qrt pcr rna  (Qiagen)


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    RNeasy Mini Kit
    Description:
    For purification of up to 100 µg total RNA from cells tissues and yeast Kit contents Qiagen RNeasy Mini Kit 50 preps 0 5 to 30mg Sample 30 to 100L Elution Volume Tissue Cells Sample Total RNA Purification Silica Technology Spin Column Format Ideal for Northern Dot and Slot Blotting End point RT PCR Quantitative Real time RT PCR For Purification of Up to 100g Total RNA from Cells Tissues and Yeast Includes 250 RNeasy Mini Spin Columns Collection Tubes 1 5mL and 2mL RNase free Reagents and Buffers Benefits Fast procedure delivering high quality total RNA in minutes Ready to use RNA for high performance in any downstream application Consistent RNA yields from small amounts of starting material No phenol chloroform extraction no CsCl gradients no LiCl or ethanol precipitatio
    Catalog Number:
    74104
    Price:
    314
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    RNeasy Mini Kit
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    Structured Review

    Qiagen qrt pcr rna
    RNeasy Mini Kit
    For purification of up to 100 µg total RNA from cells tissues and yeast Kit contents Qiagen RNeasy Mini Kit 50 preps 0 5 to 30mg Sample 30 to 100L Elution Volume Tissue Cells Sample Total RNA Purification Silica Technology Spin Column Format Ideal for Northern Dot and Slot Blotting End point RT PCR Quantitative Real time RT PCR For Purification of Up to 100g Total RNA from Cells Tissues and Yeast Includes 250 RNeasy Mini Spin Columns Collection Tubes 1 5mL and 2mL RNase free Reagents and Buffers Benefits Fast procedure delivering high quality total RNA in minutes Ready to use RNA for high performance in any downstream application Consistent RNA yields from small amounts of starting material No phenol chloroform extraction no CsCl gradients no LiCl or ethanol precipitatio
    https://www.bioz.com/result/qrt pcr rna/product/Qiagen
    Average 99 stars, based on 41058 article reviews
    Price from $9.99 to $1999.99
    qrt pcr rna - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Chemical Screening Identifies Enhancers of Mutant Oligodendrocyte Survival and Unmasks a Distinct Pathological Phase in Pelizaeus-Merzbacher Disease"

    Article Title: Chemical Screening Identifies Enhancers of Mutant Oligodendrocyte Survival and Unmasks a Distinct Pathological Phase in Pelizaeus-Merzbacher Disease

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2018.07.015

    Ro 25–6981 Mechanistic Studies Reveal UPR Modulation in Jimpy (A) qRT-PCR of Plp1 for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. n = 4 technical replicates per sample. (B) qRT-PCR of UPR-related panel for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. For all other probes n = 4 technical replicates per sample. Error bars represent mean ± SD. See also Figure S6 and Table S6 .
    Figure Legend Snippet: Ro 25–6981 Mechanistic Studies Reveal UPR Modulation in Jimpy (A) qRT-PCR of Plp1 for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. n = 4 technical replicates per sample. (B) qRT-PCR of UPR-related panel for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. For all other probes n = 4 technical replicates per sample. Error bars represent mean ± SD. See also Figure S6 and Table S6 .

    Techniques Used: Quantitative RT-PCR

    2) Product Images from "Cancer-associated fibroblasts suppress SOX2-induced dysplasia in a lung squamous cancer coculture"

    Article Title: Cancer-associated fibroblasts suppress SOX2-induced dysplasia in a lung squamous cancer coculture

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

    doi: 10.1073/pnas.1803718115

    CAFs enhance the acinar forming capacity of SOX2oe TUM622 cells and suppress dysplasia. ( A ) Quantification of spheroids formed from Vector and SOX2oe cells as monoculture/coculture with CAFs. Each column represents triplicates. Error bars represent SD. ( B ) CAFs induce the formation of acinar-like structures when in coculture with SOX2oe cells. Both Vector and SOX2oe spheroids become invasive when they are in close proximity with CAFs. (Scales bars, 100 μm.) ( C ) Immunofluorescence of SOX2oe monoculture as well as CAF cocultures stained for Vimentin (cytoplasmic)/p63 (nuclear), E-Cadherin, and ITGA6. Anti-vimentin and anti-p63 antibodies were stained with the same secondary antibody (green). ( D ) Immunofluorescence of SOX2oe monoculture as well as CAF cocultures stained for Vimentin, SOX2, and Phalloidin. (Scale bars, 50 μm.)
    Figure Legend Snippet: CAFs enhance the acinar forming capacity of SOX2oe TUM622 cells and suppress dysplasia. ( A ) Quantification of spheroids formed from Vector and SOX2oe cells as monoculture/coculture with CAFs. Each column represents triplicates. Error bars represent SD. ( B ) CAFs induce the formation of acinar-like structures when in coculture with SOX2oe cells. Both Vector and SOX2oe spheroids become invasive when they are in close proximity with CAFs. (Scales bars, 100 μm.) ( C ) Immunofluorescence of SOX2oe monoculture as well as CAF cocultures stained for Vimentin (cytoplasmic)/p63 (nuclear), E-Cadherin, and ITGA6. Anti-vimentin and anti-p63 antibodies were stained with the same secondary antibody (green). ( D ) Immunofluorescence of SOX2oe monoculture as well as CAF cocultures stained for Vimentin, SOX2, and Phalloidin. (Scale bars, 50 μm.)

    Techniques Used: Plasmid Preparation, Immunofluorescence, Staining

    CAFs increase the number and invasiveness of TUM622 acini. ( A ) Micrograph depicting coculture setups where CAFs are either overlaid or embedded together with TUM622 cells in ECM. After 6 d to 12 d in coculture, TUM622 cells are able to form more and larger acini compared with monoculture, and invade the ECM when in close proximity or direct contact with CAFs. ( B ) Bright-field images of TUM622 3D cultures in the presence or absence of overlaid CAFs after 8 d. (Scale bars, 200 μm.) ( C ) Quantification of acini number in monoculture vs. coculture. Columns represent the average of four replicates performed in technical triplicates, error bars represents SD, and P
    Figure Legend Snippet: CAFs increase the number and invasiveness of TUM622 acini. ( A ) Micrograph depicting coculture setups where CAFs are either overlaid or embedded together with TUM622 cells in ECM. After 6 d to 12 d in coculture, TUM622 cells are able to form more and larger acini compared with monoculture, and invade the ECM when in close proximity or direct contact with CAFs. ( B ) Bright-field images of TUM622 3D cultures in the presence or absence of overlaid CAFs after 8 d. (Scale bars, 200 μm.) ( C ) Quantification of acini number in monoculture vs. coculture. Columns represent the average of four replicates performed in technical triplicates, error bars represents SD, and P

    Techniques Used:

    TME is dominant over the genotype in determining the epithelial architecture of TUM622 cells in vitro. A subpopulation of TUM622 cells has the ability to form acinar-like structures when plated as single cells in ECM culture. These acini are hyperplastic but nevertheless display proper apical−basal cell polarity, similar to hyperplasia observed at the earliest stage of LUSC development. Overexpression of SOX2 in TUM622 cells increases spheroid-forming capacity of TUM622 cells and drives a hyperplastic to dysplastic change in acinar phenotype where apical−basal cell polarity is disrupted and solid spheroids are formed. SOX2oe spheroids also display reduced apoptosis and increased differentiation. Addition of CAFs to TUM622 3D culture enhances acinar formation/growth and promotes invasion toward the CAFs. CAFs could promote the formation of an acinar-like structure in SOX2oe spheroids and induce invasion. Therefore, the key stages of LUSC development, from hyperplasia to dysplasia and eventually invasion, can be observed in this model system.
    Figure Legend Snippet: TME is dominant over the genotype in determining the epithelial architecture of TUM622 cells in vitro. A subpopulation of TUM622 cells has the ability to form acinar-like structures when plated as single cells in ECM culture. These acini are hyperplastic but nevertheless display proper apical−basal cell polarity, similar to hyperplasia observed at the earliest stage of LUSC development. Overexpression of SOX2 in TUM622 cells increases spheroid-forming capacity of TUM622 cells and drives a hyperplastic to dysplastic change in acinar phenotype where apical−basal cell polarity is disrupted and solid spheroids are formed. SOX2oe spheroids also display reduced apoptosis and increased differentiation. Addition of CAFs to TUM622 3D culture enhances acinar formation/growth and promotes invasion toward the CAFs. CAFs could promote the formation of an acinar-like structure in SOX2oe spheroids and induce invasion. Therefore, the key stages of LUSC development, from hyperplasia to dysplasia and eventually invasion, can be observed in this model system.

    Techniques Used: In Vitro, Over Expression

    Cells from TUM622 acini are capable of self-renewal and recapitulate the intratumoral heterogeneity observed in the PDX model and original human tumor. ( A ) Quantification of acinar-like structures formed in an LDA. Each seeding density is plated in triplicate. ( B ) Quantification of acini formed from three serial passages of TUM622 acini in Matrigel. Each passage is plated in triplicate. Error bars represent SD. ( C ) Quantification of in vivo growth of s.c. injected TUM622 acini; x axis represents days after injection, and y axis represents tumor volume (cubic millimeters). Error bars represent SEM. ( D ) Antibody staining of acini with markers of stem/progenitor cells (CXCR4 and SOX2), mesenchyme (Vimentin), epithelial differentiation (Involucrin), apoptosis (CC-3), and proliferation (Ki-67) in green; DAPI (blue), and CDH1 and Phalloidin (red). (Scale bars, 50 μm.) ( E ) Immunohistochemistry staining on sequential sections of TUM622 PDX and the human tumor from which it is derived. Magnification: 40×. Yellow arrow heads point to Vimentin and E-Cadherin double-positive cells. Red arrows point to cells undergoing apoptosis (CC-3−positive), and black arrows point to adjacent Involucrin positive cells that are negative for CC-3. (Scale bars, 60 µm.)
    Figure Legend Snippet: Cells from TUM622 acini are capable of self-renewal and recapitulate the intratumoral heterogeneity observed in the PDX model and original human tumor. ( A ) Quantification of acinar-like structures formed in an LDA. Each seeding density is plated in triplicate. ( B ) Quantification of acini formed from three serial passages of TUM622 acini in Matrigel. Each passage is plated in triplicate. Error bars represent SD. ( C ) Quantification of in vivo growth of s.c. injected TUM622 acini; x axis represents days after injection, and y axis represents tumor volume (cubic millimeters). Error bars represent SEM. ( D ) Antibody staining of acini with markers of stem/progenitor cells (CXCR4 and SOX2), mesenchyme (Vimentin), epithelial differentiation (Involucrin), apoptosis (CC-3), and proliferation (Ki-67) in green; DAPI (blue), and CDH1 and Phalloidin (red). (Scale bars, 50 μm.) ( E ) Immunohistochemistry staining on sequential sections of TUM622 PDX and the human tumor from which it is derived. Magnification: 40×. Yellow arrow heads point to Vimentin and E-Cadherin double-positive cells. Red arrows point to cells undergoing apoptosis (CC-3−positive), and black arrows point to adjacent Involucrin positive cells that are negative for CC-3. (Scale bars, 60 µm.)

    Techniques Used: In Vivo, Injection, Staining, Immunohistochemistry, Derivative Assay

    TUM622 is capable of forming acinar-like structures in 3D ECM. ( A ) Representative bright-field images of spheroids derived from single NSCLC cells in 3D ECM culture between days 8 and 12. ( Top ) Cell lines (TUM622, TUM426, TUM449, and TUM110) are derived from either patient tumors or the corresponding PDX. ( Bottom ) Established cell lines of LUSC (H520), LUAD (HCC2935), and immortalized bronchial epithelial cells (BEAS2B and NHBE). (Scale bars, 50 μm.) ( B ) Time course images of TUM622 cell cultured in Matrigel over a 10-d period. ( Top ) Bright-field images. ( Bottom ) Green is calcein, marking live cells; red is ethidium bromide, marking dead cells. (Scale bars, 100 μm.) ( C ) Quantification of acini number (right y axis, red) and average size of acini (left y axis, blue) plated in triplicate in a 24-well plate over 23 d in culture. Error bars represent SD. ( D ) ( Left ) Immunofluorescence of TUM622 acini stained with apical−basal cell polarity markers Golgi enzyme GM130 (green, apical) and ITGA6 (CD49f, basal, red), ( Right ) in comparison with β-catenin staining (green). (Scale bars, 50 μm.)
    Figure Legend Snippet: TUM622 is capable of forming acinar-like structures in 3D ECM. ( A ) Representative bright-field images of spheroids derived from single NSCLC cells in 3D ECM culture between days 8 and 12. ( Top ) Cell lines (TUM622, TUM426, TUM449, and TUM110) are derived from either patient tumors or the corresponding PDX. ( Bottom ) Established cell lines of LUSC (H520), LUAD (HCC2935), and immortalized bronchial epithelial cells (BEAS2B and NHBE). (Scale bars, 50 μm.) ( B ) Time course images of TUM622 cell cultured in Matrigel over a 10-d period. ( Top ) Bright-field images. ( Bottom ) Green is calcein, marking live cells; red is ethidium bromide, marking dead cells. (Scale bars, 100 μm.) ( C ) Quantification of acini number (right y axis, red) and average size of acini (left y axis, blue) plated in triplicate in a 24-well plate over 23 d in culture. Error bars represent SD. ( D ) ( Left ) Immunofluorescence of TUM622 acini stained with apical−basal cell polarity markers Golgi enzyme GM130 (green, apical) and ITGA6 (CD49f, basal, red), ( Right ) in comparison with β-catenin staining (green). (Scale bars, 50 μm.)

    Techniques Used: Derivative Assay, Cell Culture, Immunofluorescence, Staining

    SOX2 overexpression enhances the spheroid forming capacity of TUM622 cells and induces dysplasia. ( A ) Western blot comparing the expression level of SOX2 in Vector and SOX2oe cells to three LUSC cell lines with no/low (SW900), medium (H2170), and high (H520) levels of SOX2 expression. ( B ) SOX2 expression level in Vector vs. SOX2oe spheroids in 3D culture as quantified by qRT-PCR. Column represents triplicates, and error bars represent SD; P
    Figure Legend Snippet: SOX2 overexpression enhances the spheroid forming capacity of TUM622 cells and induces dysplasia. ( A ) Western blot comparing the expression level of SOX2 in Vector and SOX2oe cells to three LUSC cell lines with no/low (SW900), medium (H2170), and high (H520) levels of SOX2 expression. ( B ) SOX2 expression level in Vector vs. SOX2oe spheroids in 3D culture as quantified by qRT-PCR. Column represents triplicates, and error bars represent SD; P

    Techniques Used: Over Expression, Western Blot, Expressing, Plasmid Preparation, Quantitative RT-PCR

    Gene sets enriched in TUM622 3D vs. 2D culture. ( A ) Top four enriched gene sets identified from TUM622 3D culture (in comparison with TUM622 2D culture) that belong to each of the following categories: key signaling pathways (Wnt, Notch, and Hedgehog signaling), ES cell, polarity/acinar morphogenesis, and prognosis. Horizontal axis represents the −log 10 of FDR value, and the vertical axis represents NES. See the entire list of enriched gene sets corresponding to each category in Dataset S1 . ( B – E ) Enrichment plots of the top two enriched gene sets in each of the categories represented in A .
    Figure Legend Snippet: Gene sets enriched in TUM622 3D vs. 2D culture. ( A ) Top four enriched gene sets identified from TUM622 3D culture (in comparison with TUM622 2D culture) that belong to each of the following categories: key signaling pathways (Wnt, Notch, and Hedgehog signaling), ES cell, polarity/acinar morphogenesis, and prognosis. Horizontal axis represents the −log 10 of FDR value, and the vertical axis represents NES. See the entire list of enriched gene sets corresponding to each category in Dataset S1 . ( B – E ) Enrichment plots of the top two enriched gene sets in each of the categories represented in A .

    Techniques Used:

    3) Product Images from "Faustovirus E12 Transcriptome Analysis Reveals Complex Splicing in Capsid Gene"

    Article Title: Faustovirus E12 Transcriptome Analysis Reveals Complex Splicing in Capsid Gene

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02534

    Flowchart illustrating the workflow of this study. This flowchart shows the general pipeline of this RNA-seq study, starting from sample preparation and RNA extraction to cDNA sequencing and data analyses. The biological interpretation of expression count was possible through the functional categories clustering of expressed genes.
    Figure Legend Snippet: Flowchart illustrating the workflow of this study. This flowchart shows the general pipeline of this RNA-seq study, starting from sample preparation and RNA extraction to cDNA sequencing and data analyses. The biological interpretation of expression count was possible through the functional categories clustering of expressed genes.

    Techniques Used: RNA Sequencing Assay, Sample Prep, RNA Extraction, Sequencing, Expressing, Functional Assay

    4) Product Images from "Delineating the Dynamic Transcriptome Response of mRNA and microRNA during Zebrafish Heart Regeneration"

    Article Title: Delineating the Dynamic Transcriptome Response of mRNA and microRNA during Zebrafish Heart Regeneration

    Journal: Biomolecules

    doi: 10.3390/biom9010011

    ( A ) Experimental design of the H9c2 cell line. Both mRNA and miRNA were sequenced at 4 different time points that display a differentiation into a CM-like phenotype and a reduction of the proliferative capability. ( B ) Proportions of the significant down-regulated genes (d5 vs. undiff; FDR
    Figure Legend Snippet: ( A ) Experimental design of the H9c2 cell line. Both mRNA and miRNA were sequenced at 4 different time points that display a differentiation into a CM-like phenotype and a reduction of the proliferative capability. ( B ) Proportions of the significant down-regulated genes (d5 vs. undiff; FDR

    Techniques Used:

    5) Product Images from "Constitutive Changes in Circulating Follicular Helper T Cells and Their Subsets in Patients with Graves' Disease"

    Article Title: Constitutive Changes in Circulating Follicular Helper T Cells and Their Subsets in Patients with Graves' Disease

    Journal: Journal of Immunology Research

    doi: 10.1155/2018/8972572

    mRNA expression in human CD4 + T cells from GD patients. The expression of several transcription factors (Bcl-6, T-bet, GATA-3, and ROR γ t) in peripheral CD4 + T cells from the 9 GD patients and 12 HC was detected with a real-time PCR assay. (a) Relative expression levels of Bcl-6 mRNA; (b) relative expression levels of T-bet mRNA; (c) relative expression levels of GATA-3 mRNA; (d) relative expression levels of ROR γ t mRNA. ∗ p
    Figure Legend Snippet: mRNA expression in human CD4 + T cells from GD patients. The expression of several transcription factors (Bcl-6, T-bet, GATA-3, and ROR γ t) in peripheral CD4 + T cells from the 9 GD patients and 12 HC was detected with a real-time PCR assay. (a) Relative expression levels of Bcl-6 mRNA; (b) relative expression levels of T-bet mRNA; (c) relative expression levels of GATA-3 mRNA; (d) relative expression levels of ROR γ t mRNA. ∗ p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    6) Product Images from "Loss of KLHL6 promotes diffuse large B-cell lymphoma growth and survival by stabilizing the mRNA decay factor Roquin2"

    Article Title: Loss of KLHL6 promotes diffuse large B-cell lymphoma growth and survival by stabilizing the mRNA decay factor Roquin2

    Journal: Nature cell biology

    doi: 10.1038/s41556-018-0084-5

    KLHL6 is a BCR/NF-κB target gene that links Roquin2 degradation to BCR signaling (a) Immunoblot analysis of whole cell lysates from OCI-LY10, U2932, and HBL1 cells stimulated with 10 μg/ml F(ab′)2-IgM for 3 and 6 hours (left panel). A low exposure (l.e.) and high exposure (h.e.) are shown. Right panel shows level of KLHL6 mRNA analyzed by qPCR. The value for the PCR product from U2932 was set as 1. A representative graph from two independent experiments is shown. (b) Immunoblot analysis of whole cell lysates from U2932 cells treated with 10 μg/ml of F(ab′)2-IgM for the indicated times. Where indicated, cells were pre-treated with 5μM MLN4924 for 1 hour. (c) Analysis of level of KLHL6 mRNA by qPCR in U2932 cells treated with 10 μg/ml of F(ab′)2-IgM for the indicated times. A representative graph from two independent experiments is shown. The value for PCR product without treatment was set as 1. (d) Visualization of ChIP-seq peaks using the University of California Santa Cruz (UCSD) Genome browser (GEO Series accession GSE55105). RPM, reads per million mapped. (e) Same as in (c) except that U2932 cells were treated with DMSO, 10μM of IKK inhibitor (IKK-16) or 5μM of BTK inhibitor (Ibrutinib) for 6 hours. The value for PCR product present without treatment (DMSO) was set as 100% (mean±s.d., n=3 independent experiments, one-way ANOVA, *** P value≤0.001; ****P value≤0.0001). The right panel shows immunoblot analysis of whole cell lysates for the indicated proteins. (f) Immunoblot analysis of whole cell lysates from U2932 KLHL6 +/+ and KLHL6 −/− (clone-derived) cells treated with increasing concentrations of F(ab′)2-IgM for 6 hours. (g) Immunoblot analysis of whole cell lysates from U2932 cells stably expressing HA-Roquin2(WT) or HA-Roquin2(Y691F) treated with F(ab′)2-IgM for 6 hours. Unprocessed original scan of immunoblots for (a,b,e,f,g) are shown in Supplementary Fig. 8 , and source data for (a,c) and statistical source data and exact P values for (e) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.
    Figure Legend Snippet: KLHL6 is a BCR/NF-κB target gene that links Roquin2 degradation to BCR signaling (a) Immunoblot analysis of whole cell lysates from OCI-LY10, U2932, and HBL1 cells stimulated with 10 μg/ml F(ab′)2-IgM for 3 and 6 hours (left panel). A low exposure (l.e.) and high exposure (h.e.) are shown. Right panel shows level of KLHL6 mRNA analyzed by qPCR. The value for the PCR product from U2932 was set as 1. A representative graph from two independent experiments is shown. (b) Immunoblot analysis of whole cell lysates from U2932 cells treated with 10 μg/ml of F(ab′)2-IgM for the indicated times. Where indicated, cells were pre-treated with 5μM MLN4924 for 1 hour. (c) Analysis of level of KLHL6 mRNA by qPCR in U2932 cells treated with 10 μg/ml of F(ab′)2-IgM for the indicated times. A representative graph from two independent experiments is shown. The value for PCR product without treatment was set as 1. (d) Visualization of ChIP-seq peaks using the University of California Santa Cruz (UCSD) Genome browser (GEO Series accession GSE55105). RPM, reads per million mapped. (e) Same as in (c) except that U2932 cells were treated with DMSO, 10μM of IKK inhibitor (IKK-16) or 5μM of BTK inhibitor (Ibrutinib) for 6 hours. The value for PCR product present without treatment (DMSO) was set as 100% (mean±s.d., n=3 independent experiments, one-way ANOVA, *** P value≤0.001; ****P value≤0.0001). The right panel shows immunoblot analysis of whole cell lysates for the indicated proteins. (f) Immunoblot analysis of whole cell lysates from U2932 KLHL6 +/+ and KLHL6 −/− (clone-derived) cells treated with increasing concentrations of F(ab′)2-IgM for 6 hours. (g) Immunoblot analysis of whole cell lysates from U2932 cells stably expressing HA-Roquin2(WT) or HA-Roquin2(Y691F) treated with F(ab′)2-IgM for 6 hours. Unprocessed original scan of immunoblots for (a,b,e,f,g) are shown in Supplementary Fig. 8 , and source data for (a,c) and statistical source data and exact P values for (e) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Derivative Assay, Stable Transfection, Expressing, Western Blot

    KLHL6-Roquin2 axis controls NF-κB activation (a) Immunoblot analysis of whole cell lysates from U2932 cells expressing HA-Roquin2(WT) or HA-Roquin2(Y691F) treated with 10μg/ml of F(ab′)2-IgM for the indicated times. (b) Immunoblot analysis of whole cell lysates from U2932 KLHL6 +/+ or KLHL6 −/− cells (clone-derived) treated with 10μg/ml of F(ab′)2-IgM for the indicated times. (c) Immunoblot analysis of whole cell lysates from HBL1 cells electroporated with indicated siRNAs and treated as in (a) for the indicated times. (d) Immunoblot analysis of whole cell lysates from U2932 KLHL6 +/+ or KLHL6 −/− cells (clo ne-derived) treated with 10μg/ml of F(ab′)2-IgM for the indicated times. (e) Immunoblot analysis of fractionated U2932 KLHL6 +/+ or KLHL6 −/− (clone-derived) cells. (f) RelA ChIP-qPCR for the NFBKIA promoter in U2932 KLHL6 +/+ , KLHL6 −/− or KLHL6 −/− cells infected with indicated shRNAs. Data are displayed as fold enrichment relative to IgG control. A representative graph from two independent experiments is shown. (g) Overlap of BTB-associated mutations of KLHL6 with TNFAIP3 alterations in DLBCLs. Top panel shows tumors sequenced at UNMC 6 and DCI 7 (n=1175) with deleterious mutations of KLHL6 in the BTB-domain and TNFAIP3 mutations. In the bottom panel, the TNFAIP3 subset is shown as biallelic and monoallelic deletions. (h) Cell counts of GFP-sorted RCK8 cells expressing Cas9, the indicated gRNAs and a GFP marker. Cells were grown in media containing 1μg/ml of F(ab′)2-IgM (mean±s.d., n=3 independent experiments, two-way ANOVA, n.s, not significant). Right panel shows immunoblot analysis of whole cell lysates. (i) Apoptosis analysis of cells from (h) is shown (mean±s.d., n=3 independent experiments, one-way ANOVA, n.s, not significant). (j) The percentage of GFP + and AnnexinV + HBL1 and HLY-1 cells expressing the indicated shRNAs is shown (mean±s.d., n=3 independent experiments, one-way ANOVA, *P value≤0.05; *** P value≤0.001, n.s, not significant). (k) Model of KLHL6-Roquin2 axis in ABC-DLBCL. Unprocessed original scan of immunoblots for (a,b,c,d,e,h) are shown in Supplementary Fig. 8 , and source data for (f) and statistical source data for (h,i,j) and exact P values for (j) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.
    Figure Legend Snippet: KLHL6-Roquin2 axis controls NF-κB activation (a) Immunoblot analysis of whole cell lysates from U2932 cells expressing HA-Roquin2(WT) or HA-Roquin2(Y691F) treated with 10μg/ml of F(ab′)2-IgM for the indicated times. (b) Immunoblot analysis of whole cell lysates from U2932 KLHL6 +/+ or KLHL6 −/− cells (clone-derived) treated with 10μg/ml of F(ab′)2-IgM for the indicated times. (c) Immunoblot analysis of whole cell lysates from HBL1 cells electroporated with indicated siRNAs and treated as in (a) for the indicated times. (d) Immunoblot analysis of whole cell lysates from U2932 KLHL6 +/+ or KLHL6 −/− cells (clo ne-derived) treated with 10μg/ml of F(ab′)2-IgM for the indicated times. (e) Immunoblot analysis of fractionated U2932 KLHL6 +/+ or KLHL6 −/− (clone-derived) cells. (f) RelA ChIP-qPCR for the NFBKIA promoter in U2932 KLHL6 +/+ , KLHL6 −/− or KLHL6 −/− cells infected with indicated shRNAs. Data are displayed as fold enrichment relative to IgG control. A representative graph from two independent experiments is shown. (g) Overlap of BTB-associated mutations of KLHL6 with TNFAIP3 alterations in DLBCLs. Top panel shows tumors sequenced at UNMC 6 and DCI 7 (n=1175) with deleterious mutations of KLHL6 in the BTB-domain and TNFAIP3 mutations. In the bottom panel, the TNFAIP3 subset is shown as biallelic and monoallelic deletions. (h) Cell counts of GFP-sorted RCK8 cells expressing Cas9, the indicated gRNAs and a GFP marker. Cells were grown in media containing 1μg/ml of F(ab′)2-IgM (mean±s.d., n=3 independent experiments, two-way ANOVA, n.s, not significant). Right panel shows immunoblot analysis of whole cell lysates. (i) Apoptosis analysis of cells from (h) is shown (mean±s.d., n=3 independent experiments, one-way ANOVA, n.s, not significant). (j) The percentage of GFP + and AnnexinV + HBL1 and HLY-1 cells expressing the indicated shRNAs is shown (mean±s.d., n=3 independent experiments, one-way ANOVA, *P value≤0.05; *** P value≤0.001, n.s, not significant). (k) Model of KLHL6-Roquin2 axis in ABC-DLBCL. Unprocessed original scan of immunoblots for (a,b,c,d,e,h) are shown in Supplementary Fig. 8 , and source data for (f) and statistical source data for (h,i,j) and exact P values for (j) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.

