesis, Labeling, In Vitro, Stable Transfection, Transfection, Staining

    ADAM17 cleaves the CA IX ectodomain and deletion of amino acids 393–402 in the CA IX stalk region prevents the ECD shedding. a Verification of the cleavage activity of the recombinant human TACE/ADAM17 (rhADAM17) towards the fluorogenic peptide Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Ser-Arg-NH2. The peptide was used at the final concentration of 10 μM in a total of 100 μL reaction mixture with 10 ng of the rhADAM17. Time-related increase of the fluorescence emitted from the pro-TNF-α-derived peptide proves that rhADAM17 was active. b Recombinant CA IX-SBP fusion protein attached to a streptavidin-sepharose matrix was cleaved by rhADAM17, eluted and detected by ELISA using CA IX-specific monoclonal antibodies. c Cells transiently expressing FL-CA IX were treated with ADAM17/10 inhibitor for 24 h and CA IX ECD was detected in culture media by ELISA. Results in b and c support the role of ADAM17 in cleavage of CA IX. d Schematic illustration of the CA IX domain structure and positions of deletions in the mutants. Scissors indicates extracellular domain cleavage region. Missing amino-acid residues of individual deletion variants are represented by numbers on the right side. SP signal peptide, PG proteoglycan-like domain, CA catalytic domain, TM transmembrane region, IC intracellular tail. e Immunofluorescence analysis of CHO-wt, CHO-M2 and CHO-M2-TACE cells transiently expressing the FL-CA IX and two stalk deletion variants. The cells were fixed with methanol, incubated with M75 antibody followed by ALEXA Fluor488-secondary antibody and nuclei were stained with DAPI. Deletion of the cleavage site did not affect the CA IX localisation. f , g ELISA analysis of the CA IX deletion variants for the ECD shedding. The plasmids encoding FL-CA IX and its two deletion mutants were transiently transfected to CHO-wt, CHO-M2 (ADAM17-defective) and CHO-M2-TACE (human ADAM17 expressing) cell lines. 48 h after transfection, the cells were cultivated for 3 h in equal medium volumes in the presence or absence of PMA. Undiluted conditioned media ( f ) and cell lysates diluted 1:10 ( g ) were collected and examined by ELISA using V/10 antibody as a capture and mixture of biotinylated MAbs M75 and IV/18 as a detector. h Biochemical evidence that ADAM17 can cleave FL-CA IX, but not the NS mutant was obtained by treatment of CHO cell variants expressing FL and NS, respectively, with recombinant rhADAM17 added to medium at a concentration of 50 μg/ml for 24 h. Collected media were analysed by ELISA. i ADAM17 suppression resulting from infection by lentiviruses expressing ADAM17-specific shRNA led to a decreased CA IX shedding from C33a-FL cells, but not from C33a-NS cells proving the involvement of ADAM17 in the CA IX ECD cleavage. The results (mean ± SD) represent the mean of two measurements in triplicates. (* P

    Journal: British Journal of Cancer

    Article Title: Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells

    doi: 10.1038/s41416-020-0804-z

    Figure Lengend Snippet: ADAM17 cleaves the CA IX ectodomain and deletion of amino acids 393–402 in the CA IX stalk region prevents the ECD shedding. a Verification of the cleavage activity of the recombinant human TACE/ADAM17 (rhADAM17) towards the fluorogenic peptide Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Ser-Arg-NH2. The peptide was used at the final concentration of 10 μM in a total of 100 μL reaction mixture with 10 ng of the rhADAM17. Time-related increase of the fluorescence emitted from the pro-TNF-α-derived peptide proves that rhADAM17 was active. b Recombinant CA IX-SBP fusion protein attached to a streptavidin-sepharose matrix was cleaved by rhADAM17, eluted and detected by ELISA using CA IX-specific monoclonal antibodies. c Cells transiently expressing FL-CA IX were treated with ADAM17/10 inhibitor for 24 h and CA IX ECD was detected in culture media by ELISA. Results in b and c support the role of ADAM17 in cleavage of CA IX. d Schematic illustration of the CA IX domain structure and positions of deletions in the mutants. Scissors indicates extracellular domain cleavage region. Missing amino-acid residues of individual deletion variants are represented by numbers on the right side. SP signal peptide, PG proteoglycan-like domain, CA catalytic domain, TM transmembrane region, IC intracellular tail. e Immunofluorescence analysis of CHO-wt, CHO-M2 and CHO-M2-TACE cells transiently expressing the FL-CA IX and two stalk deletion variants. The cells were fixed with methanol, incubated with M75 antibody followed by ALEXA Fluor488-secondary antibody and nuclei were stained with DAPI. Deletion of the cleavage site did not affect the CA IX localisation. f , g ELISA analysis of the CA IX deletion variants for the ECD shedding. The plasmids encoding FL-CA IX and its two deletion mutants were transiently transfected to CHO-wt, CHO-M2 (ADAM17-defective) and CHO-M2-TACE (human ADAM17 expressing) cell lines. 48 h after transfection, the cells were cultivated for 3 h in equal medium volumes in the presence or absence of PMA. Undiluted conditioned media ( f ) and cell lysates diluted 1:10 ( g ) were collected and examined by ELISA using V/10 antibody as a capture and mixture of biotinylated MAbs M75 and IV/18 as a detector. h Biochemical evidence that ADAM17 can cleave FL-CA IX, but not the NS mutant was obtained by treatment of CHO cell variants expressing FL and NS, respectively, with recombinant rhADAM17 added to medium at a concentration of 50 μg/ml for 24 h. Collected media were analysed by ELISA. i ADAM17 suppression resulting from infection by lentiviruses expressing ADAM17-specific shRNA led to a decreased CA IX shedding from C33a-FL cells, but not from C33a-NS cells proving the involvement of ADAM17 in the CA IX ECD cleavage. The results (mean ± SD) represent the mean of two measurements in triplicates. (* P

    Article Snippet: After 5 min incubation at 4 °C, lysates were mixed with the Pierce™ High-Capacity Streptavidin Agarose beads (Thermo Fisher Scientific, MA, USA) and incubated overnight at 4 °C on rotary stirrer.

    Techniques: Activity Assay, Recombinant, Concentration Assay, Fluorescence, Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Incubation, Staining, Transfection, Mutagenesis, Infection, shRNA