    Techniques Used: Activation Assay, Expressing, Derivative Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Infection, Marker, Western Blot

    KLHL6 interacts and promotes ubiquitylation and degradation of Roquin2 (a) Proteomic analysis of KLHL6 immunoprecipitations. Spectral counts for Roquin2 proteins are shown. EV, empty vector. The analysis was performed once in two different cell lines (HEK293T and ARP-1). (b) Immunoblot analysis of immunoprecipitated FLAG-F-box proteins (FBPs) or BTB proteins (BTBPs) in HEK293T cells. Lane 1 shows whole cell lysates (WCL) from cells transfected with an empty vector. (c) Immunoblot analysis of immunoprecipitated FLAG-Roquin1 or FLAG-Roquin2 in HEK293T cells. (d) Immunoblot analysis of human DLBCL cell lysates. A low exposure (l.e.) and high exposure (h.e.) are shown. A representative blot from two independent experiments is shown. (e) Immunoblot analysis of whole cell lysates from OCI-LY10 cells electroporated with indicated siRNAs and treated with cycloheximide (CHX). Quantification and statistical analysis is shown in Fig. S2d . (f) KLHL6 +/+ and KLHL6 −/− U2932 cells(clone-derived) were processed as in (e). A low exposure (l.e.) and high exposure (h.e.) are shown. Quantification and statistical analysis is shown in Fig. S2d . (g) Schematic representation of KLHL6 protein displaying endogenous mutations in VAL and SUDHL10 (top panel). Bottom panel shows immunoblot analysis from immunoprecipitated FLAG-tagged KLHL6 wild-type (WT), KLHL6 mutants (L24R, A25E, N60T, and T72R), or empty vector (EV) in HEK293T cells. (h) Immunoblot analysis of whole cell lysates from VAL and SUDHL10 cells electroporated with siRNA scramble (siCTRL) or targeting KLHL6 (siKLHL6). (i) Analysis of level of KLHL6 mRNA by quantitative PCR(qPCR). The value for the PCR product from U2932 cells was set as 1. A representative graph from two independent experiments is shown. (j) Immunoblot analysis of whole cell lysates from VAL and SUDHL10 cells stably expressing KLHL6 under a doxycycline (DOX) inducible promoter with a puryomycin cassette after 12h of DOX treatment. (k) In vitro ubiquitylation reaction of immunopurified FLAG-KLHL6 and HA-Roquin2. Unprocessed original scan of immunoblots for (b,c,d,e,f,g,h,j,k) are shown in Supplementary Fig. 8 , and source data for (i) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.
    Figure Legend Snippet: KLHL6 interacts and promotes ubiquitylation and degradation of Roquin2 (a) Proteomic analysis of KLHL6 immunoprecipitations. Spectral counts for Roquin2 proteins are shown. EV, empty vector. The analysis was performed once in two different cell lines (HEK293T and ARP-1). (b) Immunoblot analysis of immunoprecipitated FLAG-F-box proteins (FBPs) or BTB proteins (BTBPs) in HEK293T cells. Lane 1 shows whole cell lysates (WCL) from cells transfected with an empty vector. (c) Immunoblot analysis of immunoprecipitated FLAG-Roquin1 or FLAG-Roquin2 in HEK293T cells. (d) Immunoblot analysis of human DLBCL cell lysates. A low exposure (l.e.) and high exposure (h.e.) are shown. A representative blot from two independent experiments is shown. (e) Immunoblot analysis of whole cell lysates from OCI-LY10 cells electroporated with indicated siRNAs and treated with cycloheximide (CHX). Quantification and statistical analysis is shown in Fig. S2d . (f) KLHL6 +/+ and KLHL6 −/− U2932 cells(clone-derived) were processed as in (e). A low exposure (l.e.) and high exposure (h.e.) are shown. Quantification and statistical analysis is shown in Fig. S2d . (g) Schematic representation of KLHL6 protein displaying endogenous mutations in VAL and SUDHL10 (top panel). Bottom panel shows immunoblot analysis from immunoprecipitated FLAG-tagged KLHL6 wild-type (WT), KLHL6 mutants (L24R, A25E, N60T, and T72R), or empty vector (EV) in HEK293T cells. (h) Immunoblot analysis of whole cell lysates from VAL and SUDHL10 cells electroporated with siRNA scramble (siCTRL) or targeting KLHL6 (siKLHL6). (i) Analysis of level of KLHL6 mRNA by quantitative PCR(qPCR). The value for the PCR product from U2932 cells was set as 1. A representative graph from two independent experiments is shown. (j) Immunoblot analysis of whole cell lysates from VAL and SUDHL10 cells stably expressing KLHL6 under a doxycycline (DOX) inducible promoter with a puryomycin cassette after 12h of DOX treatment. (k) In vitro ubiquitylation reaction of immunopurified FLAG-KLHL6 and HA-Roquin2. Unprocessed original scan of immunoblots for (b,c,d,e,f,g,h,j,k) are shown in Supplementary Fig. 8 , and source data for (i) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.

    Techniques Used: Plasmid Preparation, Immunoprecipitation, Transfection, Derivative Assay, Polymerase Chain Reaction, Stable Transfection, Expressing, In Vitro, Western Blot

    KLHL6 functions as a tumor suppressor in ABC-DLBCL by regulating its growth and survival (a) Kaplan–Meier analysis based on gene expression data for ABC-DLBCL tumors (GSE10846, GSE34171 and GSE3131239-41) is shown (n=367 patients). Censored subjects are indicated on the Kaplan-Meier cure as tick mark. Statistical analysis was performed using the Log-rank (Mantel-Cox), two-sided test, 95% confidence interval. (b) Cell counts of U2932-, OCI-LY10- and TMD8-Cas9 cells expressing the indicated gRNAs and carrying a puromycin cassette (mean±s.d., n=3 independent experiments, two-way ANOVA, *P value≤0.05; **P value≤0.01; *** P value≤0.001; ****P value≤0.0001) (left panel). Cells were grown in media containing 1μg/ml (U2932- and OCI-LY10) or 4μg/ml (TMD8) of F(ab′)2-IgM. Right panel shows immunoblot analysis of whole cell lysates. (c) Apoptosis analysis of U2932-, OCI-LY10-, and TMD8-Cas9 cells expressing the indicated gRNAs and carrying a GFP marker. Cells were grown as in (b) . Apoptosis was quantified on GFP + and Annexin V + cells (mean±s.d., n=3 independent experiments, one-way ANOVA, **P value≤0.01; *** P value≤0.001). (d) Left panel shows cell counts of GFP-sorted U2932 KLHL6 −/− (clone-derived) cells expressing empty vector (EV), KLHL6(WT) or BTB-mutants (L65P, S94I and F97L) and carrying a GFP marker (mean±s.d., n=3 independent experiments, two-way ANOVA, **P value≤0.01; *** P value≤0.001; ****P value≤0.0001). Right panel shows the immunoblot analysis of whole cell lysates. (e) Xenograft experiments with GFP-sorted U2932 KLHL6 −/− cells expressing an empty vector (EV), KLHL6(WT) or KLHL6(S94I) and carrying a GFP marker. Top panel shows the tumors at the experimental endpoint. Tumor volume (mean±s.d., n=5 mice per group, one-way ANOVA, *P value≤0.05; n.s, not significant) and tumor weight (mean±s.d., n=5 mice per group, one-tailed t-test, *P value ≤0.05; **P value ≤0.01; n.s, not significant) are shown in the bottom left and right, respectively. Unprocessed original scan of immunoblots for (b,d) are shown in Supplementary Fig. 8 , and statistical source data and exact P values for (a,b,c,d,e) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.
    Figure Legend Snippet: KLHL6 functions as a tumor suppressor in ABC-DLBCL by regulating its growth and survival (a) Kaplan–Meier analysis based on gene expression data for ABC-DLBCL tumors (GSE10846, GSE34171 and GSE3131239-41) is shown (n=367 patients). Censored subjects are indicated on the Kaplan-Meier cure as tick mark. Statistical analysis was performed using the Log-rank (Mantel-Cox), two-sided test, 95% confidence interval. (b) Cell counts of U2932-, OCI-LY10- and TMD8-Cas9 cells expressing the indicated gRNAs and carrying a puromycin cassette (mean±s.d., n=3 independent experiments, two-way ANOVA, *P value≤0.05; **P value≤0.01; *** P value≤0.001; ****P value≤0.0001) (left panel). Cells were grown in media containing 1μg/ml (U2932- and OCI-LY10) or 4μg/ml (TMD8) of F(ab′)2-IgM. Right panel shows immunoblot analysis of whole cell lysates. (c) Apoptosis analysis of U2932-, OCI-LY10-, and TMD8-Cas9 cells expressing the indicated gRNAs and carrying a GFP marker. Cells were grown as in (b) . Apoptosis was quantified on GFP + and Annexin V + cells (mean±s.d., n=3 independent experiments, one-way ANOVA, **P value≤0.01; *** P value≤0.001). (d) Left panel shows cell counts of GFP-sorted U2932 KLHL6 −/− (clone-derived) cells expressing empty vector (EV), KLHL6(WT) or BTB-mutants (L65P, S94I and F97L) and carrying a GFP marker (mean±s.d., n=3 independent experiments, two-way ANOVA, **P value≤0.01; *** P value≤0.001; ****P value≤0.0001). Right panel shows the immunoblot analysis of whole cell lysates. (e) Xenograft experiments with GFP-sorted U2932 KLHL6 −/− cells expressing an empty vector (EV), KLHL6(WT) or KLHL6(S94I) and carrying a GFP marker. Top panel shows the tumors at the experimental endpoint. Tumor volume (mean±s.d., n=5 mice per group, one-way ANOVA, *P value≤0.05; n.s, not significant) and tumor weight (mean±s.d., n=5 mice per group, one-tailed t-test, *P value ≤0.05; **P value ≤0.01; n.s, not significant) are shown in the bottom left and right, respectively. Unprocessed original scan of immunoblots for (b,d) are shown in Supplementary Fig. 8 , and statistical source data and exact P values for (a,b,c,d,e) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.

    Techniques Used: Expressing, Marker, Derivative Assay, Plasmid Preparation, Mouse Assay, One-tailed Test, Western Blot

    A non-degradable Roquin2 mutant phenocopies loss of KLHL6 (a) Left panel shows immunoblot analysis of immunoprecipitated FLAG-tagged Roquin2 wild type (WT) or mutants in HEK293T cells stably expressing KLHL6. EV, Empty vector. Right panel shows a schematic representation of Roquin2 mutants. Roquin2 mutants that interact (+) or do not interact (-) with KLHL6 are shown. A representative blot from two independent experiments is shown. Asterisk indicates non-specific bands. (b–d) Same as in (a). (e) Immunoblot analysis of immunoprecipitated FLAG-tagged Roquin2 wild type (WT) or mutants, as indicated, in HEK293T cells stably expressing KLHL6. EV, Empty vector. (f) Immunoblot analysis of whole cell lysates from a DLBCL cell line, BJAB, retrovirally transduced with cDNAs encoding Roquin2(WT) or Roquin2(Y691F) carrying a puromycin cassette. Cells were treated with cycloheximide (CHX) for the indicated times. Quantification and statistical analysis is shown in Fig. S4e . (g) Top panel shows tumor volume from subcutaneously injected NSG mice with U2932 cells stably expressing retroviruses encoding HA-Roquin2(WT) or HA-Roquin2(Y691F) carrying a puromycin cassette (mean±s.d., n=3 mice per group, two-way ANOVA, *P value≤0.05; **P value≤0.01; *** P value≤0.001; ****P value≤0.0001). Bottom panel shows tumor weight (mean±s.d., n=3 mice per group, two-tailed t-test, *** P value≤0.001). (h) Cell counts of GFP-sorted U2932 (left panel) or OCI-LY10 (right panel) KLHL6 +/+ and KLHL6 −/− (clone-derived) cells infected with scramble shRNA(shCTRL) or shRNA targeting Roquin2 (ShRoquin2#1 or #2) carrying a GFP marker. GFP + cells were grown in media containing 1μg/ml of F(ab′)2-IgM. (mean±s.d., n=3 independent experiments, two-way ANOVA, ****P value≤0.0001). Unprocessed original scan of immunoblots for (a,b,c,d,e,f) are shown in Supplementary Fig. 8 , and statistical source data and exact P values for (g,h) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.
    Figure Legend Snippet: A non-degradable Roquin2 mutant phenocopies loss of KLHL6 (a) Left panel shows immunoblot analysis of immunoprecipitated FLAG-tagged Roquin2 wild type (WT) or mutants in HEK293T cells stably expressing KLHL6. EV, Empty vector. Right panel shows a schematic representation of Roquin2 mutants. Roquin2 mutants that interact (+) or do not interact (-) with KLHL6 are shown. A representative blot from two independent experiments is shown. Asterisk indicates non-specific bands. (b–d) Same as in (a). (e) Immunoblot analysis of immunoprecipitated FLAG-tagged Roquin2 wild type (WT) or mutants, as indicated, in HEK293T cells stably expressing KLHL6. EV, Empty vector. (f) Immunoblot analysis of whole cell lysates from a DLBCL cell line, BJAB, retrovirally transduced with cDNAs encoding Roquin2(WT) or Roquin2(Y691F) carrying a puromycin cassette. Cells were treated with cycloheximide (CHX) for the indicated times. Quantification and statistical analysis is shown in Fig. S4e . (g) Top panel shows tumor volume from subcutaneously injected NSG mice with U2932 cells stably expressing retroviruses encoding HA-Roquin2(WT) or HA-Roquin2(Y691F) carrying a puromycin cassette (mean±s.d., n=3 mice per group, two-way ANOVA, *P value≤0.05; **P value≤0.01; *** P value≤0.001; ****P value≤0.0001). Bottom panel shows tumor weight (mean±s.d., n=3 mice per group, two-tailed t-test, *** P value≤0.001). (h) Cell counts of GFP-sorted U2932 (left panel) or OCI-LY10 (right panel) KLHL6 +/+ and KLHL6 −/− (clone-derived) cells infected with scramble shRNA(shCTRL) or shRNA targeting Roquin2 (ShRoquin2#1 or #2) carrying a GFP marker. GFP + cells were grown in media containing 1μg/ml of F(ab′)2-IgM. (mean±s.d., n=3 independent experiments, two-way ANOVA, ****P value≤0.0001). Unprocessed original scan of immunoblots for (a,b,c,d,e,f) are shown in Supplementary Fig. 8 , and statistical source data and exact P values for (g,h) can be found in Supplementary Table 6 . Unless otherwise noted, immunoblots are representative of three independent experiments.

    Techniques Used: Mutagenesis, Immunoprecipitation, Stable Transfection, Expressing, Plasmid Preparation, Transduction, Injection, Mouse Assay, Two Tailed Test, Derivative Assay, Infection, shRNA, Marker, Western Blot

    Stabilization of Roquin2 down-regulates BCR responsive genes (a) Representative image of U2932 cells expressing HA-Roquin2(WT), HA-Roquin2(Y691F) or HA-Roquin2(Y691FΔROQ) with puromycin cassette plated into a matrigel (Left panel). Middle panel shows immunoblot analysis of whole cell lysates and right panel shows cell counts from the matrigel (mean±s.d., n=4 independent experiments, one-way ANOVA, ****P value≤0.0001, n.s, not significant). Scale bar 150μm. (b) Volcano plot (left panel) showing down-regulated mRNAs (blue) in U2932 cells expressing Roquin2(Y691F) vs Roquin2(WT) upon 12 hours treatment with 10 μg/ml of F(ab′)2-IgM [log2(fold-change)
    Figure Legend Snippet: Stabilization of Roquin2 down-regulates BCR responsive genes (a) Representative image of U2932 cells expressing HA-Roquin2(WT), HA-Roquin2(Y691F) or HA-Roquin2(Y691FΔROQ) with puromycin cassette plated into a matrigel (Left panel). Middle panel shows immunoblot analysis of whole cell lysates and right panel shows cell counts from the matrigel (mean±s.d., n=4 independent experiments, one-way ANOVA, ****P value≤0.0001, n.s, not significant). Scale bar 150μm. (b) Volcano plot (left panel) showing down-regulated mRNAs (blue) in U2932 cells expressing Roquin2(Y691F) vs Roquin2(WT) upon 12 hours treatment with 10 μg/ml of F(ab′)2-IgM [log2(fold-change)

    Techniques Used: Expressing

    7) Product Images from "Host suppression of quorum sensing during catheter-associated urinary tract infections"

    Article Title: Host suppression of quorum sensing during catheter-associated urinary tract infections

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06882-y

    Urine and urea suppress quorum-regulated phenotypes. P. aeruginosa grown in 1× phosphate-buffered saline + 1% tryptone (PBS-T) supplemented with increasing concentrations of human urine was quantified for a zone of clearance around bacterial colony grown on milk plates, b pyocyanin (red bars) and rhamnolipid (blue bars) production. P. aeruginosa grown in LB supplemented with increasing amounts of urea was quantified for c zone of clearance around colonies grown on milk plates, d pyocyanin (red bars) and rhamnolipid (blue bars) production. e Quantification of pyocyanin produced by P. aeruginosa after 24-h growth in the presence or absence of 0.5 M urea followed by 16-h subculture growth in the presence or absence of 0.5 M urea. Data represent the mean and standard deviation of at least three independent replicates and were analyzed by unpaired t -test using GraphPad Prism software. Asterisk (*, **, and ***) indicates p
    Figure Legend Snippet: Urine and urea suppress quorum-regulated phenotypes. P. aeruginosa grown in 1× phosphate-buffered saline + 1% tryptone (PBS-T) supplemented with increasing concentrations of human urine was quantified for a zone of clearance around bacterial colony grown on milk plates, b pyocyanin (red bars) and rhamnolipid (blue bars) production. P. aeruginosa grown in LB supplemented with increasing amounts of urea was quantified for c zone of clearance around colonies grown on milk plates, d pyocyanin (red bars) and rhamnolipid (blue bars) production. e Quantification of pyocyanin produced by P. aeruginosa after 24-h growth in the presence or absence of 0.5 M urea followed by 16-h subculture growth in the presence or absence of 0.5 M urea. Data represent the mean and standard deviation of at least three independent replicates and were analyzed by unpaired t -test using GraphPad Prism software. Asterisk (*, **, and ***) indicates p

    Techniques Used: Produced, Standard Deviation, Software

    8) Product Images from "Hedgehog signaling has a protective effect in glucocorticoid-induced mouse neonatal brain injury through an 11?HSD2-dependent mechanism"

    Article Title: Hedgehog signaling has a protective effect in glucocorticoid-induced mouse neonatal brain injury through an 11?HSD2-dependent mechanism

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI36376

    Shh signaling upregulates transcription of 11 β HSD2 in vitro and in vivo. ( A ) Whole RNA was isolated from WT and Math1cre,SmoM2 CGNP cultures treated with vehicle, Shh, or Shh plus 40 μM Dex for 24 h. Quantitative PCR analysis showed that Shh treatment potently induced the Shh targets N-myc and Gli1 as well as 11 β HSD2 expression in WT CGNP cultures. Math1cre,SmoM2 CGNPs showed increased expression of N-myc , Gli1 , and 11 β HSD2 in both vehicle and Shh groups and was unchanged after Dex treatment. 11 β HSD1 expression was below detectable levels. ( B ) In vivo, N-myc , Gli1 , and 11 β HSD2 levels were upregulated in the Math1cre,SmoM2 cerebellum (CB). ( C ) In situ hybridization showing specific expression of 11 β HSD2 in the EGL of the P7 WT and Math1cre,SmoM2 cerebellum. Original magnification, ×400.
    Figure Legend Snippet: Shh signaling upregulates transcription of 11 β HSD2 in vitro and in vivo. ( A ) Whole RNA was isolated from WT and Math1cre,SmoM2 CGNP cultures treated with vehicle, Shh, or Shh plus 40 μM Dex for 24 h. Quantitative PCR analysis showed that Shh treatment potently induced the Shh targets N-myc and Gli1 as well as 11 β HSD2 expression in WT CGNP cultures. Math1cre,SmoM2 CGNPs showed increased expression of N-myc , Gli1 , and 11 β HSD2 in both vehicle and Shh groups and was unchanged after Dex treatment. 11 β HSD1 expression was below detectable levels. ( B ) In vivo, N-myc , Gli1 , and 11 β HSD2 levels were upregulated in the Math1cre,SmoM2 cerebellum (CB). ( C ) In situ hybridization showing specific expression of 11 β HSD2 in the EGL of the P7 WT and Math1cre,SmoM2 cerebellum. Original magnification, ×400.

    Techniques Used: In Vitro, In Vivo, Isolation, Real-time Polymerase Chain Reaction, Expressing, In Situ Hybridization

    9) Product Images from "Phosphorylated and sumoylation-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression"

    Article Title: Phosphorylated and sumoylation-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr3211

    Phosphorylation of PR Ser294 drives SUMO-deficient PR gene expression and promoter selectivity in MCF-7 and T47D cells . ( A ) Relative expression level (copy number) of PR target genes in breast cancer patient cohorts. ( B ) Relative gene expression levels of selected PR target genes in MCF-7 cells stably expressing either empty vector (PR-null), WT or SUMO-deficient K388R PRs. Cells were co-treated with progestin R5020 and/or antiprogestin RU486 for six hours and mRNA levels were measured using RT-qPCR (see Materials and methods). ( C ) Relative gene expression levels of the same PR target genes (as in parts A-B) were measured using RT-qPCR in five vector-matched T47D cell lines stably expressing PRs: empty vector (null), wild-type (WT) PR, K388R mutant (KR) PR, S294A mutant (SA) PR, and K388R and S294A double mutant (KRSA) PR. Cells were treated with R5020 for six hours. ( D ) T47D cells expressing WT PR were treated cells with epidermal growth factor (EGF) for two days and treated with R5020 for 3, 24, or 48 hours. Relative MAP1A and RGS2 mRNA levels were measured using RT-qPCR. ( E ) Parental T47Dco cells were pretreated with EGF for 20 minutes prior to 24 hours of R5020 treatment. Relative RGS2 mRNA levels were measured by RT-qPCR. Data are represented as mean of n = 3 +/- SD and significance calculated using Student's t-test. n, number; PR, progesterone receptor; SD, standard deviation; SUMO, small ubiquitin-like modifier; WT, wild type.
    Figure Legend Snippet: Phosphorylation of PR Ser294 drives SUMO-deficient PR gene expression and promoter selectivity in MCF-7 and T47D cells . ( A ) Relative expression level (copy number) of PR target genes in breast cancer patient cohorts. ( B ) Relative gene expression levels of selected PR target genes in MCF-7 cells stably expressing either empty vector (PR-null), WT or SUMO-deficient K388R PRs. Cells were co-treated with progestin R5020 and/or antiprogestin RU486 for six hours and mRNA levels were measured using RT-qPCR (see Materials and methods). ( C ) Relative gene expression levels of the same PR target genes (as in parts A-B) were measured using RT-qPCR in five vector-matched T47D cell lines stably expressing PRs: empty vector (null), wild-type (WT) PR, K388R mutant (KR) PR, S294A mutant (SA) PR, and K388R and S294A double mutant (KRSA) PR. Cells were treated with R5020 for six hours. ( D ) T47D cells expressing WT PR were treated cells with epidermal growth factor (EGF) for two days and treated with R5020 for 3, 24, or 48 hours. Relative MAP1A and RGS2 mRNA levels were measured using RT-qPCR. ( E ) Parental T47Dco cells were pretreated with EGF for 20 minutes prior to 24 hours of R5020 treatment. Relative RGS2 mRNA levels were measured by RT-qPCR. Data are represented as mean of n = 3 +/- SD and significance calculated using Student's t-test. n, number; PR, progesterone receptor; SD, standard deviation; SUMO, small ubiquitin-like modifier; WT, wild type.

    Techniques Used: Expressing, Stable Transfection, Plasmid Preparation, Quantitative RT-PCR, Mutagenesis, Standard Deviation

    The SUMO-deficient PR gene expression signature is associated with HER2-positive human breast tumors and predicts reduced patient survival . ( A ) Normalized gene expression levels (for genes in our LD KR > WT gene signature) are presented for each tumor in the patient cohort [ 64 ], organized by ERBB2 status. ( B ) Gene expression levels were measured by RT-qPCR for CHN2 and RGS2 (both upregulated by SUMO-deficient PR, and members of the LD KR > WT gene signature) and the control gene ACOT6 (equally upregulated by both WT and KR receptors) in BT-474 human breast cancer cells. Cells were pre-treated with MEK kinase inhibitor U0126 prior to progestin or antiprogestin co-treatment. Protein levels were evaluated by western blotting for total PR, PR Ser294 phosphorylation, total ERK1/2, and ERK1/2 phosphorylation. ( C ) Kaplan-Meier survival curve for distant metastasis free survival for patients whose tumors expressed the combined T47D metagenes (WT or KR, -/+R5020) relative to patient tumors lacking these metagenes. Patient samples include untreated and tamoxifen-treated ER-positive tumors from the Loi et al. dataset [ 29 ]. ( D ) Survival curves as in part C for patients whose tumors expressed the combined T47D metagenes (KR -R5020, or KR +R5020) relative to patient tumors lacking these metagenes. [See also Additional files 4 and 9 ]. ER, estrogen receptor; KR, K388R PR-B mutant; LD, ligand dependent; SUMO, small ubiquitin-like modifier; WT, wild type.
    Figure Legend Snippet: The SUMO-deficient PR gene expression signature is associated with HER2-positive human breast tumors and predicts reduced patient survival . ( A ) Normalized gene expression levels (for genes in our LD KR > WT gene signature) are presented for each tumor in the patient cohort [ 64 ], organized by ERBB2 status. ( B ) Gene expression levels were measured by RT-qPCR for CHN2 and RGS2 (both upregulated by SUMO-deficient PR, and members of the LD KR > WT gene signature) and the control gene ACOT6 (equally upregulated by both WT and KR receptors) in BT-474 human breast cancer cells. Cells were pre-treated with MEK kinase inhibitor U0126 prior to progestin or antiprogestin co-treatment. Protein levels were evaluated by western blotting for total PR, PR Ser294 phosphorylation, total ERK1/2, and ERK1/2 phosphorylation. ( C ) Kaplan-Meier survival curve for distant metastasis free survival for patients whose tumors expressed the combined T47D metagenes (WT or KR, -/+R5020) relative to patient tumors lacking these metagenes. Patient samples include untreated and tamoxifen-treated ER-positive tumors from the Loi et al. dataset [ 29 ]. ( D ) Survival curves as in part C for patients whose tumors expressed the combined T47D metagenes (KR -R5020, or KR +R5020) relative to patient tumors lacking these metagenes. [See also Additional files 4 and 9 ]. ER, estrogen receptor; KR, K388R PR-B mutant; LD, ligand dependent; SUMO, small ubiquitin-like modifier; WT, wild type.

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

    Gene expression profiling of T47D cells stably expressing WT or SUMO-deficient PR, treated with or without R5020 for six hours . ( A ) Western blot showing total and phospho-Ser294 PR proteins (total ERK1/2 served as a loading control) in 12 human breast tumors. ( B ) T47D cells stably expressing either wild-type PR-B (WT), SUMO-deficient mutant K388R PR-B (KR), or empty vector (null) controls were treated without or with R5020 prior to western blotting for PR-B. ( C ) Heat map showing normalized expression values for differentially expressed transcripts (fold change > 8.0 in at least one sample, BH adjusted P
    Figure Legend Snippet: Gene expression profiling of T47D cells stably expressing WT or SUMO-deficient PR, treated with or without R5020 for six hours . ( A ) Western blot showing total and phospho-Ser294 PR proteins (total ERK1/2 served as a loading control) in 12 human breast tumors. ( B ) T47D cells stably expressing either wild-type PR-B (WT), SUMO-deficient mutant K388R PR-B (KR), or empty vector (null) controls were treated without or with R5020 prior to western blotting for PR-B. ( C ) Heat map showing normalized expression values for differentially expressed transcripts (fold change > 8.0 in at least one sample, BH adjusted P

    Techniques Used: Expressing, Stable Transfection, Western Blot, Mutagenesis, Plasmid Preparation

    Promoter selectivity is achieved through increased recruitment of SUMO-deficient KR PR, CBP, MLL2 and histone tail modification, H3K4me2, to enhancer loci . ( A ) Schematic showing the MSX2 gene PRE-containing enhancer region located 15,094 bp upstream from the transcriptional start site. ( B ) Relative recruitment of PR to the MSX2 enhancer region was measured by ChIP-qPCR assays in T47D cells expressing constitutive PR null, WT or KR PR after treatment with R5020 for one or four hours. PR recruitment values were normalized as a percentage of input chromatin DNA values. To control for background non-specific antibody binding, immunoprecipitated chromatin contained a mixture from all samples with an IgG antibody. Similar ChIP results were obtained in T47D cells expressing inducible PR (right side). ( C ) The relative recruitment of CBP to the MSX2 enhancer region was measured as described in part B . ( D ) Levels of H3K4 dimethylation at the MSX2 enhancer were measured in the inducible PR expressing cell lines (iWT and iKR). The presence of H3K4me2 was determined at the MSX2 enhancer, up/downstream from the PRE, using overlapping qPCR products that span the region. ( E ) MLL2 recruitment to the MSX2 enhancer region was determined in T47D cells expressing both constitutive PR and inducible PR, as described in part B . ( F ) MAT2A gene expression was measured by RT-qPCR in T47D cells expressing stable WT or SUMO-deficient KR PR. Additionally, PR and MLL2 recruitment was quantified in these cells, as measured by standard ChIP-qPCR assay. Data are represented as mean of n = 3 +/- SD and significance calculated using Student's t-test. [See also Additional files 7 , 5 . KR, K388R PR-B mutant]. CBP, CREB-(cAMP-response element-binding protein)-binding protein; ChIP, chromatin immunoprecipitation; H3K4me2, histone H3 lysine 4 dimethylation; IgG, immunoglobulin G; KR, K388R PR-B mutant; MLL2, mixed lineage leukemia 2; PR, progesterone receptor; PRE, progesterone receptor response element; SD, standard deviation; SUMO, small ubiquitin-like modifier; WT, wild type.
    Figure Legend Snippet: Promoter selectivity is achieved through increased recruitment of SUMO-deficient KR PR, CBP, MLL2 and histone tail modification, H3K4me2, to enhancer loci . ( A ) Schematic showing the MSX2 gene PRE-containing enhancer region located 15,094 bp upstream from the transcriptional start site. ( B ) Relative recruitment of PR to the MSX2 enhancer region was measured by ChIP-qPCR assays in T47D cells expressing constitutive PR null, WT or KR PR after treatment with R5020 for one or four hours. PR recruitment values were normalized as a percentage of input chromatin DNA values. To control for background non-specific antibody binding, immunoprecipitated chromatin contained a mixture from all samples with an IgG antibody. Similar ChIP results were obtained in T47D cells expressing inducible PR (right side). ( C ) The relative recruitment of CBP to the MSX2 enhancer region was measured as described in part B . ( D ) Levels of H3K4 dimethylation at the MSX2 enhancer were measured in the inducible PR expressing cell lines (iWT and iKR). The presence of H3K4me2 was determined at the MSX2 enhancer, up/downstream from the PRE, using overlapping qPCR products that span the region. ( E ) MLL2 recruitment to the MSX2 enhancer region was determined in T47D cells expressing both constitutive PR and inducible PR, as described in part B . ( F ) MAT2A gene expression was measured by RT-qPCR in T47D cells expressing stable WT or SUMO-deficient KR PR. Additionally, PR and MLL2 recruitment was quantified in these cells, as measured by standard ChIP-qPCR assay. Data are represented as mean of n = 3 +/- SD and significance calculated using Student's t-test. [See also Additional files 7 , 5 . KR, K388R PR-B mutant]. CBP, CREB-(cAMP-response element-binding protein)-binding protein; ChIP, chromatin immunoprecipitation; H3K4me2, histone H3 lysine 4 dimethylation; IgG, immunoglobulin G; KR, K388R PR-B mutant; MLL2, mixed lineage leukemia 2; PR, progesterone receptor; PRE, progesterone receptor response element; SD, standard deviation; SUMO, small ubiquitin-like modifier; WT, wild type.

    Techniques Used: Modification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Binding Assay, Immunoprecipitation, Quantitative RT-PCR, Mutagenesis, Protein Binding, Standard Deviation

    10) Product Images from "MMP-9 and CXCL8/IL-8 Are Potential Therapeutic Targets in Epidermolysis Bullosa Simplex"

    Article Title: MMP-9 and CXCL8/IL-8 Are Potential Therapeutic Targets in Epidermolysis Bullosa Simplex

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070123

    Reduced expression of target genes upon IL-1β depletion. NEB-1 cells and EBDM-1 cells were incubated with 2 µg/ml IL-1β neutralizing antibody for 24 h to deplete IL-1β in the culture medium. Expression of untreated EBDM-1 cells (−) was compared to treated EBDM-1 cells (+) by SQRT-PCR. We picked at least one representative target gene of each of the six functional groups identified in the microarray. All investigated targets showed significant downregulation after IL-1β depletion. No differences in geneexpression were observed in NEB-1 cells −/+ antibody incubation (n = 3). Student’s t -test was performed with p values: * ≤0.05, *** ≤0.005, ΔΔ ≤0.0005.
    Figure Legend Snippet: Reduced expression of target genes upon IL-1β depletion. NEB-1 cells and EBDM-1 cells were incubated with 2 µg/ml IL-1β neutralizing antibody for 24 h to deplete IL-1β in the culture medium. Expression of untreated EBDM-1 cells (−) was compared to treated EBDM-1 cells (+) by SQRT-PCR. We picked at least one representative target gene of each of the six functional groups identified in the microarray. All investigated targets showed significant downregulation after IL-1β depletion. No differences in geneexpression were observed in NEB-1 cells −/+ antibody incubation (n = 3). Student’s t -test was performed with p values: * ≤0.05, *** ≤0.005, ΔΔ ≤0.0005.

    Techniques Used: Expressing, Incubation, Polymerase Chain Reaction, Functional Assay, Microarray

    11) Product Images from "The oncogenic properties of EWS/WT1 of desmoplastic small round cell tumors are unmasked by loss of p53 in murine embryonic fibroblasts"

    Article Title: The oncogenic properties of EWS/WT1 of desmoplastic small round cell tumors are unmasked by loss of p53 in murine embryonic fibroblasts

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-13-585

    MEFs expressing EWS/ WT1- KTS or EWS/ WT1 + KTS have distinct expression profiles and EWS/ WT1 + KTS expression is associated with up - regulation of canonical Wnt pathway signaling. (A) Heat map of differentially expressed probes identified in primary MEFs expressing EWS/WT1 + KTS, eGFP controls and EWS/WT1-KTS. The comparison is of four independently generated pools of MEFs each infected with EWS/WT1 + KTS, EWS/WT1-KTS or GFP. (B) GSEA plot depicting enrichment of the KIM_WT1_TARGETS_UP gene set in lines expressing EWS/WT1-KTS compared to eGFP controls. (C) Wnt related gene sets enriched in EWS/WT1 + KTS expressing cells (left) and GSEA plot of PID_WNT_Signaling_Pathway geneset (right). (D) Immunocytochemistry of 293 T cells infected with doxycycline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS. Cells are shown in the absence of doxycyline (panel on top and also with the presence of doxycycline treatment (thus doxycycline repressed) for 48 hours (panel on bottom). Cells are stained with DAPI for nuclear staining (blue) and β-catenin antibody with secondary anti-goat Alexa Fluor (red). β-catenin translocation from the cellular membrane to the nucleus is a marker of canonical Wnt-pathway activation.
    Figure Legend Snippet: MEFs expressing EWS/ WT1- KTS or EWS/ WT1 + KTS have distinct expression profiles and EWS/ WT1 + KTS expression is associated with up - regulation of canonical Wnt pathway signaling. (A) Heat map of differentially expressed probes identified in primary MEFs expressing EWS/WT1 + KTS, eGFP controls and EWS/WT1-KTS. The comparison is of four independently generated pools of MEFs each infected with EWS/WT1 + KTS, EWS/WT1-KTS or GFP. (B) GSEA plot depicting enrichment of the KIM_WT1_TARGETS_UP gene set in lines expressing EWS/WT1-KTS compared to eGFP controls. (C) Wnt related gene sets enriched in EWS/WT1 + KTS expressing cells (left) and GSEA plot of PID_WNT_Signaling_Pathway geneset (right). (D) Immunocytochemistry of 293 T cells infected with doxycycline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS. Cells are shown in the absence of doxycyline (panel on top and also with the presence of doxycycline treatment (thus doxycycline repressed) for 48 hours (panel on bottom). Cells are stained with DAPI for nuclear staining (blue) and β-catenin antibody with secondary anti-goat Alexa Fluor (red). β-catenin translocation from the cellular membrane to the nucleus is a marker of canonical Wnt-pathway activation.

    Techniques Used: Expressing, Generated, Infection, Immunocytochemistry, Staining, Translocation Assay, Marker, Activation Assay

    EWS/WT1-KTS and EWS/WT1 + KTS enhances clonogenicity in p53 +/- or p53 -/- MEFs in reduced serum. (A) Western blot analysis of lysates derived from wild-type, p53 +/- , and p53 -/- background MEFs which were infected with doxycyline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS lentiviral particles, showing expression of EWS/WT1 in the absence of doxycycline. (B) Representative images from one experiment showing MEF colonies of the indicated genotype expressing eGFP, EWS/WT1-KTS or EWS/WT1 + KTS, plated at 1× 10 5 cells per plate and cultured in 1% or 2% fetal calf serum. After 14 days cells were fixed with glutaraldehyde and stained with crystal violet. (C) Mean number of colonies (from three independent experiments) greater than 5 mm of cells cultured in 1% (left panel) or 2% (right panel) serum on day 14. Error bars show ± SEM from three independent experiments. # denotes a p value of
    Figure Legend Snippet: EWS/WT1-KTS and EWS/WT1 + KTS enhances clonogenicity in p53 +/- or p53 -/- MEFs in reduced serum. (A) Western blot analysis of lysates derived from wild-type, p53 +/- , and p53 -/- background MEFs which were infected with doxycyline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS lentiviral particles, showing expression of EWS/WT1 in the absence of doxycycline. (B) Representative images from one experiment showing MEF colonies of the indicated genotype expressing eGFP, EWS/WT1-KTS or EWS/WT1 + KTS, plated at 1× 10 5 cells per plate and cultured in 1% or 2% fetal calf serum. After 14 days cells were fixed with glutaraldehyde and stained with crystal violet. (C) Mean number of colonies (from three independent experiments) greater than 5 mm of cells cultured in 1% (left panel) or 2% (right panel) serum on day 14. Error bars show ± SEM from three independent experiments. # denotes a p value of

    Techniques Used: Western Blot, Derivative Assay, Infection, Expressing, Cell Culture, Staining

    EWS/WT1-KTS and EWS/WT1 + KTS increase anchorage independent growth and confer resistance to daunorubin induced apoptosis and cell cycle arrest following radiation. (A) 1x10 4 p53 wild-type, p53 +/- or p53 -/- MEFs expressing eGFP, EWS/WT1-KTS or EWS/WT1 + KTS were plated in soft agar and the number of colonies greater than 2 mm counted after 14 days. Values are mean ± SEM of three independently generated and infected pools of MEFs for each genotype tested in three independent experiments. P values of less than 0.05 as determined by Student’s t-tests are shown. (B) Viability assay of cells treated with daunorubicin (0.5 μg/ml) for 24 hours. The data show the ratio of WST1 absorbance of treated cells to the same number of untreated cells. Values are mean ± SEM of three independent experiments. P values
    Figure Legend Snippet: EWS/WT1-KTS and EWS/WT1 + KTS increase anchorage independent growth and confer resistance to daunorubin induced apoptosis and cell cycle arrest following radiation. (A) 1x10 4 p53 wild-type, p53 +/- or p53 -/- MEFs expressing eGFP, EWS/WT1-KTS or EWS/WT1 + KTS were plated in soft agar and the number of colonies greater than 2 mm counted after 14 days. Values are mean ± SEM of three independently generated and infected pools of MEFs for each genotype tested in three independent experiments. P values of less than 0.05 as determined by Student’s t-tests are shown. (B) Viability assay of cells treated with daunorubicin (0.5 μg/ml) for 24 hours. The data show the ratio of WST1 absorbance of treated cells to the same number of untreated cells. Values are mean ± SEM of three independent experiments. P values

    Techniques Used: Expressing, Generated, Infection, Viability Assay

    EWS/WT1-KTS and EWS/WT1 + KTS expression co-operates with loss or inactivation of p53 to transform MEFs. (A) Western blots of lysates from SV40 transformed MEFs expressing either eGFP, EWS/WT1-KTS or EWS/WT1 + KTS under the control of a 4-OHT inducible promoter 48 hours after 4-OHT treatment (upper panel) or a doxycycline repressible promoter without doxycycline (lower panel). The anti-WT1 antibody detects a Cterminal-epitope in WT1. Arrow indicates EWS/WT1 to distinguish it from endogenous WT1. A non-specific band of similar size to EWS/WT1 was observed in SV40-transformed, but not untransformed MEFs. (B) qPCR analysis of mRNA expression of eGFP, EWS/WT1-KTS or EWS/WT1 + KTS in MEFs induced by either the 4-OHT inducible or tetracycline repressible expression systems. (C) Fold change in cell number 14 days after MEFs transformed by either SV40 or EIA/RAS were infected with eGFP, EWS/WT1-KTS or EWS/WT1 + KTS using the 4OHT inducible system in SV40 transformed cells, and the doxycycline repressible system in EIA/RAS transformed cells. Cells were plated at equal densities and counted and re-plated at the same dilution every 3–4 days. Data represent the mean ± SEM of three independently generated pools of MEFs tested in three independent experiments. (D) MEFs derived from littermate wildtype (upper panel), p53+/- (middle panel) and p53-/-(lower panel) mice were infected with doxycycline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS and then plated at equal density. Cells were counted and re-plated on the indicated days. Values are mean ± SEM of three independently generated and infected pools of MEFs tested over three independent experiments. # denotes a p value of 0.003 and * denotes a p value of 0.004 with Student’s t-test comparing eGFP to EWS/WT1–KTS and eGFP to EWS/WT1 + KTS. Representative images of morphology of MEFs at 22 days are shown.
    Figure Legend Snippet: EWS/WT1-KTS and EWS/WT1 + KTS expression co-operates with loss or inactivation of p53 to transform MEFs. (A) Western blots of lysates from SV40 transformed MEFs expressing either eGFP, EWS/WT1-KTS or EWS/WT1 + KTS under the control of a 4-OHT inducible promoter 48 hours after 4-OHT treatment (upper panel) or a doxycycline repressible promoter without doxycycline (lower panel). The anti-WT1 antibody detects a Cterminal-epitope in WT1. Arrow indicates EWS/WT1 to distinguish it from endogenous WT1. A non-specific band of similar size to EWS/WT1 was observed in SV40-transformed, but not untransformed MEFs. (B) qPCR analysis of mRNA expression of eGFP, EWS/WT1-KTS or EWS/WT1 + KTS in MEFs induced by either the 4-OHT inducible or tetracycline repressible expression systems. (C) Fold change in cell number 14 days after MEFs transformed by either SV40 or EIA/RAS were infected with eGFP, EWS/WT1-KTS or EWS/WT1 + KTS using the 4OHT inducible system in SV40 transformed cells, and the doxycycline repressible system in EIA/RAS transformed cells. Cells were plated at equal densities and counted and re-plated at the same dilution every 3–4 days. Data represent the mean ± SEM of three independently generated pools of MEFs tested in three independent experiments. (D) MEFs derived from littermate wildtype (upper panel), p53+/- (middle panel) and p53-/-(lower panel) mice were infected with doxycycline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS and then plated at equal density. Cells were counted and re-plated on the indicated days. Values are mean ± SEM of three independently generated and infected pools of MEFs tested over three independent experiments. # denotes a p value of 0.003 and * denotes a p value of 0.004 with Student’s t-test comparing eGFP to EWS/WT1–KTS and eGFP to EWS/WT1 + KTS. Representative images of morphology of MEFs at 22 days are shown.

    Techniques Used: Expressing, Western Blot, Transformation Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Infection, Generated, Derivative Assay, Mouse Assay

    MEFs expressing EWS/ WT1- KTS or EWS/ WT1 + KTS have distinct expression profiles and EWS/ WT1 + KTS expression is associated with up - regulation of canonical Wnt pathway signaling. (A) Heat map of differentially expressed probes identified in primary MEFs expressing EWS/WT1 + KTS, eGFP controls and EWS/WT1-KTS. The comparison is of four independently generated pools of MEFs each infected with EWS/WT1 + KTS, EWS/WT1-KTS or GFP. (B) GSEA plot depicting enrichment of the KIM_WT1_TARGETS_UP gene set in lines expressing EWS/WT1-KTS compared to eGFP controls. (C) Wnt related gene sets enriched in EWS/WT1 + KTS expressing cells (left) and GSEA plot of PID_WNT_Signaling_Pathway geneset (right). (D) Immunocytochemistry of 293 T cells infected with doxycycline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS. Cells are shown in the absence of doxycyline (panel on top and also with the presence of doxycycline treatment (thus doxycycline repressed) for 48 hours (panel on bottom). Cells are stained with DAPI for nuclear staining (blue) and β-catenin antibody with secondary anti-goat Alexa Fluor (red). β-catenin translocation from the cellular membrane to the nucleus is a marker of canonical Wnt-pathway activation.
    Figure Legend Snippet: MEFs expressing EWS/ WT1- KTS or EWS/ WT1 + KTS have distinct expression profiles and EWS/ WT1 + KTS expression is associated with up - regulation of canonical Wnt pathway signaling. (A) Heat map of differentially expressed probes identified in primary MEFs expressing EWS/WT1 + KTS, eGFP controls and EWS/WT1-KTS. The comparison is of four independently generated pools of MEFs each infected with EWS/WT1 + KTS, EWS/WT1-KTS or GFP. (B) GSEA plot depicting enrichment of the KIM_WT1_TARGETS_UP gene set in lines expressing EWS/WT1-KTS compared to eGFP controls. (C) Wnt related gene sets enriched in EWS/WT1 + KTS expressing cells (left) and GSEA plot of PID_WNT_Signaling_Pathway geneset (right). (D) Immunocytochemistry of 293 T cells infected with doxycycline repressible eGFP, EWS/WT1-KTS or EWS/WT1 + KTS. Cells are shown in the absence of doxycyline (panel on top and also with the presence of doxycycline treatment (thus doxycycline repressed) for 48 hours (panel on bottom). Cells are stained with DAPI for nuclear staining (blue) and β-catenin antibody with secondary anti-goat Alexa Fluor (red). β-catenin translocation from the cellular membrane to the nucleus is a marker of canonical Wnt-pathway activation.

    Techniques Used: Expressing, Generated, Infection, Immunocytochemistry, Staining, Translocation Assay, Marker, Activation Assay

    12) Product Images from "MiR-133b Regulates the Expression of the Actin Protein TAGLN2 during Oocyte Growth and Maturation: A Potential Target for Infertility Therapy"

    Article Title: MiR-133b Regulates the Expression of the Actin Protein TAGLN2 during Oocyte Growth and Maturation: A Potential Target for Infertility Therapy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0100751

    miR-133b targets TAGLN2 in 293T cells. A) Vertical axis is the ratio of Renilla to firefly luciferase and the horizontal axis is the experiment groups: 1. siCHECK-NP; 2. siCHECK-3UTR+microRNA-NC; 3. siCHECK-3UTR+25 nM microRNA-133b; 4. siCHECK-3UTR+50 nM microRNA-133b; 5. siCHECK-3UTR+100 nM microRA-133b; 6. siCHECK-3UTR-m+100 nM microRNA-133b. B) Protein expression of TAGLN2 in 293T cells after being treated with miR-133b. C) mRNA expression of TAGLN2 in 293T cells after being treated with miR-133b. Each sample was tested in triplicate and the data were presented in mean ± SD. The experiments were repeated twice and similar results were obtained. ** P
    Figure Legend Snippet: miR-133b targets TAGLN2 in 293T cells. A) Vertical axis is the ratio of Renilla to firefly luciferase and the horizontal axis is the experiment groups: 1. siCHECK-NP; 2. siCHECK-3UTR+microRNA-NC; 3. siCHECK-3UTR+25 nM microRNA-133b; 4. siCHECK-3UTR+50 nM microRNA-133b; 5. siCHECK-3UTR+100 nM microRA-133b; 6. siCHECK-3UTR-m+100 nM microRNA-133b. B) Protein expression of TAGLN2 in 293T cells after being treated with miR-133b. C) mRNA expression of TAGLN2 in 293T cells after being treated with miR-133b. Each sample was tested in triplicate and the data were presented in mean ± SD. The experiments were repeated twice and similar results were obtained. ** P

    Techniques Used: Luciferase, Expressing

    13) Product Images from "Two Functionally Distinct Isoforms of TL1A (TNFSF15) Generated by Differential Ectodomain Shedding"

    Article Title: Two Functionally Distinct Isoforms of TL1A (TNFSF15) Generated by Differential Ectodomain Shedding

    Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

    doi: 10.1093/gerona/glq129

    Upregulation of tumor necrosis factor–like cytokine 1A (TL1A) messenger RNA (mRNA) in replicative senescent circulating endothelial progenitor cells (CEP) as well as in elderly donors. (A) CEP of a young donor (Y2 = 35 years) were serially passaged until they reached senescence. mRNA was prepared and subjected to cDNA microarray analysis. Microarray results were normalized, and the regulation for TNFSF15 probe set (TL1A) is shown. (B) In addition, CEP of young (Y1 = 30 years) and old donors (O1 = 70 years and O2 = 79 years) were isolated and analyzed by cDNA microarray. (C) The mRNA expression observed by cDNA microarray analysis was confirmed by real-time-quantitative-PCR (RTq-PCR) using primers specific for the detection of TL1A. Results are shown as fold change compared with “young” CEP regarding replicative age or donor age, respectively. (D) CEP derived from three young donors (Y3, Y4, and Y5) were transduced with either control lentivirus or three lentiviral knockdown vectors targeting TL1A (pLKO1, pLKO99). RTq-PCR showing the lentiviral knockdown of TL1A in CEP was performed 8 and 10 days after transduction at the indicated passage numbers. Results are shown as fold change compared with control. (E) Quantification of senescence-associated β-galactosidase (SA-β-gal)–positive cells after knockdown of TL1A. CEP of young donors (Y3, Y4 and Y5) were fixed and stained for SA-β-gal 7 or 9 days after transduction. Phase contrast microscopic pictures were taken at 400× magnification. The percentage of SA-β-gal–positive cells was calculated by counting about 1,500 cells per experiment. (F) CEP were transduced with lentiviral vectors expressing green fluorescent protein (GFP) or TL1A, and cell extracts were subjected to Western blot using GFP or TL1A-specific antibodies, respectively. Wild-type CEP were used as a control. (G) Six days after transduction, cells were fixed and stained for SA-β-gal. Pictures are representative of at least three experiments.
    Figure Legend Snippet: Upregulation of tumor necrosis factor–like cytokine 1A (TL1A) messenger RNA (mRNA) in replicative senescent circulating endothelial progenitor cells (CEP) as well as in elderly donors. (A) CEP of a young donor (Y2 = 35 years) were serially passaged until they reached senescence. mRNA was prepared and subjected to cDNA microarray analysis. Microarray results were normalized, and the regulation for TNFSF15 probe set (TL1A) is shown. (B) In addition, CEP of young (Y1 = 30 years) and old donors (O1 = 70 years and O2 = 79 years) were isolated and analyzed by cDNA microarray. (C) The mRNA expression observed by cDNA microarray analysis was confirmed by real-time-quantitative-PCR (RTq-PCR) using primers specific for the detection of TL1A. Results are shown as fold change compared with “young” CEP regarding replicative age or donor age, respectively. (D) CEP derived from three young donors (Y3, Y4, and Y5) were transduced with either control lentivirus or three lentiviral knockdown vectors targeting TL1A (pLKO1, pLKO99). RTq-PCR showing the lentiviral knockdown of TL1A in CEP was performed 8 and 10 days after transduction at the indicated passage numbers. Results are shown as fold change compared with control. (E) Quantification of senescence-associated β-galactosidase (SA-β-gal)–positive cells after knockdown of TL1A. CEP of young donors (Y3, Y4 and Y5) were fixed and stained for SA-β-gal 7 or 9 days after transduction. Phase contrast microscopic pictures were taken at 400× magnification. The percentage of SA-β-gal–positive cells was calculated by counting about 1,500 cells per experiment. (F) CEP were transduced with lentiviral vectors expressing green fluorescent protein (GFP) or TL1A, and cell extracts were subjected to Western blot using GFP or TL1A-specific antibodies, respectively. Wild-type CEP were used as a control. (G) Six days after transduction, cells were fixed and stained for SA-β-gal. Pictures are representative of at least three experiments.

    Techniques Used: Microarray, Isolation, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Derivative Assay, Transduction, Staining, Western Blot

    14) Product Images from "An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice"

    Article Title: An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice

    Journal: Nature genetics

    doi: 10.1038/ng.369

    Ocm , Slc26a5 , Pitpnm1 , Ptpr1 and Gfi1 expression in diminuendo a , Quantitative real-time PCR on cDNA generated from normalised RNA from the organs of Corti of 4 day old littermates. Ocm , Slc26a5 , Ptprq and Gfi1 are downregulated in heterozygotes and homozygotes. Error bars represent standard deviation. Quantities normalised to Hprt1 levels; Ngfr is expressed in support cells adjacent to hair cells 30 and was used to assess the quantity of sensory material. Ngfr : Wildtype, n=12, mean=1.01±0.12 (s.d.); heterozygote, n=12, mean=0.89±0.35 (s.d.); homozygote, n=12, mean=0.99±0.17 (s.d.) Ocm : Wildtype, n=9, mean=1.01±0.15 (s.d.); heterozygote, n=9, mean=0.07±0.04 (s.d.); homozygote, n=9, mean=0.003±0.001 (s.d.) Slc26a5 : Wildtype, n=9, mean=1.01±0.14 (s.d.); heterozygote, n=9, mean=0.22±0.11 (s.d.); homozygote, n=9, mean=0.02±0.01 (s.d.) Gfi1 : Wildtype, n=9, mean=1.01±0.12 (s.d.); heterozygote, n=9, mean=0.88±0.12 (s.d.); homozygote, n=9, mean=0.66±0.14 (s.d.) Ptprq : Wildtype, n=9, mean=1.01±0.15 (s.d.); heterozygote, n=9, mean=0.62±0.18 (s.d.); homozygote, n=9, mean=0.56±0.20 (s.d.) Pitpnm1 : Wildtype n=8, mean=1.00±0.09 (s.d.); heterozygote, n=9, mean=0.80±0.29 (s.d.); homozygote, n=9, mean=0.78±0.27 (s.d.). Three animals were used for each genotype and DNA from each was run in triplicate. T-tests: Ngfr heterozygote p=0.25 (Welch's t-test), homozygote p=0.75 (Student's t-test); Ocm heterozygote p=1.51×10 −8 (Welch's t-test), homozygote p=3.46×10 −8 (Welch's t-test); Slc26a5 heterozygote p=7.73×10 −10 (Student's t-test), homozygote p=3.37×10 −8 (Welch's t-test); Gfi1 heterozygote p=0.038 (Student's t-test), homozygote p=3.39×10 −5 (Student's t-test); Ptprq heterozygote p=1.37×10 −4 (Student's t-test), homozygote p=6.46×10 −5 (Student's t-test); Pitpnm1 heterozygote p=0.084 (Welch's t-test), homozygote p=0.35 (Student's t-test); α=0.05. b-k , location of oncomodulin ( b, c ), prestin ( d, h ), Pitpnm1 ( e , i ), Ptprq ( f , j ) and Gfi1 ( g , k ) in 5-day old wildtype ( b, d-g ) and homozygote ( c, h-k ) littermates. Scale bars = 10μm.
    Figure Legend Snippet: Ocm , Slc26a5 , Pitpnm1 , Ptpr1 and Gfi1 expression in diminuendo a , Quantitative real-time PCR on cDNA generated from normalised RNA from the organs of Corti of 4 day old littermates. Ocm , Slc26a5 , Ptprq and Gfi1 are downregulated in heterozygotes and homozygotes. Error bars represent standard deviation. Quantities normalised to Hprt1 levels; Ngfr is expressed in support cells adjacent to hair cells 30 and was used to assess the quantity of sensory material. Ngfr : Wildtype, n=12, mean=1.01±0.12 (s.d.); heterozygote, n=12, mean=0.89±0.35 (s.d.); homozygote, n=12, mean=0.99±0.17 (s.d.) Ocm : Wildtype, n=9, mean=1.01±0.15 (s.d.); heterozygote, n=9, mean=0.07±0.04 (s.d.); homozygote, n=9, mean=0.003±0.001 (s.d.) Slc26a5 : Wildtype, n=9, mean=1.01±0.14 (s.d.); heterozygote, n=9, mean=0.22±0.11 (s.d.); homozygote, n=9, mean=0.02±0.01 (s.d.) Gfi1 : Wildtype, n=9, mean=1.01±0.12 (s.d.); heterozygote, n=9, mean=0.88±0.12 (s.d.); homozygote, n=9, mean=0.66±0.14 (s.d.) Ptprq : Wildtype, n=9, mean=1.01±0.15 (s.d.); heterozygote, n=9, mean=0.62±0.18 (s.d.); homozygote, n=9, mean=0.56±0.20 (s.d.) Pitpnm1 : Wildtype n=8, mean=1.00±0.09 (s.d.); heterozygote, n=9, mean=0.80±0.29 (s.d.); homozygote, n=9, mean=0.78±0.27 (s.d.). Three animals were used for each genotype and DNA from each was run in triplicate. T-tests: Ngfr heterozygote p=0.25 (Welch's t-test), homozygote p=0.75 (Student's t-test); Ocm heterozygote p=1.51×10 −8 (Welch's t-test), homozygote p=3.46×10 −8 (Welch's t-test); Slc26a5 heterozygote p=7.73×10 −10 (Student's t-test), homozygote p=3.37×10 −8 (Welch's t-test); Gfi1 heterozygote p=0.038 (Student's t-test), homozygote p=3.39×10 −5 (Student's t-test); Ptprq heterozygote p=1.37×10 −4 (Student's t-test), homozygote p=6.46×10 −5 (Student's t-test); Pitpnm1 heterozygote p=0.084 (Welch's t-test), homozygote p=0.35 (Student's t-test); α=0.05. b-k , location of oncomodulin ( b, c ), prestin ( d, h ), Pitpnm1 ( e , i ), Ptprq ( f , j ) and Gfi1 ( g , k ) in 5-day old wildtype ( b, d-g ) and homozygote ( c, h-k ) littermates. Scale bars = 10μm.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Generated, Standard Deviation

    15) Product Images from "Relative importance of redox buffers GSH and NAD(P)H in age-related neurodegeneration and Alzheimer disease-like mouse neurons"

    Article Title: Relative importance of redox buffers GSH and NAD(P)H in age-related neurodegeneration and Alzheimer disease-like mouse neurons

    Journal: Aging Cell

    doi: 10.1111/acel.12216

    Brain NADPH concentration and NADPH/NADP redox state decline with age and correlate with decline in brain expression of NAMPT and NNT gene expression. By HPLC analysis of cortical/hippocampal tissue homogenates from 4-, 11- and 21-month non-Tg (dashed line) and 3xTg-AD (solid line), A) NADPH concentration (nmol/mg) declines with age (ANOVA F(2,24) = 10.4, P = 0.001). B) Calculated NADPH/NADP redox state (mV) using the Nernst equation indicates a large oxidative shift in both non-Tg and 3xTg-AD brain with age (ANOVA F(2,24) = 15.7, P = 0.001). qRT PCR on non-Tg (gray filled circle, dashed line) and 3xTg-AD (black filled circle, solid line) brains indicate an age- and AD-related loss in gene expression of metabolic enzymes C) NAMPT (ANOVA, age F(3,33) = 21.8, P
    Figure Legend Snippet: Brain NADPH concentration and NADPH/NADP redox state decline with age and correlate with decline in brain expression of NAMPT and NNT gene expression. By HPLC analysis of cortical/hippocampal tissue homogenates from 4-, 11- and 21-month non-Tg (dashed line) and 3xTg-AD (solid line), A) NADPH concentration (nmol/mg) declines with age (ANOVA F(2,24) = 10.4, P = 0.001). B) Calculated NADPH/NADP redox state (mV) using the Nernst equation indicates a large oxidative shift in both non-Tg and 3xTg-AD brain with age (ANOVA F(2,24) = 15.7, P = 0.001). qRT PCR on non-Tg (gray filled circle, dashed line) and 3xTg-AD (black filled circle, solid line) brains indicate an age- and AD-related loss in gene expression of metabolic enzymes C) NAMPT (ANOVA, age F(3,33) = 21.8, P

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

    16) Product Images from "STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis"

    Article Title: STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113318

    CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent. A, WT and STAT1 −/− VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for Cxcl10 using Gapdh as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

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

    Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.
    Figure Legend Snippet: Identification of genes prone to synergistic amplification upon treatment with IFNγ and LPS. WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . On RNA isolated from untreated or IFNγ, LPS or IFNγ+LPS treated VSMCs genome-wide expression profiling was performed. A, Venn diagrams revealing number of differentially expressed genes upon stimulation. B, Heat map of the expression of synergistically amplified genes in WT and STAT1 −/− VSMCs . C, Clustering of the synergistically upregulated genes according to their expression profile. AVG, average expression in the group. For details see text.

    Techniques Used: Amplification, Isolation, Genome Wide, Expressing

    IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines. WT, STAT1 −/− and IRF8 −/− VSMCs and HMECs were treated as described in Fig. 1 . A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT , and IRF8 −/− were compared. E, Migration assay of CD45 + /CD3 + performed on conditioned medium remained after treatment of VSMCs WT and STAT1 −/ − . Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Functional Assay, Activity Assay, Amplification, Isolation, Quantitative RT-PCR, Expressing, Migration

    Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: Effect of STAT1 dependent signal integration on chemokine expression. WT and STAT1 −/− VSMCs , HMECs or WT aortic ring segments were treated as described in Fig. 1 . A, RNA from VSMCs was isolated and qRT-PCR for Ccl5 , Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10 , Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p

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

    STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p
    Figure Legend Snippet: STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production. A, WT and STAT1 −/− VSMCs were treated as described in Fig. 1 . RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1 , medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p

    Techniques Used: Isolation, Quantitative RT-PCR

    17) Product Images from "Exendin-4 enhances expression of Neurod1 and Glut2 in insulin-producing cells derived from mouse embryonic stem cells"

    Article Title: Exendin-4 enhances expression of Neurod1 and Glut2 in insulin-producing cells derived from mouse embryonic stem cells

    Journal: Archives of Medical Science : AMS

    doi: 10.5114/aoms.2016.57596

    Morphology of RI ES cells, insulin-producing cells derived from different differentiation protocols. A – Morphology of R1 ES cells. B – Alkaline phosphatase (AP) staining of R1 ES cells. C – In vitro formation of simple EBs on day 6 of stage 1. D–F – Insulin-producing cells derived from different differentiation protocols. D – low dosage group, E – high dosage group, F – control group. Scale bars = 25 µm
    Figure Legend Snippet: Morphology of RI ES cells, insulin-producing cells derived from different differentiation protocols. A – Morphology of R1 ES cells. B – Alkaline phosphatase (AP) staining of R1 ES cells. C – In vitro formation of simple EBs on day 6 of stage 1. D–F – Insulin-producing cells derived from different differentiation protocols. D – low dosage group, E – high dosage group, F – control group. Scale bars = 25 µm

    Techniques Used: Derivative Assay, Staining, In Vitro

    Immunostaining of insulin and C-peptide. Day 31 R1 ES cell-derived insulin-producing cells were immunostained with antibodies against insulin ( A ) and C-peptide ( B ). A – Positive signals for insulin were easily observed in groups C, L and H. B – C-peptide positive staining was observed only in groups H and L. Nuclei were stained with DAPI (4’,6-diamidine-2’-phenylindole dihydrochloride). Scale bars = 25 µm
    Figure Legend Snippet: Immunostaining of insulin and C-peptide. Day 31 R1 ES cell-derived insulin-producing cells were immunostained with antibodies against insulin ( A ) and C-peptide ( B ). A – Positive signals for insulin were easily observed in groups C, L and H. B – C-peptide positive staining was observed only in groups H and L. Nuclei were stained with DAPI (4’,6-diamidine-2’-phenylindole dihydrochloride). Scale bars = 25 µm

    Techniques Used: Immunostaining, Derivative Assay, Staining

    18) Product Images from "Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming"

    Article Title: Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0131288

    Ocular epithelial differentiation from different cell-of-origin iPSCs lines. (A) To compare the pluripotency nature of different types of iPSCs, OECiPSCs and OSCiPSCs were subjected to sphere culture for 7 days then monolayer culture for further 3 days. Cells were harvested for testing endodermal, mesodermal and ectodermal markers by real-time PCR. Expression level of pluripotency markers Pou5f1, Sox2 and Dnmt3a were dramatically decreased up to 1000 times in both differences in OECiPSCs and OSCiPSCs. The OSCiPSCs demonstrated a preference for endodermal differentiation while OECiPSCs show higher potential in ectodermal differ nation. iPSCs vs OECiPSCs vs OSCiPSCs: Pou5f1 (0.00±0;-3.56±0.06;-2.98±0.07); Sox2 (0.00±0;-2.83±0.04;-3.25±0.03); Dnmt3a (0.00±0;-2.42±0.03;-3.01±0.04); Sox17 (0.00±0; 1.08±0.04; 1.48±0.03); Foxa2 (0.00±0; 1.35±0.04; 2.12±0.03); AFP (0.00±0; 1.23±0.23; 1.41±0.47); T (0.00±0; 2.21±0.04; 1.41±0.03); Kit (0.00±0; 2.17±0.0; 2.62±0.03); Nestin (0.00±0; 2.65±0.20; 1.56±0.22); Pax6 (0.00±0; 2.75±0.04; 2.23±0.11); (SEM N = 3;* P
    Figure Legend Snippet: Ocular epithelial differentiation from different cell-of-origin iPSCs lines. (A) To compare the pluripotency nature of different types of iPSCs, OECiPSCs and OSCiPSCs were subjected to sphere culture for 7 days then monolayer culture for further 3 days. Cells were harvested for testing endodermal, mesodermal and ectodermal markers by real-time PCR. Expression level of pluripotency markers Pou5f1, Sox2 and Dnmt3a were dramatically decreased up to 1000 times in both differences in OECiPSCs and OSCiPSCs. The OSCiPSCs demonstrated a preference for endodermal differentiation while OECiPSCs show higher potential in ectodermal differ nation. iPSCs vs OECiPSCs vs OSCiPSCs: Pou5f1 (0.00±0;-3.56±0.06;-2.98±0.07); Sox2 (0.00±0;-2.83±0.04;-3.25±0.03); Dnmt3a (0.00±0;-2.42±0.03;-3.01±0.04); Sox17 (0.00±0; 1.08±0.04; 1.48±0.03); Foxa2 (0.00±0; 1.35±0.04; 2.12±0.03); AFP (0.00±0; 1.23±0.23; 1.41±0.47); T (0.00±0; 2.21±0.04; 1.41±0.03); Kit (0.00±0; 2.17±0.0; 2.62±0.03); Nestin (0.00±0; 2.65±0.20; 1.56±0.22); Pax6 (0.00±0; 2.75±0.04; 2.23±0.11); (SEM N = 3;* P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    Endogenous expression of OCT4A and SOX2 pluripotency reprogramming factors in OECs derived from conjunctival tissues. (A) DAB-based immunohistochemistry staining displayed the expression of OCT4A (brown, arrows) in human ocular sections. OCT4A expressed in the epithelium layer but not in stromal layers (OEL, ocular epithelial layer; OSL, ocular stromal layer). (B) Immunofluorescence staining demonstrated OCT4A expression in OECs but not in OSCs. (C) (i) Reverse transcription polymerase chain reaction (RT-PCR) results in expression of pluripotency genes (from top to bottom Panels): DPPA4 , TERT , NANOG , GDF3 , REX1 , ESG1 , OCT4A , SOX2 , KLF4 and C-MYC and Beta- actin . Human ESCs and water were included as positive and negative controls respectively. (ii) Expression of OCT4 isoforms OCT4A and OCT4B, endogenous OCT 4 and viral OCT4 detecting using 5’ and 3’ UTR sequences tracking with RT-PCR. (From left to right) Primary lines (IMR90, OEC1, OEC2 and OSCs) and their corresponding iPSCs. (D) Western Blotting for OCT4A and SOX2 protein levels in OECs, OSCs and iPSCs. Lane 1: OECiPSCs, Lane 2: OSCs, Lane 3 4: OEC1 2 respectively, Lane 5: mouse OECs (E) Expression levels of (i) OCT4A and (ii) SOX2 were represented in intensity ratios respectively.
    Figure Legend Snippet: Endogenous expression of OCT4A and SOX2 pluripotency reprogramming factors in OECs derived from conjunctival tissues. (A) DAB-based immunohistochemistry staining displayed the expression of OCT4A (brown, arrows) in human ocular sections. OCT4A expressed in the epithelium layer but not in stromal layers (OEL, ocular epithelial layer; OSL, ocular stromal layer). (B) Immunofluorescence staining demonstrated OCT4A expression in OECs but not in OSCs. (C) (i) Reverse transcription polymerase chain reaction (RT-PCR) results in expression of pluripotency genes (from top to bottom Panels): DPPA4 , TERT , NANOG , GDF3 , REX1 , ESG1 , OCT4A , SOX2 , KLF4 and C-MYC and Beta- actin . Human ESCs and water were included as positive and negative controls respectively. (ii) Expression of OCT4 isoforms OCT4A and OCT4B, endogenous OCT 4 and viral OCT4 detecting using 5’ and 3’ UTR sequences tracking with RT-PCR. (From left to right) Primary lines (IMR90, OEC1, OEC2 and OSCs) and their corresponding iPSCs. (D) Western Blotting for OCT4A and SOX2 protein levels in OECs, OSCs and iPSCs. Lane 1: OECiPSCs, Lane 2: OSCs, Lane 3 4: OEC1 2 respectively, Lane 5: mouse OECs (E) Expression levels of (i) OCT4A and (ii) SOX2 were represented in intensity ratios respectively.

    Techniques Used: Expressing, Derivative Assay, Immunohistochemistry, Staining, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot

    19) Product Images from "Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210"

    Article Title: Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-015-0974-3

    Global analysis of differentially expressed miRNAs in L. m. -infected BMDM, bioinformatical prediction of miRNA interactions with LISA, and infection rates of L. m. -infected BMDM after transfection with mmu-miR-101c or mmu-miR-129-5p mimics as well as mmu-miR-155-5p or mmu-miR-210-5p inhibitors. Methods: ( a , b , d ) BMDM from BALB/c mice were infected with L. m. promastigotes for 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. Affymetrix® chips were hybridized with RNA samples from 2 independent experiments and analyzed densitometrically. Putative interactions between differentially expressed miRNAs and LISA members were predicted bioinformatically and represented in MONA-of-LISA. c Additionally, L. m. -infected BMDM were transfected with miRNA mimics or inhibitors 20 h p.i.. L. m. -infected control BMDM were transfected with a negative control of miRNA mimics or inhibitors. The infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a , b ) Differentially expressed miRNAs were detected in L. m. -infected BMDM 24 h p.i.. The results were presented in MA plots. Large dots represent probe sets, which were significantly and differentially expressed (FDR
    Figure Legend Snippet: Global analysis of differentially expressed miRNAs in L. m. -infected BMDM, bioinformatical prediction of miRNA interactions with LISA, and infection rates of L. m. -infected BMDM after transfection with mmu-miR-101c or mmu-miR-129-5p mimics as well as mmu-miR-155-5p or mmu-miR-210-5p inhibitors. Methods: ( a , b , d ) BMDM from BALB/c mice were infected with L. m. promastigotes for 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. Affymetrix® chips were hybridized with RNA samples from 2 independent experiments and analyzed densitometrically. Putative interactions between differentially expressed miRNAs and LISA members were predicted bioinformatically and represented in MONA-of-LISA. c Additionally, L. m. -infected BMDM were transfected with miRNA mimics or inhibitors 20 h p.i.. L. m. -infected control BMDM were transfected with a negative control of miRNA mimics or inhibitors. The infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a , b ) Differentially expressed miRNAs were detected in L. m. -infected BMDM 24 h p.i.. The results were presented in MA plots. Large dots represent probe sets, which were significantly and differentially expressed (FDR

    Techniques Used: Infection, Transfection, Mouse Assay, Incubation, Negative Control

    BNIP3 and CTSE transcriptomic and western blot analyses with RNAs and protein extracts from L. m. -infected and HBSS-starved BMDM as well as determination of the infection rates of L. m. -infected BMDM after BNIP3 and CTSE downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 h or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. The Proteins were harvested and subjected to western blot analysis with specific antibodies against BNIP3 and CTSE. Western blots from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. Affymetrix® chips were hybridized with RNA samples from 2 independent experiments and analyzed densitometrically. c Additionally, L. m. -infected BMDM were transfected with specific siRNAs 20 h p.i. to downregulate the expression of BNIP3 and CTSE. L. m. -infected control BMDM were transfected with negative control siRNA. Infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a ) BNIP3 and CTSE were significantly overexpressed in L. m. -infected BMDM 24 h p.i. compared to uninfected control BMDM. b Densitometric analyses of western blot experiments confirmed this overexpression 24 h p.i. and showed that CTSE was also overexpressed in L. m. -infected BMDM 1 h p.i.. At the mRNA level, Bnip3 was overexpressed in L. m. -infected BMDM 1 and 24 h p.i. and Ctse was downregulated in L. m. -infected BMDM 24 h p.i.. c A significant increase in the infection rates was detected in L. m. -infected BMDM after downregulation of protein expression of BNIP3 or CTSE compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, RFU = relative fluorescence units, * p ≤ 0.05, ** p ≤ 0.01,*** p ≤ 0.001
    Figure Legend Snippet: BNIP3 and CTSE transcriptomic and western blot analyses with RNAs and protein extracts from L. m. -infected and HBSS-starved BMDM as well as determination of the infection rates of L. m. -infected BMDM after BNIP3 and CTSE downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 h or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. The Proteins were harvested and subjected to western blot analysis with specific antibodies against BNIP3 and CTSE. Western blots from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. Affymetrix® chips were hybridized with RNA samples from 2 independent experiments and analyzed densitometrically. c Additionally, L. m. -infected BMDM were transfected with specific siRNAs 20 h p.i. to downregulate the expression of BNIP3 and CTSE. L. m. -infected control BMDM were transfected with negative control siRNA. Infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a ) BNIP3 and CTSE were significantly overexpressed in L. m. -infected BMDM 24 h p.i. compared to uninfected control BMDM. b Densitometric analyses of western blot experiments confirmed this overexpression 24 h p.i. and showed that CTSE was also overexpressed in L. m. -infected BMDM 1 h p.i.. At the mRNA level, Bnip3 was overexpressed in L. m. -infected BMDM 1 and 24 h p.i. and Ctse was downregulated in L. m. -infected BMDM 24 h p.i.. c A significant increase in the infection rates was detected in L. m. -infected BMDM after downregulation of protein expression of BNIP3 or CTSE compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, RFU = relative fluorescence units, * p ≤ 0.05, ** p ≤ 0.01,*** p ≤ 0.001

    Techniques Used: Western Blot, Infection, Mouse Assay, Incubation, Transfection, Expressing, Negative Control, Over Expression, Fluorescence

    MTOR and RPS6 transcriptomic and western blot analyses with RNAs and protein extracts from L. m. -infected and HBSS-starved BMDM, and determination of infection rates of L. m. -infected BMDM after MTOR downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. Proteins were harvested and subjected to western blot analyses with specific antibodies against MTOR, p-MTOR, RPS6, and p-RPS6. Western blots with proteins from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. The Affymetrix® chips were hybridized with RNA samples from 2 independent experiments were analyzed densitometrically. c Additionally, BMDM were transfected with specific siRNA 4 h prior to infection to downregulate the expression of MTOR, and the cells were finally infected with L. m. promastigotes. L. m. -infected controls were transfected with negative control siRNA. The infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a ) A significant hyperphosphorylation was observed for MTOR and RPS6 in samples from L. m. -infected BMDM 1 h p.i. compared to uninfected control BMDM. b Results of densitometric analyses of western blot experiments and Affymetrix® chip analyses showed that MTOR and RPS6 expressions were not regulated at the mRNA or the protein level. However, MTOR and RPS6 were significantly hyperphosphorylated in L. m. -infected BMDM 1 h p.i.. c A significant decrease in the infection rate was detected in L. m. -infected BMDM after downregulation of the protein expression of MTOR compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, RFU = relative fluorescence units, * p ≤ 0.05, ** p ≤ 0.01
    Figure Legend Snippet: MTOR and RPS6 transcriptomic and western blot analyses with RNAs and protein extracts from L. m. -infected and HBSS-starved BMDM, and determination of infection rates of L. m. -infected BMDM after MTOR downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium or starved for 1 h in HBSS. Proteins were harvested and subjected to western blot analyses with specific antibodies against MTOR, p-MTOR, RPS6, and p-RPS6. Western blots with proteins from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Total RNA was harvested from L. m. -infected BMDM and uninfected control BMDM. The Affymetrix® chips were hybridized with RNA samples from 2 independent experiments were analyzed densitometrically. c Additionally, BMDM were transfected with specific siRNA 4 h prior to infection to downregulate the expression of MTOR, and the cells were finally infected with L. m. promastigotes. L. m. -infected controls were transfected with negative control siRNA. The infection rates were determined 48 h p.i. in 2 independent experiments. Results: ( a ) A significant hyperphosphorylation was observed for MTOR and RPS6 in samples from L. m. -infected BMDM 1 h p.i. compared to uninfected control BMDM. b Results of densitometric analyses of western blot experiments and Affymetrix® chip analyses showed that MTOR and RPS6 expressions were not regulated at the mRNA or the protein level. However, MTOR and RPS6 were significantly hyperphosphorylated in L. m. -infected BMDM 1 h p.i.. c A significant decrease in the infection rate was detected in L. m. -infected BMDM after downregulation of the protein expression of MTOR compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, RFU = relative fluorescence units, * p ≤ 0.05, ** p ≤ 0.01

    Techniques Used: Western Blot, Infection, Mouse Assay, Incubation, Transfection, Expressing, Negative Control, Chromatin Immunoprecipitation, Fluorescence

    Schematic summary of results. a 0 h p.i.: macrophages were infected with L. m. promastigotes. b 10 min p.i.: promastigotes attached to macrophages and were phagocytosed by the cells. c 1 h p.i. (early infection phase): promastigotes differentiated intracellularly into amastigotes. At this point, the differentiation was not complete. Hyperphosphorylation of MTOR and RPS6 suggested autophagy inhibition. d 24 h p.i. (late infection phase): amastigote differentiation was completed. ATG5, BNIP3, CTSE, MIF, UB, and miRNAs mmu-miR-155-5p and mmu-miR-210-5p, were overexpressed. Expression of miRNAs mmu-miR-101c and mmu-miR-129-5p was downregulated. The LC3B-II/LC3B-I ratio was elevated and suggested an increased autophagic flux. Glycolytic genes were upregulated. Overexpressed MIF might have attracted new uninfected host macrophages. Putative regulatory mechanisms at the RNA level were identified, which were summarized in LISA and MONA-of-LISA. Additionally, inflammatory functions (e.g., the immune response and chemokine signaling pathway) were upregulated, which indicates L. m. -infected BMDM had an inflammatory phenotype. e 48 h p.i.: amastigotes were autophagically digested, which resulted in a decline in the infection rate. p.i. = post infection
    Figure Legend Snippet: Schematic summary of results. a 0 h p.i.: macrophages were infected with L. m. promastigotes. b 10 min p.i.: promastigotes attached to macrophages and were phagocytosed by the cells. c 1 h p.i. (early infection phase): promastigotes differentiated intracellularly into amastigotes. At this point, the differentiation was not complete. Hyperphosphorylation of MTOR and RPS6 suggested autophagy inhibition. d 24 h p.i. (late infection phase): amastigote differentiation was completed. ATG5, BNIP3, CTSE, MIF, UB, and miRNAs mmu-miR-155-5p and mmu-miR-210-5p, were overexpressed. Expression of miRNAs mmu-miR-101c and mmu-miR-129-5p was downregulated. The LC3B-II/LC3B-I ratio was elevated and suggested an increased autophagic flux. Glycolytic genes were upregulated. Overexpressed MIF might have attracted new uninfected host macrophages. Putative regulatory mechanisms at the RNA level were identified, which were summarized in LISA and MONA-of-LISA. Additionally, inflammatory functions (e.g., the immune response and chemokine signaling pathway) were upregulated, which indicates L. m. -infected BMDM had an inflammatory phenotype. e 48 h p.i.: amastigotes were autophagically digested, which resulted in a decline in the infection rate. p.i. = post infection

    Techniques Used: Infection, Inhibition, Expressing

    Global analysis of differentially expressed mRNAs in L. m. -infected BMDM and MIF western blot analyses with protein extracts from L. m. -infected and HBSS-starved BMDM. Methods: ( a – e ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium. Total RNA from 2 independent experiments was harvested from L. m. -infected BMDM or uninfected controls and hybridized with Affymetrix® chips. The BMDM were additionally incubated for 1 h in HBSS. Proteins were harvested and subjected to western blot analyses with specific antibodies against MIF. The western blots from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Results: ( a ) Differentially expressed genes were detected in L. m. -infected BMDM 1 and 24 h p.i.. The results were presented in MA plots. Large dots represent probe sets, which had significant differential expression (FDR
    Figure Legend Snippet: Global analysis of differentially expressed mRNAs in L. m. -infected BMDM and MIF western blot analyses with protein extracts from L. m. -infected and HBSS-starved BMDM. Methods: ( a – e ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 or 24 h. Uninfected control BMDM were incubated for the same amount of time in RPMI medium. Total RNA from 2 independent experiments was harvested from L. m. -infected BMDM or uninfected controls and hybridized with Affymetrix® chips. The BMDM were additionally incubated for 1 h in HBSS. Proteins were harvested and subjected to western blot analyses with specific antibodies against MIF. The western blots from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. Results: ( a ) Differentially expressed genes were detected in L. m. -infected BMDM 1 and 24 h p.i.. The results were presented in MA plots. Large dots represent probe sets, which had significant differential expression (FDR

    Techniques Used: Infection, Western Blot, Mouse Assay, Incubation, Expressing

    ATG5 and UB western blot analyses with protein extracts from L. m. -infected and HBSS-starved BMDM as well as determination of the infection rates of L. m. -infected BMDM after ATG5 and UB downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 and 24 h. Uninfected control BMDM were incubated for the same time in RPMI medium or starved for 1 h in HBSS. The proteins were harvested and subjected to western blot analysis with specific antibodies against ATG5 and UB. Western blots with proteins from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. c Additionally, BMDM were transfected with specific siRNAs 4 h prior to infection to downregulate the expression of ATG5 and UB. The cells were finally infected with L. m. promastigotes. L. m. -infected control BMDM were transfected with negative control siRNA. The infection rates were determined 48 h p.i.. Diagram shows the result of 2 independent experiments. Results: ( a ) ATG5 and UB levels in samples from L. m. -infected BMDM 24 h p.i. were increased compared to uninfected control BMDM. b Results of the densitometric analyses confirmed that ATG5 and UB were significantly increased in L. m. -infected BMDM 24 h p.i. compared to uninfected control BMDM. No upregulation was detected in L. m. -infected BMDM 1 h p.i. compared to the respective controls. c A significant increase in the infection rate was found in L. m. -infected BMDM after downregulation of the protein expression of ATG5 or UB compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, * p ≤ 0.05, *** p ≤ 0.001
    Figure Legend Snippet: ATG5 and UB western blot analyses with protein extracts from L. m. -infected and HBSS-starved BMDM as well as determination of the infection rates of L. m. -infected BMDM after ATG5 and UB downregulation by RNA interference. Methods: ( a , b ) BMDM from BALB/c mice were infected with L. m. promastigotes for 1 and 24 h. Uninfected control BMDM were incubated for the same time in RPMI medium or starved for 1 h in HBSS. The proteins were harvested and subjected to western blot analysis with specific antibodies against ATG5 and UB. Western blots with proteins from 3 independent experiments were analyzed densitometrically. ACTB served as the internal loading control. c Additionally, BMDM were transfected with specific siRNAs 4 h prior to infection to downregulate the expression of ATG5 and UB. The cells were finally infected with L. m. promastigotes. L. m. -infected control BMDM were transfected with negative control siRNA. The infection rates were determined 48 h p.i.. Diagram shows the result of 2 independent experiments. Results: ( a ) ATG5 and UB levels in samples from L. m. -infected BMDM 24 h p.i. were increased compared to uninfected control BMDM. b Results of the densitometric analyses confirmed that ATG5 and UB were significantly increased in L. m. -infected BMDM 24 h p.i. compared to uninfected control BMDM. No upregulation was detected in L. m. -infected BMDM 1 h p.i. compared to the respective controls. c A significant increase in the infection rate was found in L. m. -infected BMDM after downregulation of the protein expression of ATG5 or UB compared to L. m. -infected BMDM transfected with negative control siRNA. L. m. -inf. = L. m. -infected, neg. control = negative control, n.s. = not significant, * p ≤ 0.05, *** p ≤ 0.001

    Techniques Used: Western Blot, Infection, Mouse Assay, Incubation, Transfection, Expressing, Negative Control

    20) Product Images from "Exploring regulatory networks of miR-96 in the developing inner ear"

    Article Title: Exploring regulatory networks of miR-96 in the developing inner ear

    Journal: Scientific Reports

    doi: 10.1038/srep23363

    Testing predicted targets of miR-96 and important nodes from the regulatory network. qRTPCR was carried out on cDNA from P4 organs of Corti in wildtype (green) and diminuendo homozygote (red) littermates to test gene expression changes. Error bars are standard deviation (*adjusted P
    Figure Legend Snippet: Testing predicted targets of miR-96 and important nodes from the regulatory network. qRTPCR was carried out on cDNA from P4 organs of Corti in wildtype (green) and diminuendo homozygote (red) littermates to test gene expression changes. Error bars are standard deviation (*adjusted P

    Techniques Used: Expressing, Standard Deviation

    Testing gene expression in diminuendo homozygotes. qRTPCR was carried out on cDNA from P4 organs of Corti in wildtype (green) and diminuendo homozygote (red) littermates to test gene expression changes. Error bars are standard deviation (*adjusted P
    Figure Legend Snippet: Testing gene expression in diminuendo homozygotes. qRTPCR was carried out on cDNA from P4 organs of Corti in wildtype (green) and diminuendo homozygote (red) littermates to test gene expression changes. Error bars are standard deviation (*adjusted P

    Techniques Used: Expressing, Standard Deviation

    21) Product Images from "Proinflammatory isoforms of IL-32 as novel and robust biomarkers for control failure in HIV-infected slow progressors"

    Article Title: Proinflammatory isoforms of IL-32 as novel and robust biomarkers for control failure in HIV-infected slow progressors

    Journal: Scientific Reports

    doi: 10.1038/srep22902

    HIV infection induces expression of IL-32 in human PBMCs. ( a ) Correlation between total IL-32 and VLs from HIV-infected SP (n = 53) (EC, VC, NVC) and TP subjects (n = 16) (subjects shown in Fig. 3c ). Spearman correlation test was used to assess the significance correlations between IL-32 and HIV VL. ( b ) Human PBMCs from n = 9 HIV-uninfected donors were either stimulated with PHA (0.25μg/ml) and IL-2 (100 units/ml) and infected with HIV-BaL (Left panel) or resting cells were infected without stimulation (Right panel). Total IL-32 was measured in the supernatant of activated cells (Left panel) or from cell lysate of non-stimulated cells (Right panel). ( c ) Total plasma IL-32 was measured in n = 10 subjects within 3 mos of HIV infection and after 1 year of ART treatment (Left panel). Total plasma IL-32 was measured in the same 10 subjects treated with ART for 1 yr and in 12 HIV neg donors (n = 12) (Right panel). The significance of between-group differences was assessed using a Wilcoxon test in panel ( b ) and the Left panel of ( c ). A Mann-Whitney test was used to assess the significance of between-group differences in panel ( c ) (Right panel).
    Figure Legend Snippet: HIV infection induces expression of IL-32 in human PBMCs. ( a ) Correlation between total IL-32 and VLs from HIV-infected SP (n = 53) (EC, VC, NVC) and TP subjects (n = 16) (subjects shown in Fig. 3c ). Spearman correlation test was used to assess the significance correlations between IL-32 and HIV VL. ( b ) Human PBMCs from n = 9 HIV-uninfected donors were either stimulated with PHA (0.25μg/ml) and IL-2 (100 units/ml) and infected with HIV-BaL (Left panel) or resting cells were infected without stimulation (Right panel). Total IL-32 was measured in the supernatant of activated cells (Left panel) or from cell lysate of non-stimulated cells (Right panel). ( c ) Total plasma IL-32 was measured in n = 10 subjects within 3 mos of HIV infection and after 1 year of ART treatment (Left panel). Total plasma IL-32 was measured in the same 10 subjects treated with ART for 1 yr and in 12 HIV neg donors (n = 12) (Right panel). The significance of between-group differences was assessed using a Wilcoxon test in panel ( b ) and the Left panel of ( c ). A Mann-Whitney test was used to assess the significance of between-group differences in panel ( c ) (Right panel).

    Techniques Used: Infection, Expressing, MANN-WHITNEY

    22) Product Images from "Direct conversion of mouse embryonic fibroblasts into functional keratinocytes through transient expression of pluripotency-related genes"

    Article Title: Direct conversion of mouse embryonic fibroblasts into functional keratinocytes through transient expression of pluripotency-related genes

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-016-0357-5

    Morphological and molecular characterization of induced keratinocytes. a Induced keratinocytes ( iKCs ) exhibit keratinocyte-like morphological properties, closely resembling primary keratinocytes ( 1°KCs ) in culture. iKCs displayed a round shape and formed colonies with a cobblestone-like appearance, a characteristic of primary keratinocytes in culture. In contrast, parental mouse embryonic fibroblasts ( MEFs ) exhibit mesenchymal characteristics, as they appear spindle shaped and do not form colonies. Scale bar s = 25 μm. ( b ) Expression levels of keratinocyte and fibroblast markers were measured by semi-quantitative RT-PCR, and agarose gel electrophoresis showing the product of each PCR reaction is depicted. The relative levels of each product from three independent experiments were normalized to actin. Results are depicted as fold-change of expression compared to expression in either primary keratinocytes (K14, K5, and p63) or in MEFs (vimentin). Error bars represent standard error between experiments. In contrast to MEFs, iKCs expressed undifferentiated keratinocyte markers (K14 and K5) at levels comparable to those of primary keratinocytes; p63 expression was threefold higher in iKCs compared to MEFs, even though KCs expressed p63 at much higher levels; vimentin expression was reduced twofold in iKCs, albeit not completely abolished. c Immunofluorescence staining against vimentin ( Vim ) and keratin 14 ( K14 ) in MEFs, primary KCs, and iKCs. DAPI staining is shown in blue , and Vim or K14 staining is shown in green. Scale bars = 20 μm. ( d ) Western blot analysis to measure vimentin and K14 protein levels in MEFs and iKCs. Total cellular protein lysates were run and blotted with anti-vimentin and anti-K14 antibodies, as well as with anti-actin as a control. e Endogenous and total expression levels of pluripotency factors Klf4, Sox2, and Oct4 were assessed by RT-PCR in MEFs, KCs, and iKCs. ESCs were included as a positive control. Expression of these factors is silenced in iKCs
    Figure Legend Snippet: Morphological and molecular characterization of induced keratinocytes. a Induced keratinocytes ( iKCs ) exhibit keratinocyte-like morphological properties, closely resembling primary keratinocytes ( 1°KCs ) in culture. iKCs displayed a round shape and formed colonies with a cobblestone-like appearance, a characteristic of primary keratinocytes in culture. In contrast, parental mouse embryonic fibroblasts ( MEFs ) exhibit mesenchymal characteristics, as they appear spindle shaped and do not form colonies. Scale bar s = 25 μm. ( b ) Expression levels of keratinocyte and fibroblast markers were measured by semi-quantitative RT-PCR, and agarose gel electrophoresis showing the product of each PCR reaction is depicted. The relative levels of each product from three independent experiments were normalized to actin. Results are depicted as fold-change of expression compared to expression in either primary keratinocytes (K14, K5, and p63) or in MEFs (vimentin). Error bars represent standard error between experiments. In contrast to MEFs, iKCs expressed undifferentiated keratinocyte markers (K14 and K5) at levels comparable to those of primary keratinocytes; p63 expression was threefold higher in iKCs compared to MEFs, even though KCs expressed p63 at much higher levels; vimentin expression was reduced twofold in iKCs, albeit not completely abolished. c Immunofluorescence staining against vimentin ( Vim ) and keratin 14 ( K14 ) in MEFs, primary KCs, and iKCs. DAPI staining is shown in blue , and Vim or K14 staining is shown in green. Scale bars = 20 μm. ( d ) Western blot analysis to measure vimentin and K14 protein levels in MEFs and iKCs. Total cellular protein lysates were run and blotted with anti-vimentin and anti-K14 antibodies, as well as with anti-actin as a control. e Endogenous and total expression levels of pluripotency factors Klf4, Sox2, and Oct4 were assessed by RT-PCR in MEFs, KCs, and iKCs. ESCs were included as a positive control. Expression of these factors is silenced in iKCs

    Techniques Used: Expressing, Quantitative RT-PCR, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Immunofluorescence, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Positive Control

    Induced keratinocytes ( iKCs ) can regenerate normal skin in vivo . ( a ) iKCs or mouse embryonic fibroblasts ( MEFs ) were transplanted in the back skin of nude mice. Nude mice transplanted with iKCs showed de novo hair formation at the graft site after 6 weeks ( a ), whereas hair development was not observed on mice transplanted with MEFs ( b ). All layers of the epidermis, including de novo hair follicles and sebaceous glands, are apparent in H E stained skin sections from iKC-transplanted mice ( c ) but not MEF-grafted skin ( d ). EYFP staining ( green ) marks iKCs and de novo iKC-generated tissue ( e ), which is not present in MEF-transplanted epidermis ( f ). Scale bars = 50 μm ( c and d ) and 10 μm ( e and f ). ( b ) Epidermal thickness comparing iKC-derived vs MEF-transplanted skin. Blindfolded comparison was performed by measuring epidermal thickness in 25 images (two measurements/image) captured from five independent tissue sections (five images/section) from iKC-derived or MEF-derived skin. Note a nearly twofold increase in iKC-generated epidermis. Error bars represent standard error amongst 50 independent measurements for each sample. ( c ) Immunofluorescence staining for K14 (a marker of undifferentiated keratinocytes), K10 (an intermediate differentiation marker), and loricrin (a terminal differentiation marker). Tissue sections were stained for different keratinocyte stratification/differentiation markers in iKC- or MEF-transplanted tissue sections. IKC-transplanted epidermis expressed both K14 ( green ) and K10 ( red ), whereas MEF-transplanted tissue only displayed some K14-positive cells, and no K10 staining ( b ). Loricrin ( red ) was expressed on the outer epidermal layer of the iKC-derived skin ( c ) and indicates the presence of iKC-derived terminally differentiated cells. In contrast, MEF-derived skin was negative for loricrin expression ( d ). Scale bars = 10 μm
    Figure Legend Snippet: Induced keratinocytes ( iKCs ) can regenerate normal skin in vivo . ( a ) iKCs or mouse embryonic fibroblasts ( MEFs ) were transplanted in the back skin of nude mice. Nude mice transplanted with iKCs showed de novo hair formation at the graft site after 6 weeks ( a ), whereas hair development was not observed on mice transplanted with MEFs ( b ). All layers of the epidermis, including de novo hair follicles and sebaceous glands, are apparent in H E stained skin sections from iKC-transplanted mice ( c ) but not MEF-grafted skin ( d ). EYFP staining ( green ) marks iKCs and de novo iKC-generated tissue ( e ), which is not present in MEF-transplanted epidermis ( f ). Scale bars = 50 μm ( c and d ) and 10 μm ( e and f ). ( b ) Epidermal thickness comparing iKC-derived vs MEF-transplanted skin. Blindfolded comparison was performed by measuring epidermal thickness in 25 images (two measurements/image) captured from five independent tissue sections (five images/section) from iKC-derived or MEF-derived skin. Note a nearly twofold increase in iKC-generated epidermis. Error bars represent standard error amongst 50 independent measurements for each sample. ( c ) Immunofluorescence staining for K14 (a marker of undifferentiated keratinocytes), K10 (an intermediate differentiation marker), and loricrin (a terminal differentiation marker). Tissue sections were stained for different keratinocyte stratification/differentiation markers in iKC- or MEF-transplanted tissue sections. IKC-transplanted epidermis expressed both K14 ( green ) and K10 ( red ), whereas MEF-transplanted tissue only displayed some K14-positive cells, and no K10 staining ( b ). Loricrin ( red ) was expressed on the outer epidermal layer of the iKC-derived skin ( c ) and indicates the presence of iKC-derived terminally differentiated cells. In contrast, MEF-derived skin was negative for loricrin expression ( d ). Scale bars = 10 μm

    Techniques Used: In Vivo, Mouse Assay, Staining, Generated, Derivative Assay, Immunofluorescence, Marker, Expressing

    Conversion of mouse embryonic fibroblasts ( MEFs ) to functional keratinocytes ( KCs ), without isolating an induced pluripotent stem cell ( iPSC ) intermediate. a The hypothesis behind the experimental design. b After a brief initiation of pluripotency-inducing transcriptional reprogramming by transduction with Sox2, Oct4, and Klf4, induced MEFs are subsequently pushed towards the epidermal lineage by retinoic acid ( RA ) and bone morphogenetic protein-4 ( BMP-4 ) treatment. DKSF defined keratinocyte serum free, IKC induced keratinocyte
    Figure Legend Snippet: Conversion of mouse embryonic fibroblasts ( MEFs ) to functional keratinocytes ( KCs ), without isolating an induced pluripotent stem cell ( iPSC ) intermediate. a The hypothesis behind the experimental design. b After a brief initiation of pluripotency-inducing transcriptional reprogramming by transduction with Sox2, Oct4, and Klf4, induced MEFs are subsequently pushed towards the epidermal lineage by retinoic acid ( RA ) and bone morphogenetic protein-4 ( BMP-4 ) treatment. DKSF defined keratinocyte serum free, IKC induced keratinocyte

    Techniques Used: Functional Assay, Transduction

    23) Product Images from "HIRA Is Required for Heart Development and Directly Regulates Tnni2 and Tnnt3"

    Article Title: HIRA Is Required for Heart Development and Directly Regulates Tnni2 and Tnnt3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0161096

    HIRA is required for the regulation of a subset of genes expressed during cardiac morphogenesis. A. Venn diagram indicating the number of genes whose expression is up or downregulated in Mesp1Cre ; Hira -/fl compared to Mesp1Cre ; Hira +/fl hearts at E11.5 and E12.5. The table indicates the level of fold change observed at E11.5 and at E12.5 on a subset of genes. B. Heatmap displaying the differential gene expression in mutant (Mesp1Cre ; Hira -/fl ) vs control (Mesp1Cre ; Hira +/fl ) in triplicates at E12.5. C. Heatmap of the gene ontology analysis revealed a trend in upregulation of sarcomeric contractile fibre genes (aside from Tnnt1 and Slc4a1 which were downregulated). D. qRT-PCR and RNASeq of the indicated genes within E12.5 hearts displayed as the fold induction in the mutants compared to their WT littermates (n = 3). Unpaired t-test: p
    Figure Legend Snippet: HIRA is required for the regulation of a subset of genes expressed during cardiac morphogenesis. A. Venn diagram indicating the number of genes whose expression is up or downregulated in Mesp1Cre ; Hira -/fl compared to Mesp1Cre ; Hira +/fl hearts at E11.5 and E12.5. The table indicates the level of fold change observed at E11.5 and at E12.5 on a subset of genes. B. Heatmap displaying the differential gene expression in mutant (Mesp1Cre ; Hira -/fl ) vs control (Mesp1Cre ; Hira +/fl ) in triplicates at E12.5. C. Heatmap of the gene ontology analysis revealed a trend in upregulation of sarcomeric contractile fibre genes (aside from Tnnt1 and Slc4a1 which were downregulated). D. qRT-PCR and RNASeq of the indicated genes within E12.5 hearts displayed as the fold induction in the mutants compared to their WT littermates (n = 3). Unpaired t-test: p

    Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR

    NKX2.5 shows diminished binding at the TTe locus in conditionally HIRA depleted hearts. NKX2.5 qChIP on 20 WT and Mesp1Cre ; Hira -/fl E12.5 hearts. Enrichment at the TTe was confirmed and found to be reduced in mutant hearts. Slc9a3r1 and Abca4 were enriched for both HIRA and NKX2.5 in WT hearts and displayed an increased expression in both Hira and Nkx2 . 5 mutant hearts. SLc9a3r1 , but not Abca4 , showed a significantly reduced binding of NKX2.5 in the absence of HIRA. Three different negative regions were tested. A region known to be enriched for NKX2.5, but not HIRA, binding ( Pop dc2) was used as a NKX2.5 qChIp positive control. Data is normalized to the input (= 100). Errors bars represent the min/max from technical duplicates. Unpaired t-test: p
    Figure Legend Snippet: NKX2.5 shows diminished binding at the TTe locus in conditionally HIRA depleted hearts. NKX2.5 qChIP on 20 WT and Mesp1Cre ; Hira -/fl E12.5 hearts. Enrichment at the TTe was confirmed and found to be reduced in mutant hearts. Slc9a3r1 and Abca4 were enriched for both HIRA and NKX2.5 in WT hearts and displayed an increased expression in both Hira and Nkx2 . 5 mutant hearts. SLc9a3r1 , but not Abca4 , showed a significantly reduced binding of NKX2.5 in the absence of HIRA. Three different negative regions were tested. A region known to be enriched for NKX2.5, but not HIRA, binding ( Pop dc2) was used as a NKX2.5 qChIp positive control. Data is normalized to the input (= 100). Errors bars represent the min/max from technical duplicates. Unpaired t-test: p

    Techniques Used: Binding Assay, Mutagenesis, Expressing, Positive Control

    HIRA is required in the developing heart. A. Lateral view of littermate embryos with the indicated genotype. Mesp1Cre ; Hira -/fl embryos had a low penetrance external phenotype at E12.5: exencephaly (E) and light haemorrhage (H) are indicated in the mutants. At E15.5, all mutants showed severe oedema (O) as indicated. B. Transverse OPT reconstructions followed by virtual reslicing of E15.5 embryo trunks with the indicated genotype. VSD (V) and ASD (A) are indicated in the mutants. H E of transverse sections of E14.5 embryos with the indicated genotype also showed a common atrioventricular junction (CJ) in the mutants as indicated. C . H E staining of transverse sections from E12.5 embryos reveals a disruption of the endocardial cushion (EC) fusion (two controls and two littermate mutants shown). The muscular septum is deficient ( * , top) and the relatively flat rather than crescentic cushion shape in the mutant are indicated (arrows). Scale bars represent 2mm (A) , 0 . 5mm (B-C) . D . Transverse sections of E12.5 embryonic hearts of the indicated genotype immunostained with DAPI and Troponin C captured on confocal showing disrupted sarcomeric structure in the mutant ventricular free wall. Scale bar : 10μm .
    Figure Legend Snippet: HIRA is required in the developing heart. A. Lateral view of littermate embryos with the indicated genotype. Mesp1Cre ; Hira -/fl embryos had a low penetrance external phenotype at E12.5: exencephaly (E) and light haemorrhage (H) are indicated in the mutants. At E15.5, all mutants showed severe oedema (O) as indicated. B. Transverse OPT reconstructions followed by virtual reslicing of E15.5 embryo trunks with the indicated genotype. VSD (V) and ASD (A) are indicated in the mutants. H E of transverse sections of E14.5 embryos with the indicated genotype also showed a common atrioventricular junction (CJ) in the mutants as indicated. C . H E staining of transverse sections from E12.5 embryos reveals a disruption of the endocardial cushion (EC) fusion (two controls and two littermate mutants shown). The muscular septum is deficient ( * , top) and the relatively flat rather than crescentic cushion shape in the mutant are indicated (arrows). Scale bars represent 2mm (A) , 0 . 5mm (B-C) . D . Transverse sections of E12.5 embryonic hearts of the indicated genotype immunostained with DAPI and Troponin C captured on confocal showing disrupted sarcomeric structure in the mutant ventricular free wall. Scale bar : 10μm .

    Techniques Used: Staining, Mutagenesis

    24) Product Images from "Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4"

    Article Title: Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4

    Journal: Scientific Reports

    doi: 10.1038/srep30529

    Viperin expression inhibits RABV replication. ( A ) Viperin inhibits RABV replication in viperin-eGFP-transfected BHK-21 cells. The viperin stably expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.1. Virus titres were determined at 24, 36, and 48 hpi. ( B ) RABV proteins in the infected viperin stably expressing BHK-21 cells were detected by Western blotting. ( C ) The N protein/actin, P protein/actin and M protein/actin ratios in Figure 2F were measured using Li-Cor Odyssey 3.0 analytical software version 29. ( D ) RNA expression levels of viperin. rRC-HL vRNA and N mRNA expression levels were detected by qRT-PCR at 24, 36, and 48 hpi. Viperin-expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.01. Data were normalized to β-actin expression and are presented as relative fold expression values to each control cell population infected with rRC-HL.
    Figure Legend Snippet: Viperin expression inhibits RABV replication. ( A ) Viperin inhibits RABV replication in viperin-eGFP-transfected BHK-21 cells. The viperin stably expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.1. Virus titres were determined at 24, 36, and 48 hpi. ( B ) RABV proteins in the infected viperin stably expressing BHK-21 cells were detected by Western blotting. ( C ) The N protein/actin, P protein/actin and M protein/actin ratios in Figure 2F were measured using Li-Cor Odyssey 3.0 analytical software version 29. ( D ) RNA expression levels of viperin. rRC-HL vRNA and N mRNA expression levels were detected by qRT-PCR at 24, 36, and 48 hpi. Viperin-expressing BHK-21 cells were infected with rRC-HL at an MOI of 0.01. Data were normalized to β-actin expression and are presented as relative fold expression values to each control cell population infected with rRC-HL.

    Techniques Used: Expressing, Transfection, Stable Transfection, Infection, Western Blot, Software, RNA Expression, Quantitative RT-PCR

    25) Product Images from "Glycyrrhizin protects against focal cerebral ischemia via inhibition of T cell activity and HMGB1-mediated mechanisms"

    Article Title: Glycyrrhizin protects against focal cerebral ischemia via inhibition of T cell activity and HMGB1-mediated mechanisms

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0705-5

    Gly inhibits IFNγ gene expression in CD4 T cells. Leukocytes were isolated from the ischemic brain 3 days after stroke, and CD4 T cells were purified by using FACS sorter. Gene expression of IFNγ, IL-4, and IL-10 were measured by RT-qPCR. However, IL-4 and IL-10 gene expressions were not detectable. Gene expression of IFNγ was significantly inhibited by Gly administration. N = 3, * P
    Figure Legend Snippet: Gly inhibits IFNγ gene expression in CD4 T cells. Leukocytes were isolated from the ischemic brain 3 days after stroke, and CD4 T cells were purified by using FACS sorter. Gene expression of IFNγ, IL-4, and IL-10 were measured by RT-qPCR. However, IL-4 and IL-10 gene expressions were not detectable. Gene expression of IFNγ was significantly inhibited by Gly administration. N = 3, * P

    Techniques Used: Expressing, Isolation, Purification, FACS, Quantitative RT-PCR

    26) Product Images from "FTO Is a Relevant Factor for the Development of the Metabolic Syndrome in Mice"

    Article Title: FTO Is a Relevant Factor for the Development of the Metabolic Syndrome in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105349

    Detailed analysis of adipose tissue. All data are collected from 30 weeks old mice. *indicate significant p-values between Lep ob/ob ;Fto +/+ and Lep ob/ob ; Fto −/− . a) Weights of different fat pads from female mice (n = 13, 16, 18, 16). b) Area size of epigonadal fat cells from female mice (n = 4, 4, 8, 7). c+d) Expression analysis for different marker genes of epigonadal adipose tissue (n = 6, 4, 5, 5, 5). Following p-values were calculated: between Lep ob/ob ;Fto +/− and Lep ob/ob ; Fto −/− : PPARγ2: p = 0,08, Adiponectin: p = 0,21, TNFα:p = 0,06, IL-6:p = 0,03. Data are presented as mean. Error bars indicate the SEM.
    Figure Legend Snippet: Detailed analysis of adipose tissue. All data are collected from 30 weeks old mice. *indicate significant p-values between Lep ob/ob ;Fto +/+ and Lep ob/ob ; Fto −/− . a) Weights of different fat pads from female mice (n = 13, 16, 18, 16). b) Area size of epigonadal fat cells from female mice (n = 4, 4, 8, 7). c+d) Expression analysis for different marker genes of epigonadal adipose tissue (n = 6, 4, 5, 5, 5). Following p-values were calculated: between Lep ob/ob ;Fto +/− and Lep ob/ob ; Fto −/− : PPARγ2: p = 0,08, Adiponectin: p = 0,21, TNFα:p = 0,06, IL-6:p = 0,03. Data are presented as mean. Error bars indicate the SEM.

    Techniques Used: Mouse Assay, Expressing, Marker

    27) Product Images from "Oncogene-triggered suppression of DNA repair leads to DNA instability in cancer"

    Article Title: Oncogene-triggered suppression of DNA repair leads to DNA instability in cancer

    Journal: Oncotarget

    doi:

    NeuT oncogene suppresses expression of H2AX A Expression of NeuT in MCF10A cells. MCF10A cells were infected with NeuT- expressing virus and two days post-infection selected with puromicin (0.75μg/ml). Control cells were infected with a corresponding empty virus. After 6 days of selection, cells were harvested, lysed, and analyzed by immunoblotting. B. NeuT expression does not affect stability of H2AX. MCF10A cells were infected as above. Cells were treated with emetine (10μM) and levels of H2AX at corresponding time points were assayed by immunoblotting. C. NeuT expression doesn't suppress translation of H2AX mRNA. MCF10A cells were infected as above. On day 6, post-infection cells were transfected with plasmid expressing renilla luciferase under H2AX 3'UTR control and the firefly luciferase used as an internal control. Translational efficiency was measured as renilla/luciferase ration (relative units). D. NeuT oncogene suppresses transcription of H2AX mRNA. MCF10A cells were infected as above, and Q-PCR was performed as described in Materials and Methods to measure H2AX mRNA. Each sample was analyzed in triplicates. E. NeuT expression does not affect stability of H2AX mRNA. MCF10A cells were infected as above. Cells were treated with actinomycin D (7μg/ml) and samples for RNA extraction were collected at corresponding time points. Q-PCR was performed as described in Materials and Methods. Each point represents three independent experiments. F. Inhibition of CDKs suppresses expression of H2AX. MCF10A cells were treated with indicated doses of roscovitine overnight. Cells were lysed and H2AX levels were assayed by immunoblotting. G. Rb silencing reverses suppression of H2AX levels caused by expression of NeuT. MCF10A cells were simultaneously infected with NeuT- expressing retrovirus (under blasticidin selection) and siRB1-expressing lentivirus (under puromycin selection); control cells were infected with corresponding empty viruses. Two days post-infection puromycin (0.75μg/ml) and blastocidin (10μg/ml) were added and after another 7 days cells were harvested, lysed, and analyzed by immunoblotting to assay H2AX levels (left panel) or by Q-PCR to assay level of Rb1 silencing (right panel).
    Figure Legend Snippet: NeuT oncogene suppresses expression of H2AX A Expression of NeuT in MCF10A cells. MCF10A cells were infected with NeuT- expressing virus and two days post-infection selected with puromicin (0.75μg/ml). Control cells were infected with a corresponding empty virus. After 6 days of selection, cells were harvested, lysed, and analyzed by immunoblotting. B. NeuT expression does not affect stability of H2AX. MCF10A cells were infected as above. Cells were treated with emetine (10μM) and levels of H2AX at corresponding time points were assayed by immunoblotting. C. NeuT expression doesn't suppress translation of H2AX mRNA. MCF10A cells were infected as above. On day 6, post-infection cells were transfected with plasmid expressing renilla luciferase under H2AX 3'UTR control and the firefly luciferase used as an internal control. Translational efficiency was measured as renilla/luciferase ration (relative units). D. NeuT oncogene suppresses transcription of H2AX mRNA. MCF10A cells were infected as above, and Q-PCR was performed as described in Materials and Methods to measure H2AX mRNA. Each sample was analyzed in triplicates. E. NeuT expression does not affect stability of H2AX mRNA. MCF10A cells were infected as above. Cells were treated with actinomycin D (7μg/ml) and samples for RNA extraction were collected at corresponding time points. Q-PCR was performed as described in Materials and Methods. Each point represents three independent experiments. F. Inhibition of CDKs suppresses expression of H2AX. MCF10A cells were treated with indicated doses of roscovitine overnight. Cells were lysed and H2AX levels were assayed by immunoblotting. G. Rb silencing reverses suppression of H2AX levels caused by expression of NeuT. MCF10A cells were simultaneously infected with NeuT- expressing retrovirus (under blasticidin selection) and siRB1-expressing lentivirus (under puromycin selection); control cells were infected with corresponding empty viruses. Two days post-infection puromycin (0.75μg/ml) and blastocidin (10μg/ml) were added and after another 7 days cells were harvested, lysed, and analyzed by immunoblotting to assay H2AX levels (left panel) or by Q-PCR to assay level of Rb1 silencing (right panel).

    Techniques Used: Expressing, Infection, Selection, Transfection, Plasmid Preparation, Luciferase, Polymerase Chain Reaction, RNA Extraction, Inhibition

    28) Product Images from "Effects of Cdh23 single nucleotide substitutions on age-related hearing loss in C57BL/6 and 129S1/Sv mice and comparisons with congenic strains"

    Article Title: Effects of Cdh23 single nucleotide substitutions on age-related hearing loss in C57BL/6 and 129S1/Sv mice and comparisons with congenic strains

    Journal: Scientific Reports

    doi: 10.1038/srep44450

    DNA sequence validation, PCR identification of targeted SNVs and assessment of exon skpping. ( A ) Sequence chromatograms of PCR amplified DNA surrounding the targeted Cdh23 c.753 nucleotide (indicated by the red downward-pointing arrow) confirm that C57BL/6 NJ (B6N) and 129S- Cdh23 c.753A (129S-SNV) mice are homozygous for the Cdh23 c.753 A nucleotide, while 129S1/SvImJ (129S1) and B6N- Cdh23 c.753G (B6N-SNV) mice are homozygous for the Cdh23 c.753 G nucleotide. ( B ) Identification of Cdh23 alleles with targeted SNVs by PCR amplification of the closely linked PGK-Neo insertion remnant. Primers flanking the PGK-Neo cassette insertion site were used to amplify PCR products that differ in size between the wildtype allele and the targeted SNV allele, which retains an intronic 104 bp remnant of the PGK-Neo cassette after Cre deletion. Because of its close proximity (178 bp) to the targeted SNV, the presence or absence of the PGK-Neo remnant can be used to distinguish the wildtype allele (+, 112 bp) from the targeted SNV allele ( v , 216 bp). Lane 1, 100-bp DNA size ladder; lanes 2–5, genotypes of individual mice. ( C ) RT-PCR to evaluate the extent of exon skipping related to the Cdh23 c.753A variant. cDNA primers flanking the alternatively spliced exon of Cdh23 (containing the c.753 nucleotide) were used to amplify alternatively spliced products. The PCR product size from the wild-type Cdh23 transcript (395 bp) is larger than the PCR product from the alternatively spliced, in-frame transcript (266 bp), which lacks the 129 bp skipped exon. Lane 1, 100-bp DNA size ladder; lane 2, B6N; lane 3, 129S1; lane 4, B6.129S1- Cdh23 Ahl + congenic (B6-con); lane 5, 129S1.B6- Cdh23 ahl congenic (129S-con); lane 6, B6- Cdh23 c.753G SNV (B6-SNV); and lane 7, 129S- Cdh23 c.753A SNV (129S-SNV). Lanes 2, 5, and 7 show alternatively spliced transcripts caused by the Cdh23 c.753A variant.
    Figure Legend Snippet: DNA sequence validation, PCR identification of targeted SNVs and assessment of exon skpping. ( A ) Sequence chromatograms of PCR amplified DNA surrounding the targeted Cdh23 c.753 nucleotide (indicated by the red downward-pointing arrow) confirm that C57BL/6 NJ (B6N) and 129S- Cdh23 c.753A (129S-SNV) mice are homozygous for the Cdh23 c.753 A nucleotide, while 129S1/SvImJ (129S1) and B6N- Cdh23 c.753G (B6N-SNV) mice are homozygous for the Cdh23 c.753 G nucleotide. ( B ) Identification of Cdh23 alleles with targeted SNVs by PCR amplification of the closely linked PGK-Neo insertion remnant. Primers flanking the PGK-Neo cassette insertion site were used to amplify PCR products that differ in size between the wildtype allele and the targeted SNV allele, which retains an intronic 104 bp remnant of the PGK-Neo cassette after Cre deletion. Because of its close proximity (178 bp) to the targeted SNV, the presence or absence of the PGK-Neo remnant can be used to distinguish the wildtype allele (+, 112 bp) from the targeted SNV allele ( v , 216 bp). Lane 1, 100-bp DNA size ladder; lanes 2–5, genotypes of individual mice. ( C ) RT-PCR to evaluate the extent of exon skipping related to the Cdh23 c.753A variant. cDNA primers flanking the alternatively spliced exon of Cdh23 (containing the c.753 nucleotide) were used to amplify alternatively spliced products. The PCR product size from the wild-type Cdh23 transcript (395 bp) is larger than the PCR product from the alternatively spliced, in-frame transcript (266 bp), which lacks the 129 bp skipped exon. Lane 1, 100-bp DNA size ladder; lane 2, B6N; lane 3, 129S1; lane 4, B6.129S1- Cdh23 Ahl + congenic (B6-con); lane 5, 129S1.B6- Cdh23 ahl congenic (129S-con); lane 6, B6- Cdh23 c.753G SNV (B6-SNV); and lane 7, 129S- Cdh23 c.753A SNV (129S-SNV). Lanes 2, 5, and 7 show alternatively spliced transcripts caused by the Cdh23 c.753A variant.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Variant Assay

    29) Product Images from "Defective protein repair under methionine sulfoxide A deletion drives autophagy and ARE-dependent gene transcription"

    Article Title: Defective protein repair under methionine sulfoxide A deletion drives autophagy and ARE-dependent gene transcription

    Journal: Redox Biology

    doi: 10.1016/j.redox.2018.04.001

    Expression and activity of Nrf2-regulated genes are elevated under MsrA deletion. (A, B) GCLC (A) and GR (B) mRNA levels in MsrA-/- and WT VSMC by qRT-PCR, normalized to ARP; n = 3 biological replicates. (C-E) Representative immunoblot (C) and summary data for GCLC (D) and GR (E) protein levels in whole cell lysates from MsrA-/- and WT VSMC. Data were normalized to GAPDH; n = 6–7 biological replicates. (F) GR activity, normalized to total protein concentration; n = 4 biological replicates. (G, H) Levels of reduced glutathione (GSH, G) and oxidized glutathione (GSSG, H), normalized to total protein; n = 4 biological replicates. (I) Ratio of reduced/oxidized glutathione. (J) Glutathione transferase (GST) activity, normalized to total protein; n = 7 biological replicates. * p
    Figure Legend Snippet: Expression and activity of Nrf2-regulated genes are elevated under MsrA deletion. (A, B) GCLC (A) and GR (B) mRNA levels in MsrA-/- and WT VSMC by qRT-PCR, normalized to ARP; n = 3 biological replicates. (C-E) Representative immunoblot (C) and summary data for GCLC (D) and GR (E) protein levels in whole cell lysates from MsrA-/- and WT VSMC. Data were normalized to GAPDH; n = 6–7 biological replicates. (F) GR activity, normalized to total protein concentration; n = 4 biological replicates. (G, H) Levels of reduced glutathione (GSH, G) and oxidized glutathione (GSSG, H), normalized to total protein; n = 4 biological replicates. (I) Ratio of reduced/oxidized glutathione. (J) Glutathione transferase (GST) activity, normalized to total protein; n = 7 biological replicates. * p

    Techniques Used: Expressing, Activity Assay, Quantitative RT-PCR, Protein Concentration

    p62 is elevated in MsrA-deficient VSMC and arteries. (A, B) Representative immunoblot (A) and summary data (B) for p62 in whole cell lysates of VSMC isolated from MsrA-/- and WT mice. Data were normalized to GAPDH loading control and expressed relative to p62 levels in WT VSMC (n = 9 biological replicates). (C, D) Representative immunoblot (C) and summary data (D) for p62 in whole cell lysates of carotid arteries isolated from MsrA-/- and WT mice (n = 10 biological replicates). E) Representative immunofluorescent images of p62 (green) and nuclei (TOPRO, blue) in VSMC from MsrA-/- and WT mice with or without treatment with bafilomycin a1 (Baf) for 24 h. Scale bars 20 µm. (F) Quantification of p62 aggregates from (E). Arbitrary aggregate score was calculated as the mean GFP fluorescence intensity per cell in at least 5 images per biological replicate (1–5 cells/image; n = 5 biological replicates). (G) Representative immunofluorescent images of p62 (green), smooth muscle actin (red) and nuclei (TOPRO, blue) in carotid artery sections from MsrA-/- and WT mice. 100 × , scale bar 10 µm. NC denotes negative control without primary antibody, p62 inset with p62 (green) only. Arrows denote p62 aggregates. (H) mRNA expression of p62 in VSMC from MsrA-/- and WT mice by qRT-PCR; data were normalized to ARP and expressed relative to p62 in WT VSMC (n = 5 biological replicates). * p
    Figure Legend Snippet: p62 is elevated in MsrA-deficient VSMC and arteries. (A, B) Representative immunoblot (A) and summary data (B) for p62 in whole cell lysates of VSMC isolated from MsrA-/- and WT mice. Data were normalized to GAPDH loading control and expressed relative to p62 levels in WT VSMC (n = 9 biological replicates). (C, D) Representative immunoblot (C) and summary data (D) for p62 in whole cell lysates of carotid arteries isolated from MsrA-/- and WT mice (n = 10 biological replicates). E) Representative immunofluorescent images of p62 (green) and nuclei (TOPRO, blue) in VSMC from MsrA-/- and WT mice with or without treatment with bafilomycin a1 (Baf) for 24 h. Scale bars 20 µm. (F) Quantification of p62 aggregates from (E). Arbitrary aggregate score was calculated as the mean GFP fluorescence intensity per cell in at least 5 images per biological replicate (1–5 cells/image; n = 5 biological replicates). (G) Representative immunofluorescent images of p62 (green), smooth muscle actin (red) and nuclei (TOPRO, blue) in carotid artery sections from MsrA-/- and WT mice. 100 × , scale bar 10 µm. NC denotes negative control without primary antibody, p62 inset with p62 (green) only. Arrows denote p62 aggregates. (H) mRNA expression of p62 in VSMC from MsrA-/- and WT mice by qRT-PCR; data were normalized to ARP and expressed relative to p62 in WT VSMC (n = 5 biological replicates). * p

    Techniques Used: Isolation, Mouse Assay, Fluorescence, Negative Control, Expressing, Quantitative RT-PCR

    Elevated Nrf2 protein expression under MsrA deficiency is due to protein stabilization rather than increased transcription. (A, B) Representative immunoblot (A) and quantification (B) for Nrf2 protein levels in whole cell lysates from MsrA-/- and WT VSMC; n = 3 biological replicates. (C) Nrf2 mRNA levels in VSMC by qRT-PCR; n = 5 biological replicates. (D) Representative immunoprecipitation of Nrf2 followed by immunoblot for ubiquitin in MsrA-/- and WT VSMC. IgG: IP with IgG, WT + MC132: IP with anti-Nrf2 in WT VSMC incubated with MG132, WCL: whole cell lysate of WT VSMC as controls. (E) Quantification of (D); n = 7 biological replicates. (F) p62 mRNA levels by qRT-PCR in aortic samples WT, MsrA-/- and MsrA-/- x Nrf2-/- mice; n = 7, 9 biological replicates. (G) Representative Immunoblots for Nrf2 and GAPDH in aortic samples from WT, MsrA-/- and MsrA-/- x Nrf2-/- mice. (H) Quantification of (G) n = 7 biological replicates. (E) * p
    Figure Legend Snippet: Elevated Nrf2 protein expression under MsrA deficiency is due to protein stabilization rather than increased transcription. (A, B) Representative immunoblot (A) and quantification (B) for Nrf2 protein levels in whole cell lysates from MsrA-/- and WT VSMC; n = 3 biological replicates. (C) Nrf2 mRNA levels in VSMC by qRT-PCR; n = 5 biological replicates. (D) Representative immunoprecipitation of Nrf2 followed by immunoblot for ubiquitin in MsrA-/- and WT VSMC. IgG: IP with IgG, WT + MC132: IP with anti-Nrf2 in WT VSMC incubated with MG132, WCL: whole cell lysate of WT VSMC as controls. (E) Quantification of (D); n = 7 biological replicates. (F) p62 mRNA levels by qRT-PCR in aortic samples WT, MsrA-/- and MsrA-/- x Nrf2-/- mice; n = 7, 9 biological replicates. (G) Representative Immunoblots for Nrf2 and GAPDH in aortic samples from WT, MsrA-/- and MsrA-/- x Nrf2-/- mice. (H) Quantification of (G) n = 7 biological replicates. (E) * p

    Techniques Used: Expressing, Quantitative RT-PCR, Immunoprecipitation, Incubation, Mouse Assay, Western Blot

    30) Product Images from "Histone chaperone HIRA deposits histone H3.3 onto foreign viral DNA and contributes to anti-viral intrinsic immunity"

    Article Title: Histone chaperone HIRA deposits histone H3.3 onto foreign viral DNA and contributes to anti-viral intrinsic immunity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx771

    HIRA contributes to efficient suppression of viral infection. ( A ) Virus yield from HIRA-depleted and control IMR90 cells infected at MOI 0.01 with ICP0-null HSV-1 mutant dl 1403 CMV lacZ or wt HSV-1 variant in 1863. Supernatant was harvested at indicated times post infection (h p.i.) and virus titres determined by plaque assay. Data are mean +/- SD (error bars) (n = 3 biological repeats) with indicated P values. ( B ) HIRA-depleted and control IMR90 cells infected with ICP0-null HSV-1 mutant dl 1403 at MOI 2.0. Lysates were harvested and processed at indicated time points post infection (h p.i.) ( C – E ) Control ( CAGG-Cre-ER , WT, +tamoxifen) or Hira-deficient ( CAGG-Cre-ER, Hirafl/fl , +tamoxifen) mice were infected with MCMV and spleen harvested 4 days later. Each spleen was divided into three pieces for downstream analysis. (C) Western blot analysis of WT or HIRA −/− animals showing knock out of Hira. Shown are representative western blot results from two different gels with 4 WT and 8 HIRA −/- mice. (D) mRNA abundance of IFN-β target genes by qRT-PCR. Bar chart displays mean of each IFN-β target gene mRNA abundance in WT mice compared to HIRA −/- mice, normalized to β - actin as housekeeping control. Data are mean ± SEM (error bars) ( n = 4 for WT mice and n = 8 for HIRA −/- mice). * P
    Figure Legend Snippet: HIRA contributes to efficient suppression of viral infection. ( A ) Virus yield from HIRA-depleted and control IMR90 cells infected at MOI 0.01 with ICP0-null HSV-1 mutant dl 1403 CMV lacZ or wt HSV-1 variant in 1863. Supernatant was harvested at indicated times post infection (h p.i.) and virus titres determined by plaque assay. Data are mean +/- SD (error bars) (n = 3 biological repeats) with indicated P values. ( B ) HIRA-depleted and control IMR90 cells infected with ICP0-null HSV-1 mutant dl 1403 at MOI 2.0. Lysates were harvested and processed at indicated time points post infection (h p.i.) ( C – E ) Control ( CAGG-Cre-ER , WT, +tamoxifen) or Hira-deficient ( CAGG-Cre-ER, Hirafl/fl , +tamoxifen) mice were infected with MCMV and spleen harvested 4 days later. Each spleen was divided into three pieces for downstream analysis. (C) Western blot analysis of WT or HIRA −/− animals showing knock out of Hira. Shown are representative western blot results from two different gels with 4 WT and 8 HIRA −/- mice. (D) mRNA abundance of IFN-β target genes by qRT-PCR. Bar chart displays mean of each IFN-β target gene mRNA abundance in WT mice compared to HIRA −/- mice, normalized to β - actin as housekeeping control. Data are mean ± SEM (error bars) ( n = 4 for WT mice and n = 8 for HIRA −/- mice). * P

    Techniques Used: Infection, Mutagenesis, Variant Assay, Plaque Assay, Mouse Assay, Western Blot, Knock-Out, Quantitative RT-PCR

    31) Product Images from "Prolactin signaling through focal adhesion complexes is amplified by stiff extracellular matrices in breast cancer cells"

    Article Title: Prolactin signaling through focal adhesion complexes is amplified by stiff extracellular matrices in breast cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10137

    Inhibiting SFKs decreases PRL signals to pFAK Y925 only in stiff environments A-B. T47D cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24h, then treated with vehicle (−) or SFK inhibitor, PP-2 (+) for 1 h prior to ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 3. Different letters represent significant differences within each stiffness determined by paired t-tests (lower case, 12kPa; upper case, 75kPa), p
    Figure Legend Snippet: Inhibiting SFKs decreases PRL signals to pFAK Y925 only in stiff environments A-B. T47D cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24h, then treated with vehicle (−) or SFK inhibitor, PP-2 (+) for 1 h prior to ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 3. Different letters represent significant differences within each stiffness determined by paired t-tests (lower case, 12kPa; upper case, 75kPa), p

    Techniques Used: Western Blot

    Collagen ligand density does not modulate PRL signals to ERK1/2 or FAK A-C. T47D were cells plated on 25 kPa polyacrylamide gels coated with either 50, 200, or 800 μg/ml collagen-I, serum starved for 24h, then treated ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 3. Different letters represent significant differences between treatments, p
    Figure Legend Snippet: Collagen ligand density does not modulate PRL signals to ERK1/2 or FAK A-C. T47D were cells plated on 25 kPa polyacrylamide gels coated with either 50, 200, or 800 μg/ml collagen-I, serum starved for 24h, then treated ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 3. Different letters represent significant differences between treatments, p

    Techniques Used: Western Blot

    Blocking β1-integrin decreases PRL signals to pERK1/2 and pFAK Y925 in stiff environments A-B. T47D cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24h, then treated with isotype control antibody (−) or β1-integrin blocking antibody mAb13 (+) for 1 h prior to ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M., n = 4. Different letters represent significant differences within each stiffness (lower case, 12kPa; upper case, 75kPa), p
    Figure Legend Snippet: Blocking β1-integrin decreases PRL signals to pERK1/2 and pFAK Y925 in stiff environments A-B. T47D cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24h, then treated with isotype control antibody (−) or β1-integrin blocking antibody mAb13 (+) for 1 h prior to ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M., n = 4. Different letters represent significant differences within each stiffness (lower case, 12kPa; upper case, 75kPa), p

    Techniques Used: Blocking Assay, Western Blot

    Inhibiting integrin activated FAK at Y397 more efficiently decreases PRL signals to pFAK Y925 in stiff environments A-B. T47D cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24h, then treated with vehicle (−) or FAK Y397 inhibitor PF-573228 (+) for 1 h prior to ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 4. Different letters represent significant differences within each stiffness (lower case, 12kPa; upper case, 75kPa), p
    Figure Legend Snippet: Inhibiting integrin activated FAK at Y397 more efficiently decreases PRL signals to pFAK Y925 in stiff environments A-B. T47D cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24h, then treated with vehicle (−) or FAK Y397 inhibitor PF-573228 (+) for 1 h prior to ± PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 4. Different letters represent significant differences within each stiffness (lower case, 12kPa; upper case, 75kPa), p

    Techniques Used: Western Blot

    Stiff environments increase FAK-mediated hormone induced proliferation T47D and MCF-7 cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I in phenol-red free 5% charcoal stripped FBS for 24 h, serum starved for 24 h, and then treated with vehicle (DMSO 1:1000) or the FAK inhibitor, PF-573228 (1μM), for 1 h prior to ± PRL (4 nM), ± E2 (1nM) for 24 h. Cells were then stained with DAPI and Ki-67 antibody as described in Experimental Procedures. A, C. Effect of hormones on Ki67 staining, assessed by percentage of Ki-67 positive T47D (A) and MCF-7 (C) cells. B, D. Inhibition of proliferation by PF-573,228 compared to vehicle treated T47D cells (B) and MCF7 cells (D). Different letters represent significant differences within each stiffness (lower case, 12 kPa; upper case, 75 kPa). * represent significant differences between the same treatments at different stiffnesses: *p
    Figure Legend Snippet: Stiff environments increase FAK-mediated hormone induced proliferation T47D and MCF-7 cells were plated on 12 or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I in phenol-red free 5% charcoal stripped FBS for 24 h, serum starved for 24 h, and then treated with vehicle (DMSO 1:1000) or the FAK inhibitor, PF-573228 (1μM), for 1 h prior to ± PRL (4 nM), ± E2 (1nM) for 24 h. Cells were then stained with DAPI and Ki-67 antibody as described in Experimental Procedures. A, C. Effect of hormones on Ki67 staining, assessed by percentage of Ki-67 positive T47D (A) and MCF-7 (C) cells. B, D. Inhibition of proliferation by PF-573,228 compared to vehicle treated T47D cells (B) and MCF7 cells (D). Different letters represent significant differences within each stiffness (lower case, 12 kPa; upper case, 75 kPa). * represent significant differences between the same treatments at different stiffnesses: *p

    Techniques Used: Staining, Inhibition

    Stiffer environments robustly increase PRL signals to pERK1/2 and pFAK Y925, but only slightly increase signals to pSTAT5 A-C. T47D cells were plated on 12, 25, or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24 h, and treated ± PRL (4nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 5. Different letters represent significant differences between treatments, p
    Figure Legend Snippet: Stiffer environments robustly increase PRL signals to pERK1/2 and pFAK Y925, but only slightly increase signals to pSTAT5 A-C. T47D cells were plated on 12, 25, or 75 kPa polyacrylamide gels coated with 200 μg/ml collagen-I, serum starved for 24 h, and treated ± PRL (4nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means ± S.E.M. n = 5. Different letters represent significant differences between treatments, p

    Techniques Used: Western Blot

    32) Product Images from "Changes of circulating Th22 cells in children with hand, foot, and mouth disease caused by enterovirus 71 infection"

    Article Title: Changes of circulating Th22 cells in children with hand, foot, and mouth disease caused by enterovirus 71 infection

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14083

    Increased mRNA expression of cTh22 cells is associated with cytokines and transcription factors in patients with EV71-associated HFMD A .- C . Human peripheral blood mononuclear cells (PBMCs) from 18 mild patients and 11 severe patients with EV71-associated HFMD and 11 healthy controls (HC) were isolated and analyzed using the PCR assay for IL-6, IL-23 and TNF-α mRNA expression. D .- G . The mRNA levels of IL-22, IL-17A, AHR and RORγt in CD4 + T cells were analyzed using the PCR assay as described in the Methods section. *, p
    Figure Legend Snippet: Increased mRNA expression of cTh22 cells is associated with cytokines and transcription factors in patients with EV71-associated HFMD A .- C . Human peripheral blood mononuclear cells (PBMCs) from 18 mild patients and 11 severe patients with EV71-associated HFMD and 11 healthy controls (HC) were isolated and analyzed using the PCR assay for IL-6, IL-23 and TNF-α mRNA expression. D .- G . The mRNA levels of IL-22, IL-17A, AHR and RORγt in CD4 + T cells were analyzed using the PCR assay as described in the Methods section. *, p

    Techniques Used: Expressing, Isolation, Polymerase Chain Reaction

    33) Product Images from "Changes of circulating Th22 cells in children with hand, foot, and mouth disease caused by enterovirus 71 infection"

    Article Title: Changes of circulating Th22 cells in children with hand, foot, and mouth disease caused by enterovirus 71 infection

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14083

    Circulating Th22 cells, a principal source of intracellular IL-22 in peripheral blood A . The cells were gated initially on IL-22 + cells and then on CD4 + T cells and IL-17A cells. B . Circulating IL-22 + IL-17A − CD4 + Th22 (cTh22) cells. C . Circulating IL-22 + cTh17 cells. D . IL-17A − CD4 − cells in the total IL-22 + cells were analyzed in HC and mild and severe patients. *, p
    Figure Legend Snippet: Circulating Th22 cells, a principal source of intracellular IL-22 in peripheral blood A . The cells were gated initially on IL-22 + cells and then on CD4 + T cells and IL-17A cells. B . Circulating IL-22 + IL-17A − CD4 + Th22 (cTh22) cells. C . Circulating IL-22 + cTh17 cells. D . IL-17A − CD4 − cells in the total IL-22 + cells were analyzed in HC and mild and severe patients. *, p

    Techniques Used:

    Increased frequencies of cTh22 and cTh17 cells in human CD4 + T cells of peripheral blood from patients with EV71-associated HFMD Human peripheral blood mononuclear cells (PBMCs) from 32 mild and 24 severe patients with EV71-associated HFMD and 46 healthy controls (HC) were isolated and stained with labeled antibodies and analyzed by flow cytometry as described in the Methods section. A . The cells were gated initially on lymphocytes and then on CD4 + T cells, circulating IL-22 + IL-17A − CD4 + Th22 (cTh22) cells, circulating IL-17A + CD4 + Th17 (cTh17) cells, and IL-22 + IL-17A + CD4 + T (IL-22 + cTh17)cells in the total number of CD4 + T cells. B .- D . The analysis of the percentages of cTh22 cells, cTh17 cells and IL-22 + cTh17 cells in CD4 + T cells from HC and mild and severe patients. E .- F . The proportion of circulating IL-22 + cells in cTh17 cells. G .- H . The correlation of cTh17 cells and IL-22 + cTh17 cells in mild and severe cases. *, p
    Figure Legend Snippet: Increased frequencies of cTh22 and cTh17 cells in human CD4 + T cells of peripheral blood from patients with EV71-associated HFMD Human peripheral blood mononuclear cells (PBMCs) from 32 mild and 24 severe patients with EV71-associated HFMD and 46 healthy controls (HC) were isolated and stained with labeled antibodies and analyzed by flow cytometry as described in the Methods section. A . The cells were gated initially on lymphocytes and then on CD4 + T cells, circulating IL-22 + IL-17A − CD4 + Th22 (cTh22) cells, circulating IL-17A + CD4 + Th17 (cTh17) cells, and IL-22 + IL-17A + CD4 + T (IL-22 + cTh17)cells in the total number of CD4 + T cells. B .- D . The analysis of the percentages of cTh22 cells, cTh17 cells and IL-22 + cTh17 cells in CD4 + T cells from HC and mild and severe patients. E .- F . The proportion of circulating IL-22 + cells in cTh17 cells. G .- H . The correlation of cTh17 cells and IL-22 + cTh17 cells in mild and severe cases. *, p

    Techniques Used: Isolation, Staining, Labeling, Flow Cytometry, Cytometry

    Increased mRNA expression of cTh22 cells is associated with cytokines and transcription factors in patients with EV71-associated HFMD A .- C . Human peripheral blood mononuclear cells (PBMCs) from 18 mild patients and 11 severe patients with EV71-associated HFMD and 11 healthy controls (HC) were isolated and analyzed using the PCR assay for IL-6, IL-23 and TNF-α mRNA expression. D .- G . The mRNA levels of IL-22, IL-17A, AHR and RORγt in CD4 + T cells were analyzed using the PCR assay as described in the Methods section. *, p
    Figure Legend Snippet: Increased mRNA expression of cTh22 cells is associated with cytokines and transcription factors in patients with EV71-associated HFMD A .- C . Human peripheral blood mononuclear cells (PBMCs) from 18 mild patients and 11 severe patients with EV71-associated HFMD and 11 healthy controls (HC) were isolated and analyzed using the PCR assay for IL-6, IL-23 and TNF-α mRNA expression. D .- G . The mRNA levels of IL-22, IL-17A, AHR and RORγt in CD4 + T cells were analyzed using the PCR assay as described in the Methods section. *, p

    Techniques Used: Expressing, Isolation, Polymerase Chain Reaction

    34) Product Images from "Changes of circulating Th22 cells in children with hand, foot, and mouth disease caused by enterovirus 71 infection"

    Article Title: Changes of circulating Th22 cells in children with hand, foot, and mouth disease caused by enterovirus 71 infection

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14083

    Increased mRNA expression of cTh22 cells is associated with cytokines and transcription factors in patients with EV71-associated HFMD A .- C . Human peripheral blood mononuclear cells (PBMCs) from 18 mild patients and 11 severe patients with EV71-associated HFMD and 11 healthy controls (HC) were isolated and analyzed using the PCR assay for IL-6, IL-23 and TNF-α mRNA expression. D .- G . The mRNA levels of IL-22, IL-17A, AHR and RORγt in CD4 + T cells were analyzed using the PCR assay as described in the Methods section. *, p
    Figure Legend Snippet: Increased mRNA expression of cTh22 cells is associated with cytokines and transcription factors in patients with EV71-associated HFMD A .- C . Human peripheral blood mononuclear cells (PBMCs) from 18 mild patients and 11 severe patients with EV71-associated HFMD and 11 healthy controls (HC) were isolated and analyzed using the PCR assay for IL-6, IL-23 and TNF-α mRNA expression. D .- G . The mRNA levels of IL-22, IL-17A, AHR and RORγt in CD4 + T cells were analyzed using the PCR assay as described in the Methods section. *, p

    Techniques Used: Expressing, Isolation, Polymerase Chain Reaction

    35) Product Images from "Chronic Oxidative Stress, Mitochondrial Dysfunction, Nrf2 Activation and Inflammation in the Hippocampus Accompany Heightened Systemic Inflammation and Oxidative Stress in an Animal Model of Gulf War Illness"

    Article Title: Chronic Oxidative Stress, Mitochondrial Dysfunction, Nrf2 Activation and Inflammation in the Hippocampus Accompany Heightened Systemic Inflammation and Oxidative Stress in an Animal Model of Gulf War Illness

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00182

    The hippocampus of rats with chronic gulf war illness-like symptoms (GWI-rats) showed a higher expression of multiple antioxidant genes. (A) Cluster diagram comparing the relative expression of antioxidant genes between animals exposed to vehicle ( n = 5), GWI-related chemicals (GWIR-Cs), and stress ( n = 5), which was measured 6 months after exposure using quantitative real-time polymerase chain reaction. Bar charts comparing the expression of Apc , Gsr , Gstk1 , Gstp1 , Prdx5 , Ptgs1 , and Srxn1 , between the vehicle and GWI groups are illustrated in B1–B7 . The bar charts comparing the expression of Cat , Ctsb , Gpx3-4 , Gpx6-7 , Prdx1-2 , Prdx6 , Txnrd1 , and Txnrd2 , between the vehicle and GWI groups are illustrated in Figure 1 . ∗ p
    Figure Legend Snippet: The hippocampus of rats with chronic gulf war illness-like symptoms (GWI-rats) showed a higher expression of multiple antioxidant genes. (A) Cluster diagram comparing the relative expression of antioxidant genes between animals exposed to vehicle ( n = 5), GWI-related chemicals (GWIR-Cs), and stress ( n = 5), which was measured 6 months after exposure using quantitative real-time polymerase chain reaction. Bar charts comparing the expression of Apc , Gsr , Gstk1 , Gstp1 , Prdx5 , Ptgs1 , and Srxn1 , between the vehicle and GWI groups are illustrated in B1–B7 . The bar charts comparing the expression of Cat , Ctsb , Gpx3-4 , Gpx6-7 , Prdx1-2 , Prdx6 , Txnrd1 , and Txnrd2 , between the vehicle and GWI groups are illustrated in Figure 1 . ∗ p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    The hippocampus of rats with chronic gulf war illness-like symptoms (GWI-rats) exhibited an enhanced expression of genes encoding proteins involved in reactive oxygen species (ROS) metabolism and oxygen transport. (A,C) Are cluster diagrams comparing the relative expression of genes between animals exposed to vehicle ( n = 5), GWI-related chemicals (GWIR-Cs), and stress ( n = 5), which was measured 6 months after exposure using quantitative real-time polymerase chain reaction. Bar charts in B1–B6 and D1–D4 illustrate the elevated expression of genes involved in ROS metabolism and oxygen transport in animals exposed to GWIR-Cs and stress. ∗ p
    Figure Legend Snippet: The hippocampus of rats with chronic gulf war illness-like symptoms (GWI-rats) exhibited an enhanced expression of genes encoding proteins involved in reactive oxygen species (ROS) metabolism and oxygen transport. (A,C) Are cluster diagrams comparing the relative expression of genes between animals exposed to vehicle ( n = 5), GWI-related chemicals (GWIR-Cs), and stress ( n = 5), which was measured 6 months after exposure using quantitative real-time polymerase chain reaction. Bar charts in B1–B6 and D1–D4 illustrate the elevated expression of genes involved in ROS metabolism and oxygen transport in animals exposed to GWIR-Cs and stress. ∗ p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    The hippocampus of rats with chronic gulf war illness-like symptoms (GWI-rats) presented an increased expression of multiple genes ( n = 26) involved in mitochondrial respiration. (A,C) Are cluster diagrams comparing the relative expression of mitochondria-related genes between animals exposed to vehicle ( n = 5), GWI-related chemicals (GWIR-Cs), and stress ( n = 4), which was measured 6 months after exposure using quantitative real-time polymerase chain reaction. Bar charts in B1–B9,C,D1–D5,E1,E2 illustrate the elevated expression of genes involved in mitochondrial complex I ( Ndufa8, Ndufa9, Ndufa11, Ndufb3, Ndufb5, Ndufs6, Ndufs7, Ndufs8 , and Ndufv1 ; B1–B9 ), complex II ( Sdhb ; C ), complex IV ( Cox5b, Cox6a1, Cox7a2L, Surf1 , and Cyc1 ; D1–D5 ), and complex V ( Atp5a1 and Atp5b ; E1,E2 ) of GWI-rats. ∗ p
    Figure Legend Snippet: The hippocampus of rats with chronic gulf war illness-like symptoms (GWI-rats) presented an increased expression of multiple genes ( n = 26) involved in mitochondrial respiration. (A,C) Are cluster diagrams comparing the relative expression of mitochondria-related genes between animals exposed to vehicle ( n = 5), GWI-related chemicals (GWIR-Cs), and stress ( n = 4), which was measured 6 months after exposure using quantitative real-time polymerase chain reaction. Bar charts in B1–B9,C,D1–D5,E1,E2 illustrate the elevated expression of genes involved in mitochondrial complex I ( Ndufa8, Ndufa9, Ndufa11, Ndufb3, Ndufb5, Ndufs6, Ndufs7, Ndufs8 , and Ndufv1 ; B1–B9 ), complex II ( Sdhb ; C ), complex IV ( Cox5b, Cox6a1, Cox7a2L, Surf1 , and Cyc1 ; D1–D5 ), and complex V ( Atp5a1 and Atp5b ; E1,E2 ) of GWI-rats. ∗ p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    The hippocampus of rats with chronic gulf war illness-like symptoms (GWI-rats) presented an elevated expression of multiple oxidative stress-response genes. (A) Cluster diagram comparing the relative expression of genes that classically respond to enhanced oxidative stress, between animals exposed to vehicle ( n = 5), GWI-related chemicals (GWIR-Cs), and stress ( n = 5) when measured with 6 months after exposure using quantitative real-time polymerase chain reaction. Bar charts in B1–B27 illustrate the elevated expression of 27 oxidative stress-response genes in animals exposed to GWIR-Cs and stress. ∗ p
    Figure Legend Snippet: The hippocampus of rats with chronic gulf war illness-like symptoms (GWI-rats) presented an elevated expression of multiple oxidative stress-response genes. (A) Cluster diagram comparing the relative expression of genes that classically respond to enhanced oxidative stress, between animals exposed to vehicle ( n = 5), GWI-related chemicals (GWIR-Cs), and stress ( n = 5) when measured with 6 months after exposure using quantitative real-time polymerase chain reaction. Bar charts in B1–B27 illustrate the elevated expression of 27 oxidative stress-response genes in animals exposed to GWIR-Cs and stress. ∗ p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    36) Product Images from "CTCF orchestrates the germinal centre transcriptional program and prevents premature plasma cell differentiation"

    Article Title: CTCF orchestrates the germinal centre transcriptional program and prevents premature plasma cell differentiation

    Journal: Nature Communications

    doi: 10.1038/ncomms16067

    In vitro B cell stimulation through T cells recapitulates the GC reaction. RNA-seq analysis was performed in triplicate samples from Fas + GL7 + GC B cells from Peyer’s Patches, from sorted hCD2 + B cells from control mice stimulated for 48 h in CD3/CD28 T–B co-cultures or from hCD2 + B cells from wild-type mice stimulated for 48 h with LPS/IL4. Pairwise comparison of each data set with RNA-seq data from resting B cells is depicted as GC, CD3/CD28 and LPS/IL4, respectively. ( a , b ) Pairwise comparison of each data set with RNA-seq data from resting (R) B cells are depicted as GCvsR, CD3vsR and LPSvsR, respectively. Overlaps of downregulated ( a ) or upregulated ( b ) genes in all three conditions are depicted as Venn diagrams. Percentages of the common genes between GC and CD3/CD28 samples (blue dotted areas) or between GC and LPS/IL4 (white-dotted areas) are shown as bar graphs on the right. Statistical analyses were done with two-tailed χ 2 analysis. P
    Figure Legend Snippet: In vitro B cell stimulation through T cells recapitulates the GC reaction. RNA-seq analysis was performed in triplicate samples from Fas + GL7 + GC B cells from Peyer’s Patches, from sorted hCD2 + B cells from control mice stimulated for 48 h in CD3/CD28 T–B co-cultures or from hCD2 + B cells from wild-type mice stimulated for 48 h with LPS/IL4. Pairwise comparison of each data set with RNA-seq data from resting B cells is depicted as GC, CD3/CD28 and LPS/IL4, respectively. ( a , b ) Pairwise comparison of each data set with RNA-seq data from resting (R) B cells are depicted as GCvsR, CD3vsR and LPSvsR, respectively. Overlaps of downregulated ( a ) or upregulated ( b ) genes in all three conditions are depicted as Venn diagrams. Percentages of the common genes between GC and CD3/CD28 samples (blue dotted areas) or between GC and LPS/IL4 (white-dotted areas) are shown as bar graphs on the right. Statistical analyses were done with two-tailed χ 2 analysis. P

    Techniques Used: In Vitro, Cell Stimulation, RNA Sequencing Assay, Mouse Assay, Two Tailed Test

    Activation context determines the requirement of activated B cells for CTCF. ( a ) hCD2 expression in spleen B cells from CTCF fl/+ and CTCF fl/fl mice after 48 and 72 h of LPS/IL4 stimulation. ( b ) Quantification of hCD2 + cells from CTCF fl/+ and CTCF fl/fl mice after 48, 72 and 96 h of LPS/IL4 stimulation. n (CTCF fl/+ )=11; n (CTCF fl/fl )=10. ( c ) Western blot analysis of CTCF in isolated hCD2 + B cells from CTCF fl/+ and CTCF fl/fl mice after 72 h of LPS/IL4 stimulation. CTCF amount normalized to GAPDH is shown underneath. ( d ) CTCF mRNA quantification by qRT-PCR ( P =0.0283) and by RNA-seq ( P =0.0003). ( e ) hCD2 expression in spleen B cells from CTCF fl/+ and CTCF fl/fl mice after 48 and 72 h of stimulation in CD3/CD28 T–B co-cultures. Histograms show hCD2 expression gated on B220+ B cells. ( f ) Quantification of hCD2 + cells from CTCF fl/+ and CTCF fl/fl mice after 48, 72 and 96 h of CD3/CD28 T-cell stimulation. Percentages of hCD2+ cells gated on B220+ B cells are shown. n (CTCF fl/+ )=7; n (CTCF fl/fl )=7. P (72 h)=0.0438; P (96 h)=0.0187. ( g ) Western blot analysis of CTCF in isolated hCD2 + B cells from CTCF fl/+ and CTCF fl/fl mice after 72 h of stimulation with CD3/CD28 and T cells. CTCF amount normalized to GAPDH is shown underneath. ( h ) mRNA CTCF quantification by qRT-PCR ( P =0.0059) and by RNA-seq ( P =0.0002). Mean values (a and e) ±s.d. are shown. CTCF fl/+ , white dots; CTCF fl/fl ; black dots. Statistical analysis was done with two-tailed unpaired Student’s t -test.
    Figure Legend Snippet: Activation context determines the requirement of activated B cells for CTCF. ( a ) hCD2 expression in spleen B cells from CTCF fl/+ and CTCF fl/fl mice after 48 and 72 h of LPS/IL4 stimulation. ( b ) Quantification of hCD2 + cells from CTCF fl/+ and CTCF fl/fl mice after 48, 72 and 96 h of LPS/IL4 stimulation. n (CTCF fl/+ )=11; n (CTCF fl/fl )=10. ( c ) Western blot analysis of CTCF in isolated hCD2 + B cells from CTCF fl/+ and CTCF fl/fl mice after 72 h of LPS/IL4 stimulation. CTCF amount normalized to GAPDH is shown underneath. ( d ) CTCF mRNA quantification by qRT-PCR ( P =0.0283) and by RNA-seq ( P =0.0003). ( e ) hCD2 expression in spleen B cells from CTCF fl/+ and CTCF fl/fl mice after 48 and 72 h of stimulation in CD3/CD28 T–B co-cultures. Histograms show hCD2 expression gated on B220+ B cells. ( f ) Quantification of hCD2 + cells from CTCF fl/+ and CTCF fl/fl mice after 48, 72 and 96 h of CD3/CD28 T-cell stimulation. Percentages of hCD2+ cells gated on B220+ B cells are shown. n (CTCF fl/+ )=7; n (CTCF fl/fl )=7. P (72 h)=0.0438; P (96 h)=0.0187. ( g ) Western blot analysis of CTCF in isolated hCD2 + B cells from CTCF fl/+ and CTCF fl/fl mice after 72 h of stimulation with CD3/CD28 and T cells. CTCF amount normalized to GAPDH is shown underneath. ( h ) mRNA CTCF quantification by qRT-PCR ( P =0.0059) and by RNA-seq ( P =0.0002). Mean values (a and e) ±s.d. are shown. CTCF fl/+ , white dots; CTCF fl/fl ; black dots. Statistical analysis was done with two-tailed unpaired Student’s t -test.

    Techniques Used: Activation Assay, Expressing, Mouse Assay, Western Blot, Isolation, Quantitative RT-PCR, RNA Sequencing Assay, Cell Stimulation, Two Tailed Test

    CTCF deficiency recapitulates key features of plasma cells. ( a ) FACS cell-cycle analysis of hCD2 + cells from CTCF fl/+ ( n =4) and CTCF fl/fl ( n =5) mice after 48 h of stimulation with CD3/CD28 and T cells. Numbers indicate percentages ±s.d. Quantification of G1 and S/G2 phase proportions is shown on the right. p(G1)=0.0242; p(S/G2)=0.0221, two-tailed Student’s t -test. ( b ) Two-dimension expression plot. X axis, log2 fold values of significantly changed genes upon CTCF deficiency (CTCF fl/fl versus CTCF fl/+ , CD3/CD28-stimulated B cells). Y axis, log2 fold values of significantly changed genes in PC versus GC B cells (data extracted from ref. 33 . Coloured dots highlight genes of the PC differentiation programme. ( c ) Quantification of significantly changed cell-cycle-related genes. Log fold change (LogFC) in PC versus GC B cells (purple) and in CTCF fl/fl versus CTCF fl/+ CD3/CD28 T-stimulated B cells (orange). ( d ) RNA-seq analysis of CTCF expression in GC B cells and PC (data extracted from ref. 33 ). ( e ) qRT-PCR analysis of Blimp-1 ( Prdm1 ) and Bcl-6 expression in CD3/CD28 T-stimulated CTCF fl/+ ( n =12) and CTCF fl/fl ( n =9) hCD2 + cells. P (Blimp-1)
    Figure Legend Snippet: CTCF deficiency recapitulates key features of plasma cells. ( a ) FACS cell-cycle analysis of hCD2 + cells from CTCF fl/+ ( n =4) and CTCF fl/fl ( n =5) mice after 48 h of stimulation with CD3/CD28 and T cells. Numbers indicate percentages ±s.d. Quantification of G1 and S/G2 phase proportions is shown on the right. p(G1)=0.0242; p(S/G2)=0.0221, two-tailed Student’s t -test. ( b ) Two-dimension expression plot. X axis, log2 fold values of significantly changed genes upon CTCF deficiency (CTCF fl/fl versus CTCF fl/+ , CD3/CD28-stimulated B cells). Y axis, log2 fold values of significantly changed genes in PC versus GC B cells (data extracted from ref. 33 . Coloured dots highlight genes of the PC differentiation programme. ( c ) Quantification of significantly changed cell-cycle-related genes. Log fold change (LogFC) in PC versus GC B cells (purple) and in CTCF fl/fl versus CTCF fl/+ CD3/CD28 T-stimulated B cells (orange). ( d ) RNA-seq analysis of CTCF expression in GC B cells and PC (data extracted from ref. 33 ). ( e ) qRT-PCR analysis of Blimp-1 ( Prdm1 ) and Bcl-6 expression in CD3/CD28 T-stimulated CTCF fl/+ ( n =12) and CTCF fl/fl ( n =9) hCD2 + cells. P (Blimp-1)

    Techniques Used: FACS, Cell Cycle Assay, Mouse Assay, Two Tailed Test, Expressing, RNA Sequencing Assay, Quantitative RT-PCR

    CTCF transcriptionally regulates B cell signalling and cell cycle. RNA-seq analysis was performed in triplicates from hCD2 + sorted B cells from CTCF fl/fl and CTCF fl/+ mice after 48 h stimulation in the presence of CD3/CD28 T or LPS/IL4. Differentially expressed genes (adjusted P
    Figure Legend Snippet: CTCF transcriptionally regulates B cell signalling and cell cycle. RNA-seq analysis was performed in triplicates from hCD2 + sorted B cells from CTCF fl/fl and CTCF fl/+ mice after 48 h stimulation in the presence of CD3/CD28 T or LPS/IL4. Differentially expressed genes (adjusted P

    Techniques Used: RNA Sequencing Assay, Mouse Assay

    37) Product Images from "The therapeutic properties of resminostat for hepatocellular carcinoma"

    Article Title: The therapeutic properties of resminostat for hepatocellular carcinoma

    Journal: Oncoscience

    doi: 10.18632/oncoscience.420

    Resminostat reduced metastatic progression in HCC under SGBS CM treatment (A) Hep3B cells were cultured in 30% SGBS CM alone or in combination with Resminostat (80 nM) for 24 hours and their total RNAs were extracted for gene analysis with RT-PCR. A panel of oncogenes including VEGF, leptin, STAT3, TNFα, p16 and IL-10 were measured at the RNA level (n = 3). (B) Hep3B cells were cultured in 30% SGBS CM alone or in combination with Resminostat (80 nM) for 24 hours. Cells were then fixed and stained with primary antibody (anti-Vimentin mouse monoclonal) and secondary antibody (anti-mouse green immunofluorescence). Scale bar = 20 μm.
    Figure Legend Snippet: Resminostat reduced metastatic progression in HCC under SGBS CM treatment (A) Hep3B cells were cultured in 30% SGBS CM alone or in combination with Resminostat (80 nM) for 24 hours and their total RNAs were extracted for gene analysis with RT-PCR. A panel of oncogenes including VEGF, leptin, STAT3, TNFα, p16 and IL-10 were measured at the RNA level (n = 3). (B) Hep3B cells were cultured in 30% SGBS CM alone or in combination with Resminostat (80 nM) for 24 hours. Cells were then fixed and stained with primary antibody (anti-Vimentin mouse monoclonal) and secondary antibody (anti-mouse green immunofluorescence). Scale bar = 20 μm.

    Techniques Used: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Staining, Immunofluorescence

    38) Product Images from "Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule"

    Article Title: Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule

    Journal: Journal of Medicinal Chemistry

    doi: 10.1021/acs.jmedchem.7b01781

    Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC
    Figure Legend Snippet: Differentially down-regulated genes common to both PANC-1 and MIA PaCa-2 are enriched in PQs after treatment with 400 nM CM03. (a,b) MIA PaCa-2 and PANC-1 cells were treated with 400 nM CM03 for 6 and 24 h and mRNA extracted for analysis by RNA-Seq. Genes were split into four subgroups according to their fold change upon CM03 treatment versus untreated: Down (Log 2 FC

    Techniques Used: RNA Sequencing Assay

    Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p
    Figure Legend Snippet: Validation of mRNA down regulation by qRT-PCR for a subset of down-regulated genes, selected from RNA-Seq experiments. (a–d) MIA PaCa-2 and PANC-1 cells were treated (a and b) with 400 nM CM03 and (c and d) with 400 nM gemcitabine, all for 6 and 24 h. Total mRNA was extracted, reverse transcribed into cDNA, and then qRT-PCR was performed. The C t values were normalized to the genomic mean of three housekeeping genes ( ACTB , GAPDH , and TUBB ), and the relative gene expression was determined using the Livak method, 2 –ΔΔ C t . The log-fold expression changes (Log 2 FC) for each gene are shown relative to vehicle-treated controls (PBS for CM03 and DMSO for gemcitabine). Student’s t test along with 2 –Δ C t values were used to determine the statistical significance of the observed changes, which are the mean of in each case at least three determinations. Those genes with changes in expression with p

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

    39) Product Images from "Cigarette smoke promotes HIV infection of primary bronchial epithelium and additively suppresses CFTR function"

    Article Title: Cigarette smoke promotes HIV infection of primary bronchial epithelium and additively suppresses CFTR function

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26095-z

    Effect of CS exposure on CCR5 Cell surface receptor quantified by Single Cell Imaging flow cytometry. NHBE ALI cultures were exposed to CS. At the end of smoking, cells were immuno-stained with surface markers anti-human CCR5 labeled with APC. Percent Gated CCR5+ or APC+ cells for air NHBE (8.58 ± 2.85%) and smoke NHBE (20.25 ± 4.5%, P = 0.03) are represented (panel a). MFI of APC from in total population of cell images acquired (10,000 per sample) for air NHBE cells (818.74 ± 274.02) and smoked NHBE cells (1494.16 ± 447.95) are represented (panel b). Representative overlay histogram of intensity of APC for isotype control (yellow), air (blue) and smoke (green) (panel c). A representative single cell image for each sample is shown (panel d). Experiments were carried out from at least 3 different lungs. *Significant (p
    Figure Legend Snippet: Effect of CS exposure on CCR5 Cell surface receptor quantified by Single Cell Imaging flow cytometry. NHBE ALI cultures were exposed to CS. At the end of smoking, cells were immuno-stained with surface markers anti-human CCR5 labeled with APC. Percent Gated CCR5+ or APC+ cells for air NHBE (8.58 ± 2.85%) and smoke NHBE (20.25 ± 4.5%, P = 0.03) are represented (panel a). MFI of APC from in total population of cell images acquired (10,000 per sample) for air NHBE cells (818.74 ± 274.02) and smoked NHBE cells (1494.16 ± 447.95) are represented (panel b). Representative overlay histogram of intensity of APC for isotype control (yellow), air (blue) and smoke (green) (panel c). A representative single cell image for each sample is shown (panel d). Experiments were carried out from at least 3 different lungs. *Significant (p

    Techniques Used: Cell Surface Receptor Assay, Imaging, Flow Cytometry, Cytometry, Staining, Labeling

    CS increases expression of HIV receptors CD4 and CCR5 in NHBE ALI cultures. NHBE ALI cultures were exposed to CS and total protein was extracted. Cells were lysed with RIPA buffer containing protease inhibitor cocktail and protein expression was quantified by Western blot analysis and normalized using β-actin. CS significantly enhances expression of CD4 (panel a) and CCR5 protein expression (panel b) when compared to air-exposed controls. CS does not increase CXCR4 protein expression (panel c). Western blot images ( a – c ) are representative of NHBE ALI cultures from three independent lungs. Relative density of the detected protein band was measured by using the ImageJ software and the values obtained were averaged. n = 3 lungs (unless stated otherwise). *Significant (p
    Figure Legend Snippet: CS increases expression of HIV receptors CD4 and CCR5 in NHBE ALI cultures. NHBE ALI cultures were exposed to CS and total protein was extracted. Cells were lysed with RIPA buffer containing protease inhibitor cocktail and protein expression was quantified by Western blot analysis and normalized using β-actin. CS significantly enhances expression of CD4 (panel a) and CCR5 protein expression (panel b) when compared to air-exposed controls. CS does not increase CXCR4 protein expression (panel c). Western blot images ( a – c ) are representative of NHBE ALI cultures from three independent lungs. Relative density of the detected protein band was measured by using the ImageJ software and the values obtained were averaged. n = 3 lungs (unless stated otherwise). *Significant (p

    Techniques Used: Expressing, Protease Inhibitor, Western Blot, Software

    Effect of CS exposure on CD4 Cell surface receptor quantified by single cell imaging flow cytometry. NHBE ALI cultures were exposed to CS. At the end of smoking, cells were immuno-stained with surface markers anti-Human CD4 labeled with FITC. % Gated CD4+ or FITC+ cells for air NHBE (22.71 ± 2.44%) and smoke NHBE (32.78 ± 2.71%) are represented (panel a). MFI of FITC from in total population of cell images acquired (10,000 per sample) for air NHBE cells (1597.65 ± 633.43) and smoked NHBE cells (3208.24 ± 1030.86) are represented (panel b). A representative overlay histogram of intensity of FITC for isotype control (yellow), air (blue) and smoke (green) (panel c). A representative single cell image for each sample (panel d). Experiments were carried out from at least 3 different lungs. *Significant (p
    Figure Legend Snippet: Effect of CS exposure on CD4 Cell surface receptor quantified by single cell imaging flow cytometry. NHBE ALI cultures were exposed to CS. At the end of smoking, cells were immuno-stained with surface markers anti-Human CD4 labeled with FITC. % Gated CD4+ or FITC+ cells for air NHBE (22.71 ± 2.44%) and smoke NHBE (32.78 ± 2.71%) are represented (panel a). MFI of FITC from in total population of cell images acquired (10,000 per sample) for air NHBE cells (1597.65 ± 633.43) and smoked NHBE cells (3208.24 ± 1030.86) are represented (panel b). A representative overlay histogram of intensity of FITC for isotype control (yellow), air (blue) and smoke (green) (panel c). A representative single cell image for each sample (panel d). Experiments were carried out from at least 3 different lungs. *Significant (p

    Techniques Used: Cell Surface Receptor Assay, Imaging, Flow Cytometry, Cytometry, Staining, Labeling

    CS increases CCR5 expression by TGF-β mediated suppression of miR-141-5p. NHBE ALI cultures were exposed to CS. Total RNA was extracted and analyzed to quantify CCR5 mRNA expression by qRT-PCR. CS enhances CCR5 mRNA levels (panel a). Lung and age-matched NHBE ALI cultures were treated with recombinant TGF-β1(10 ng/ml apically and basolaterally). After 20 hours, cells were lysed and total RNA was extracted and analyzed for CCR5 mRNA expression by qRT-PCR. TGF-β1 increases CCR5 expression comparable to that observed in CS exposed cells (panel b). Total RNA was analyzed for expression of miR-141-5p from CS exposed and TGF-β treated cells by qRT-PCR. We found the miR-141-5p expression is suppressed by CS as well as TGF-β1 treatment (panels c,d). n = 3 different lungs. *Significant (p
    Figure Legend Snippet: CS increases CCR5 expression by TGF-β mediated suppression of miR-141-5p. NHBE ALI cultures were exposed to CS. Total RNA was extracted and analyzed to quantify CCR5 mRNA expression by qRT-PCR. CS enhances CCR5 mRNA levels (panel a). Lung and age-matched NHBE ALI cultures were treated with recombinant TGF-β1(10 ng/ml apically and basolaterally). After 20 hours, cells were lysed and total RNA was extracted and analyzed for CCR5 mRNA expression by qRT-PCR. TGF-β1 increases CCR5 expression comparable to that observed in CS exposed cells (panel b). Total RNA was analyzed for expression of miR-141-5p from CS exposed and TGF-β treated cells by qRT-PCR. We found the miR-141-5p expression is suppressed by CS as well as TGF-β1 treatment (panels c,d). n = 3 different lungs. *Significant (p

    Techniques Used: Expressing, Quantitative RT-PCR, Recombinant

    CS enhances replication of R5-tropic HIV. NHBE ALI cultures were exposed to CS prior to infecting apically and basolaterally with 5 ng p24 equivalent of either R5-tropic (HIV BaL) or X4-tropic (HIV IIIB) virus. After 16 hours cells were washed apically and basolaterally with PBS four times to remove any residual input virus. The fourth wash was collected for p24 analysis and measured as day 0 to confirm that all input virus had been removed. On day 3, culture supernatant was collected and analyzed for p24. HIV increases infection of NHBE ALI cultures by the R5-tropic HIV BaL strain (panel a) but not by X4-tropic IIIB strain (panel b). NHBE ALI cultures were infected with single cycle R5-tropic RGH virus. Cells were washed four times with PBS and infection was allowed to proceed. Three days post-infection, experiments were terminated and total DNA was quantitated by qPCR as an index of viral entry. CS increases HIV entry in NHBE ALI cultures (panel c). n = 3 different lungs *significant (p
    Figure Legend Snippet: CS enhances replication of R5-tropic HIV. NHBE ALI cultures were exposed to CS prior to infecting apically and basolaterally with 5 ng p24 equivalent of either R5-tropic (HIV BaL) or X4-tropic (HIV IIIB) virus. After 16 hours cells were washed apically and basolaterally with PBS four times to remove any residual input virus. The fourth wash was collected for p24 analysis and measured as day 0 to confirm that all input virus had been removed. On day 3, culture supernatant was collected and analyzed for p24. HIV increases infection of NHBE ALI cultures by the R5-tropic HIV BaL strain (panel a) but not by X4-tropic IIIB strain (panel b). NHBE ALI cultures were infected with single cycle R5-tropic RGH virus. Cells were washed four times with PBS and infection was allowed to proceed. Three days post-infection, experiments were terminated and total DNA was quantitated by qPCR as an index of viral entry. CS increases HIV entry in NHBE ALI cultures (panel c). n = 3 different lungs *significant (p

    Techniques Used: Infection, Real-time Polymerase Chain Reaction

    CS and HIV individually and additively suppress CFTR biogenesis and function. NHBE ALI cultures were infected with either R5- (HIV BaL) or X4- (HIV IIIB) tropic strain of HIV and exposed to chronic smoke exposure on days 0, 3, 6 and 9 (air as control). On day 9, total RNA was analyzed for CFTR mRNA by qRT-PCR. Another set of age and lung-matched cultures grown on snapwells were treated similarly and mounted in Ussing chambers on day 9. Cells were mounted in Ussing chambers and Cl - efflux in response to albuterol addition was determined in the presence of amiloride as reported by us earlier 5 . HIV infection and CS individually and additively suppresses CFTR mRNA and function (panels a,b). n = 3 lungs (unless stated otherwise) *significant; S = significant from each other (p
    Figure Legend Snippet: CS and HIV individually and additively suppress CFTR biogenesis and function. NHBE ALI cultures were infected with either R5- (HIV BaL) or X4- (HIV IIIB) tropic strain of HIV and exposed to chronic smoke exposure on days 0, 3, 6 and 9 (air as control). On day 9, total RNA was analyzed for CFTR mRNA by qRT-PCR. Another set of age and lung-matched cultures grown on snapwells were treated similarly and mounted in Ussing chambers on day 9. Cells were mounted in Ussing chambers and Cl - efflux in response to albuterol addition was determined in the presence of amiloride as reported by us earlier 5 . HIV infection and CS individually and additively suppresses CFTR mRNA and function (panels a,b). n = 3 lungs (unless stated otherwise) *significant; S = significant from each other (p

    Techniques Used: Infection, Quantitative RT-PCR

    40) Product Images from "Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines"

    Article Title: Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines

    Journal: Viruses

    doi: 10.3390/v10040145

    The LMP2 and GLuc detection result in TC-1-GLUC-LMP2 cells. ( A ) PCR results of LMP2 and GLuc amplification using the TC-1-GLUC-LMP2 cells genomic DNA as a template. M: DNA ladder; 1: TC-1-GLUC-LMP2 cells as template using primers for LMP2 ; 2: DNA of pVR-LMP2 plasmid; 3: genomic DNA of TC-1 cells; 4: TC-1-GLUC-LMP2 cells as template using primers for GLuc; 5: DNA of pCMV-Gaussia Luc; 6: genomic DNA of TC-1 cells; ( B ) RT-PCR results to verify GLuc and LMP2 mRNA expression. M: RNA ladder; 1: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying LMP2 ; 2: 293 cells transfected with pVR-LMP2 as template amplifying LMP2 ; 3: TC-1 cells as template amplifying LMP2; 4: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying GLuc ; 5: 293 cells transfected with pCMV-Gaussia Luc as template amplifying GLuc ; 6: TC-1 cells as template amplifying GLuc ; ( C ) Western blot results of LMP2 protein expression in TC-1-GLUC-LMP2 cells at different passages. M: PageRuler Prestained Protein Ladder; 1: Passage 1 TC-1-GLUC-LMP2 cells, LMP2 with a molecular weight of 55 KD; 2: Passage 10 TC-1-GLUC-LMP2 cells; 3: Passage 30 TC-1-GLUC-LMP2 cells; 4: TC-1 cells; ( D ) Identifying the stable expression of GLuc . The mean relative light unit (RLU) value of 10 4 TC-1-GLUC-LMP2 cells at passages 1, 10 and 30 were 7.44 × 10 5 , 7.63 × 10 5 and 7.34 × 10 5 . NS, no significant difference; each column represents mean ± SD ( n = 5) in ( D ).
    Figure Legend Snippet: The LMP2 and GLuc detection result in TC-1-GLUC-LMP2 cells. ( A ) PCR results of LMP2 and GLuc amplification using the TC-1-GLUC-LMP2 cells genomic DNA as a template. M: DNA ladder; 1: TC-1-GLUC-LMP2 cells as template using primers for LMP2 ; 2: DNA of pVR-LMP2 plasmid; 3: genomic DNA of TC-1 cells; 4: TC-1-GLUC-LMP2 cells as template using primers for GLuc; 5: DNA of pCMV-Gaussia Luc; 6: genomic DNA of TC-1 cells; ( B ) RT-PCR results to verify GLuc and LMP2 mRNA expression. M: RNA ladder; 1: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying LMP2 ; 2: 293 cells transfected with pVR-LMP2 as template amplifying LMP2 ; 3: TC-1 cells as template amplifying LMP2; 4: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying GLuc ; 5: 293 cells transfected with pCMV-Gaussia Luc as template amplifying GLuc ; 6: TC-1 cells as template amplifying GLuc ; ( C ) Western blot results of LMP2 protein expression in TC-1-GLUC-LMP2 cells at different passages. M: PageRuler Prestained Protein Ladder; 1: Passage 1 TC-1-GLUC-LMP2 cells, LMP2 with a molecular weight of 55 KD; 2: Passage 10 TC-1-GLUC-LMP2 cells; 3: Passage 30 TC-1-GLUC-LMP2 cells; 4: TC-1 cells; ( D ) Identifying the stable expression of GLuc . The mean relative light unit (RLU) value of 10 4 TC-1-GLUC-LMP2 cells at passages 1, 10 and 30 were 7.44 × 10 5 , 7.63 × 10 5 and 7.34 × 10 5 . NS, no significant difference; each column represents mean ± SD ( n = 5) in ( D ).

    Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Western Blot, Molecular Weight

    Comparing the TC-1-GLUC-LMP2 and TC-1 growth curves. TC-1-GLUC-LMP2 and TC-1 cells (5 × 10 3 ) were added to E-plate L 8 plates, and cell index (CI) was measured by iCELLigence. After 120 h, both cell lines grew to 2.1 × 10 5 /cm 2 . The growth rate and adhesion were not significantly different.
    Figure Legend Snippet: Comparing the TC-1-GLUC-LMP2 and TC-1 growth curves. TC-1-GLUC-LMP2 and TC-1 cells (5 × 10 3 ) were added to E-plate L 8 plates, and cell index (CI) was measured by iCELLigence. After 120 h, both cell lines grew to 2.1 × 10 5 /cm 2 . The growth rate and adhesion were not significantly different.

    Techniques Used:

    Detecting subcutaneously injected TC-1-GLUC-LMP2 cells. ( A ) The tumor in vivo was observed by the in vivo imaging system (IVIS) at 3 days post-inoculation; ( B ) The tumor was observed by the IVIS at 7 days post-inoculation; ( C ) The tumor was observed by the IVIS at 14 days post-inoculation; ( D ) IVIS results showed that the mean number of photons in the mice were 2.43 × 10 4 , 9.67 × 10 4 and 7.09 × 10 5 at three, seven, and 14 days post-inoculation, respectively; ( E ) The tumor sizes and weights of mice were recorded at 14 days after inoculation with the tumor cells; the average tumor weight and volume of the tumor-bearing mice were 0.41 g and 220 mm 3 , respectively; ( F ) Immunohistochemistry results for TC-1-GLUC-LMP2 tumors showed nuclei (blue) and cytoplasm (reddish brown), indicating that EBV LMP2 was well expressed; ( G ) Immunohistochemistry results for normal muscle tissue of mice; ( H ) HE staining results of TC-1-GLUC-LMP2 tumors showed that nuclei became larger (blue), while the cytoplasm shrank or even disappeared (pink); ( I ) HE staining results of normal muscle tissue of mice. # means the number of each mouse. *** p
    Figure Legend Snippet: Detecting subcutaneously injected TC-1-GLUC-LMP2 cells. ( A ) The tumor in vivo was observed by the in vivo imaging system (IVIS) at 3 days post-inoculation; ( B ) The tumor was observed by the IVIS at 7 days post-inoculation; ( C ) The tumor was observed by the IVIS at 14 days post-inoculation; ( D ) IVIS results showed that the mean number of photons in the mice were 2.43 × 10 4 , 9.67 × 10 4 and 7.09 × 10 5 at three, seven, and 14 days post-inoculation, respectively; ( E ) The tumor sizes and weights of mice were recorded at 14 days after inoculation with the tumor cells; the average tumor weight and volume of the tumor-bearing mice were 0.41 g and 220 mm 3 , respectively; ( F ) Immunohistochemistry results for TC-1-GLUC-LMP2 tumors showed nuclei (blue) and cytoplasm (reddish brown), indicating that EBV LMP2 was well expressed; ( G ) Immunohistochemistry results for normal muscle tissue of mice; ( H ) HE staining results of TC-1-GLUC-LMP2 tumors showed that nuclei became larger (blue), while the cytoplasm shrank or even disappeared (pink); ( I ) HE staining results of normal muscle tissue of mice. # means the number of each mouse. *** p

    Techniques Used: Injection, In Vivo, In Vivo Imaging, Mouse Assay, Immunohistochemistry, Staining

    TC-1-GLUC-LMP2 tumor challenge with LMP2-associated vaccine in vivo. ( A ) The number of tumor photons in the vaccine-NULL or vaccine-LMP2-immunized mice were detected at three, seven, 14 days with the in vivo imaging system (IVIS); ( B ) The LMP2-specific immune response was analyzed by ELISPOT after TC-1-GLUC-LMP2 tumor cells inoculation 14 days. *** p
    Figure Legend Snippet: TC-1-GLUC-LMP2 tumor challenge with LMP2-associated vaccine in vivo. ( A ) The number of tumor photons in the vaccine-NULL or vaccine-LMP2-immunized mice were detected at three, seven, 14 days with the in vivo imaging system (IVIS); ( B ) The LMP2-specific immune response was analyzed by ELISPOT after TC-1-GLUC-LMP2 tumor cells inoculation 14 days. *** p

    Techniques Used: In Vivo, Mouse Assay, In Vivo Imaging, Enzyme-linked Immunospot

    Schematic demonstrating TC-1-GLUC-LMP2 cell construction. Latent membrane protein 2 ( LMP2 ) genes, internal ribosome entry site ( IRES ) and Gaussia luciferase ( GLuc ) sequences were inserted into TC-1 cells.
    Figure Legend Snippet: Schematic demonstrating TC-1-GLUC-LMP2 cell construction. Latent membrane protein 2 ( LMP2 ) genes, internal ribosome entry site ( IRES ) and Gaussia luciferase ( GLuc ) sequences were inserted into TC-1 cells.

    Techniques Used: Luciferase

    Flow cytometry detection results. ( A ) The proportions of fluorescein isothiocyanate (FITC)-labeled cells in TC-1 was 0.2%; ( B ) The proportions of FITC-labeled cells in TC-1-GLUC-LMP2 was 98.7%. FITC was fluorescein isothiocyanate. The red area represented the detected cells.
    Figure Legend Snippet: Flow cytometry detection results. ( A ) The proportions of fluorescein isothiocyanate (FITC)-labeled cells in TC-1 was 0.2%; ( B ) The proportions of FITC-labeled cells in TC-1-GLUC-LMP2 was 98.7%. FITC was fluorescein isothiocyanate. The red area represented the detected cells.

    Techniques Used: Flow Cytometry, Cytometry, Labeling

    Cytotoxicity result of TC-1-GLUC-LMP2 cells. The ten groups of mixed cells were inoculated on E-plate L8 plates and incubated for 100 h. Line 1: 5 × 10 3 TC-1-GLUC-LMP2 target cells; Line 2: 5 × 10 3 TC-1-GLUC-LMP2 target cells mixed with 8 × 10 5 spleens without peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 3: 5 × 10 3 target cells mixed with 8 × 10 5 spleens were spiked with LMP2-specific peptide isolated from mice immunized with vaccine-NULL; Line 4: 5 × 10 3 TC-1-GLUC-LMP2 target cells mixed with 5 × 10 4 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 5: 5 × 10 3 target cells mixed with 1 × 10 5 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 6: 5 × 10 3 target cells mixed with 2 × 10 5 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 7: 5 × 10 3 target cells mixed with 4 × 10 5 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 8: 5 × 10 3 target cells mixed with 8 × 10 5 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 9: Roswell Park Memorial Institute 1640 (RPMI 1640); Line 10: 8 × 10 5 LMP2-specific mouse spleen lymphocytes.
    Figure Legend Snippet: Cytotoxicity result of TC-1-GLUC-LMP2 cells. The ten groups of mixed cells were inoculated on E-plate L8 plates and incubated for 100 h. Line 1: 5 × 10 3 TC-1-GLUC-LMP2 target cells; Line 2: 5 × 10 3 TC-1-GLUC-LMP2 target cells mixed with 8 × 10 5 spleens without peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 3: 5 × 10 3 target cells mixed with 8 × 10 5 spleens were spiked with LMP2-specific peptide isolated from mice immunized with vaccine-NULL; Line 4: 5 × 10 3 TC-1-GLUC-LMP2 target cells mixed with 5 × 10 4 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 5: 5 × 10 3 target cells mixed with 1 × 10 5 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 6: 5 × 10 3 target cells mixed with 2 × 10 5 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 7: 5 × 10 3 target cells mixed with 4 × 10 5 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 8: 5 × 10 3 target cells mixed with 8 × 10 5 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 9: Roswell Park Memorial Institute 1640 (RPMI 1640); Line 10: 8 × 10 5 LMP2-specific mouse spleen lymphocytes.

    Techniques Used: Incubation, Isolation, Mouse Assay

    41) Product Images from "Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines"

    Article Title: Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines

    Journal: Viruses

    doi: 10.3390/v10040145

    The LMP2 and GLuc detection result in TC-1-GLUC-LMP2 cells. ( A ) PCR results of LMP2 and GLuc amplification using the TC-1-GLUC-LMP2 cells genomic DNA as a template. M: DNA ladder; 1: TC-1-GLUC-LMP2 cells as template using primers for LMP2 ; 2: DNA of pVR-LMP2 plasmid; 3: genomic DNA of TC-1 cells; 4: TC-1-GLUC-LMP2 cells as template using primers for GLuc; 5: DNA of pCMV-Gaussia Luc; 6: genomic DNA of TC-1 cells; ( B ) RT-PCR results to verify GLuc and LMP2 mRNA expression. M: RNA ladder; 1: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying LMP2 ; 2: 293 cells transfected with pVR-LMP2 as template amplifying LMP2 ; 3: TC-1 cells as template amplifying LMP2; 4: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying GLuc ; 5: 293 cells transfected with pCMV-Gaussia Luc as template amplifying GLuc ; 6: TC-1 cells as template amplifying GLuc ; ( C ) Western blot results of LMP2 protein expression in TC-1-GLUC-LMP2 cells at different passages. M: PageRuler Prestained Protein Ladder; 1: Passage 1 TC-1-GLUC-LMP2 cells, LMP2 with a molecular weight of 55 KD; 2: Passage 10 TC-1-GLUC-LMP2 cells; 3: Passage 30 TC-1-GLUC-LMP2 cells; 4: TC-1 cells; ( D ) Identifying the stable expression of GLuc . The mean relative light unit (RLU) value of 10 4 TC-1-GLUC-LMP2 cells at passages 1, 10 and 30 were 7.44 × 10 5 , 7.63 × 10 5 and 7.34 × 10 5 . NS, no significant difference; each column represents mean ± SD ( n = 5) in ( D ).
    Figure Legend Snippet: The LMP2 and GLuc detection result in TC-1-GLUC-LMP2 cells. ( A ) PCR results of LMP2 and GLuc amplification using the TC-1-GLUC-LMP2 cells genomic DNA as a template. M: DNA ladder; 1: TC-1-GLUC-LMP2 cells as template using primers for LMP2 ; 2: DNA of pVR-LMP2 plasmid; 3: genomic DNA of TC-1 cells; 4: TC-1-GLUC-LMP2 cells as template using primers for GLuc; 5: DNA of pCMV-Gaussia Luc; 6: genomic DNA of TC-1 cells; ( B ) RT-PCR results to verify GLuc and LMP2 mRNA expression. M: RNA ladder; 1: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying LMP2 ; 2: 293 cells transfected with pVR-LMP2 as template amplifying LMP2 ; 3: TC-1 cells as template amplifying LMP2; 4: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying GLuc ; 5: 293 cells transfected with pCMV-Gaussia Luc as template amplifying GLuc ; 6: TC-1 cells as template amplifying GLuc ; ( C ) Western blot results of LMP2 protein expression in TC-1-GLUC-LMP2 cells at different passages. M: PageRuler Prestained Protein Ladder; 1: Passage 1 TC-1-GLUC-LMP2 cells, LMP2 with a molecular weight of 55 KD; 2: Passage 10 TC-1-GLUC-LMP2 cells; 3: Passage 30 TC-1-GLUC-LMP2 cells; 4: TC-1 cells; ( D ) Identifying the stable expression of GLuc . The mean relative light unit (RLU) value of 10 4 TC-1-GLUC-LMP2 cells at passages 1, 10 and 30 were 7.44 × 10 5 , 7.63 × 10 5 and 7.34 × 10 5 . NS, no significant difference; each column represents mean ± SD ( n = 5) in ( D ).

    Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Western Blot, Molecular Weight

    42) Product Images from "Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines"

    Article Title: Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines

    Journal: Viruses

    doi: 10.3390/v10040145

    The LMP2 and GLuc detection result in TC-1-GLUC-LMP2 cells. ( A ) PCR results of LMP2 and GLuc amplification using the TC-1-GLUC-LMP2 cells genomic DNA as a template. M: DNA ladder; 1: TC-1-GLUC-LMP2 cells as template using primers for LMP2 ; 2: DNA of pVR-LMP2 plasmid; 3: genomic DNA of TC-1 cells; 4: TC-1-GLUC-LMP2 cells as template using primers for GLuc; 5: DNA of pCMV-Gaussia Luc; 6: genomic DNA of TC-1 cells; ( B ) RT-PCR results to verify GLuc and LMP2 mRNA expression. M: RNA ladder; 1: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying LMP2 ; 2: 293 cells transfected with pVR-LMP2 as template amplifying LMP2 ; 3: TC-1 cells as template amplifying LMP2; 4: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying GLuc ; 5: 293 cells transfected with pCMV-Gaussia Luc as template amplifying GLuc ; 6: TC-1 cells as template amplifying GLuc ; ( C ) Western blot results of LMP2 protein expression in TC-1-GLUC-LMP2 cells at different passages. M: PageRuler Prestained Protein Ladder; 1: Passage 1 TC-1-GLUC-LMP2 cells, LMP2 with a molecular weight of 55 KD; 2: Passage 10 TC-1-GLUC-LMP2 cells; 3: Passage 30 TC-1-GLUC-LMP2 cells; 4: TC-1 cells; ( D ) Identifying the stable expression of GLuc . The mean relative light unit (RLU) value of 10 4 TC-1-GLUC-LMP2 cells at passages 1, 10 and 30 were 7.44 × 10 5 , 7.63 × 10 5 and 7.34 × 10 5 . NS, no significant difference; each column represents mean ± SD ( n = 5) in ( D ).
    Figure Legend Snippet: The LMP2 and GLuc detection result in TC-1-GLUC-LMP2 cells. ( A ) PCR results of LMP2 and GLuc amplification using the TC-1-GLUC-LMP2 cells genomic DNA as a template. M: DNA ladder; 1: TC-1-GLUC-LMP2 cells as template using primers for LMP2 ; 2: DNA of pVR-LMP2 plasmid; 3: genomic DNA of TC-1 cells; 4: TC-1-GLUC-LMP2 cells as template using primers for GLuc; 5: DNA of pCMV-Gaussia Luc; 6: genomic DNA of TC-1 cells; ( B ) RT-PCR results to verify GLuc and LMP2 mRNA expression. M: RNA ladder; 1: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying LMP2 ; 2: 293 cells transfected with pVR-LMP2 as template amplifying LMP2 ; 3: TC-1 cells as template amplifying LMP2; 4: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying GLuc ; 5: 293 cells transfected with pCMV-Gaussia Luc as template amplifying GLuc ; 6: TC-1 cells as template amplifying GLuc ; ( C ) Western blot results of LMP2 protein expression in TC-1-GLUC-LMP2 cells at different passages. M: PageRuler Prestained Protein Ladder; 1: Passage 1 TC-1-GLUC-LMP2 cells, LMP2 with a molecular weight of 55 KD; 2: Passage 10 TC-1-GLUC-LMP2 cells; 3: Passage 30 TC-1-GLUC-LMP2 cells; 4: TC-1 cells; ( D ) Identifying the stable expression of GLuc . The mean relative light unit (RLU) value of 10 4 TC-1-GLUC-LMP2 cells at passages 1, 10 and 30 were 7.44 × 10 5 , 7.63 × 10 5 and 7.34 × 10 5 . NS, no significant difference; each column represents mean ± SD ( n = 5) in ( D ).

    Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Western Blot, Molecular Weight

    43) Product Images from "CAPN1 is a novel binding partner and regulator of the tumor suppressor NF1 in melanoma"

    Article Title: CAPN1 is a novel binding partner and regulator of the tumor suppressor NF1 in melanoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25805

    NF1 mutant and wild-type cell lines show enhanced sensitivity to combined CAPN1 and MEKi inhibition (A) Representative dose response curves were generated using NF1 mutant cell lines (108T and 76T) treated with constant concentration of Calpain inhibitor I (6 μM) and increasing concentrations of Trametinib (1 pM - 10 μM) for 72 hours before assessing viability by Cell Titer-Glo Luminescent Cell viability assay (n=3). The relative cell number after cells were treated with Calpain inhibitor I and Trametinib is plotted as percent survival, as compared to Trametinib-treated control, versus log Trametinib concentration in pM. (B) Representative dose response curves were generated using NF1 wild-type cell lines (A375 and 74T) treated with constant concentration of Calpain inhibitor I (6 μM and 4 μM, respectively) and increasing concentrations of Trametinib (1 pM - 10 μM) for 72 hours before assessing viability by Cell Titer-Glo Luminescent Cell viability assay (n=3). The relative cell number after cells were treated with Calpain inhibitor I and Trametinib is plotted as percent survival, as compared to Trametinib-treated control, versus log Trametinib concentration in pM. ** P
    Figure Legend Snippet: NF1 mutant and wild-type cell lines show enhanced sensitivity to combined CAPN1 and MEKi inhibition (A) Representative dose response curves were generated using NF1 mutant cell lines (108T and 76T) treated with constant concentration of Calpain inhibitor I (6 μM) and increasing concentrations of Trametinib (1 pM - 10 μM) for 72 hours before assessing viability by Cell Titer-Glo Luminescent Cell viability assay (n=3). The relative cell number after cells were treated with Calpain inhibitor I and Trametinib is plotted as percent survival, as compared to Trametinib-treated control, versus log Trametinib concentration in pM. (B) Representative dose response curves were generated using NF1 wild-type cell lines (A375 and 74T) treated with constant concentration of Calpain inhibitor I (6 μM and 4 μM, respectively) and increasing concentrations of Trametinib (1 pM - 10 μM) for 72 hours before assessing viability by Cell Titer-Glo Luminescent Cell viability assay (n=3). The relative cell number after cells were treated with Calpain inhibitor I and Trametinib is plotted as percent survival, as compared to Trametinib-treated control, versus log Trametinib concentration in pM. ** P

    Techniques Used: Mutagenesis, Inhibition, Generated, Concentration Assay, Cell Viability Assay

    CAPN1 is responsible for NF1 degradation in vitro Cell lysates of A375, 74T and 293T stably over expressing NF1 (293T-NF1) were incubated with the indicated units of purified CAPN1 with 10 mM CaCl 2 for 1 hour, resolved by SDS-PAGE and blotted with anti-NF1 (polyclonal antibody) and anti-GAPDH. Anti-Tubulin served as a positive control for the assay. Molecular weights are given in kilodaltons on the left. The arrows indicate the detection of full length NF1 and the black boxes indicate an approximately 40 kDa proteolytic fragment of NF1. Addition of 45 μM of Calpain inhibitor I blocked this degradation (last lane on each blot).
    Figure Legend Snippet: CAPN1 is responsible for NF1 degradation in vitro Cell lysates of A375, 74T and 293T stably over expressing NF1 (293T-NF1) were incubated with the indicated units of purified CAPN1 with 10 mM CaCl 2 for 1 hour, resolved by SDS-PAGE and blotted with anti-NF1 (polyclonal antibody) and anti-GAPDH. Anti-Tubulin served as a positive control for the assay. Molecular weights are given in kilodaltons on the left. The arrows indicate the detection of full length NF1 and the black boxes indicate an approximately 40 kDa proteolytic fragment of NF1. Addition of 45 μM of Calpain inhibitor I blocked this degradation (last lane on each blot).

    Techniques Used: In Vitro, Stable Transfection, Expressing, Incubation, Purification, SDS Page, Positive Control

    CAPN1 inhibition stabilizes NF1 levels, affecting RAS signaling and cell proliferation (A) NF1 wild type cells (A375, 74T) were treated with increasing concentrations of Calpain inhibitor I (μM) for 6 hours or with DMSO as control, and NF1 levels were tested by immunoblot. (B) NF1 mutant cells (108T, 76T) were treated with increasing concentrations of Calpain inhibitor I (μM) for 6 hours or with DMSO as control, and NF1 levels were tested by immunoblot. Ratios of NF1 levels to GAPDH were generated using Image lab (BioRad) and Microsoft Excel analysis. (C) 74T and A375 cells were treated with 3 μM or 5 μM of Calpain inhibitor I for 16 hours, respectively. Cell lysates were analyzed by western blot with the indicated antibodies. RAS-GTP levels were assessed by RAS pulldown assay after treatment with 50 μM of Calpain inhibitor I, respectively for 6 hours. (D) A375 and 74T cells were seeded in the 96 well plates with 10% FBS and cells were treatedwith 2.5 or 5 μM of Calpain inhibitor I, respectively. DMSO was used as a control for this experiment. The average cell number was measured by assessing DNA content using SYBR green I in two independent experiments with six replicates each. Error bars, s.e.m.
    Figure Legend Snippet: CAPN1 inhibition stabilizes NF1 levels, affecting RAS signaling and cell proliferation (A) NF1 wild type cells (A375, 74T) were treated with increasing concentrations of Calpain inhibitor I (μM) for 6 hours or with DMSO as control, and NF1 levels were tested by immunoblot. (B) NF1 mutant cells (108T, 76T) were treated with increasing concentrations of Calpain inhibitor I (μM) for 6 hours or with DMSO as control, and NF1 levels were tested by immunoblot. Ratios of NF1 levels to GAPDH were generated using Image lab (BioRad) and Microsoft Excel analysis. (C) 74T and A375 cells were treated with 3 μM or 5 μM of Calpain inhibitor I for 16 hours, respectively. Cell lysates were analyzed by western blot with the indicated antibodies. RAS-GTP levels were assessed by RAS pulldown assay after treatment with 50 μM of Calpain inhibitor I, respectively for 6 hours. (D) A375 and 74T cells were seeded in the 96 well plates with 10% FBS and cells were treatedwith 2.5 or 5 μM of Calpain inhibitor I, respectively. DMSO was used as a control for this experiment. The average cell number was measured by assessing DNA content using SYBR green I in two independent experiments with six replicates each. Error bars, s.e.m.

    Techniques Used: Inhibition, Mutagenesis, Generated, Western Blot, SYBR Green Assay

    44) Product Images from "Combinatorial Smad2/3 Activities Downstream of Nodal Signaling Maintain Embryonic/Extra-Embryonic Cell Identities during Lineage Priming"

    Article Title: Combinatorial Smad2/3 Activities Downstream of Nodal Signaling Maintain Embryonic/Extra-Embryonic Cell Identities during Lineage Priming

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.07.077

    Smad2/3 Governs Embryonic Cell Fate Specification (A) Heatmap showing the log2 fold change (log2FC) in expression of the top 20 genes upregulated in day 3 WT EBs relative to WT ESCs (left) in comparison with their expression changes in Smad2/3 DKO (right). See also Table S1 . (B) Anti-AP2γ and Oct4 immunofluorescence staining of WT and Smad2/3 DKO day 2 and 4 PGCLCs. (C) WT and Smad2/3 DKO NPCs at day 7 stained with anti-Tuj1 and counterstained with DAPI. (D) Bright-field images of control WT or Smad2/3 DKO NPCs grown in the absence or presence of BMP4 (5 ng/mL) at days 3, 5, and 7. (E) WT and Smad2/3 DKO NPCs grown in the absence or presence of BMP4 (5 ng/mL) stained with anti-Sox1 and Ap2γ and counterstained with DAPI at day 5.
    Figure Legend Snippet: Smad2/3 Governs Embryonic Cell Fate Specification (A) Heatmap showing the log2 fold change (log2FC) in expression of the top 20 genes upregulated in day 3 WT EBs relative to WT ESCs (left) in comparison with their expression changes in Smad2/3 DKO (right). See also Table S1 . (B) Anti-AP2γ and Oct4 immunofluorescence staining of WT and Smad2/3 DKO day 2 and 4 PGCLCs. (C) WT and Smad2/3 DKO NPCs at day 7 stained with anti-Tuj1 and counterstained with DAPI. (D) Bright-field images of control WT or Smad2/3 DKO NPCs grown in the absence or presence of BMP4 (5 ng/mL) at days 3, 5, and 7. (E) WT and Smad2/3 DKO NPCs grown in the absence or presence of BMP4 (5 ng/mL) stained with anti-Sox1 and Ap2γ and counterstained with DAPI at day 5.

    Techniques Used: Expressing, Immunofluorescence, Staining

    Enhanced Bmp Signaling Caused by the Absence of Smad2/3 Disturbs Embryonic Patterning (A) Heatmap showing the log2FC in expression of selected Bmp target genes and those involved in DNA methylation for Smad2/3 DKO ESCs, EpiLCs, and day 3 EBs compared to WT controls (n = 3 or 4). (B) Western blot analysis of WT and Smad2/3 DKO day 3 EBs treated with DMSO or LDN-193189 (250 nM, 24 hr from day 2 to day 3) or untreated. Blots were probed with the indicated antibodies. (C and D) Anti-p-Smad1/5/8 and Oct4 (C) or Eomes and Otx2 (D) immunofluorescence staining of E5.5 WT or Smad2/3 DKO mouse embryos. (E) Heatmap showing the log2FC in expression of the top 20 upregulated genes (p
    Figure Legend Snippet: Enhanced Bmp Signaling Caused by the Absence of Smad2/3 Disturbs Embryonic Patterning (A) Heatmap showing the log2FC in expression of selected Bmp target genes and those involved in DNA methylation for Smad2/3 DKO ESCs, EpiLCs, and day 3 EBs compared to WT controls (n = 3 or 4). (B) Western blot analysis of WT and Smad2/3 DKO day 3 EBs treated with DMSO or LDN-193189 (250 nM, 24 hr from day 2 to day 3) or untreated. Blots were probed with the indicated antibodies. (C and D) Anti-p-Smad1/5/8 and Oct4 (C) or Eomes and Otx2 (D) immunofluorescence staining of E5.5 WT or Smad2/3 DKO mouse embryos. (E) Heatmap showing the log2FC in expression of the top 20 upregulated genes (p

    Techniques Used: Expressing, DNA Methylation Assay, Western Blot, Immunofluorescence, Staining

    Smad2/3 Repress Expression of Extra-Embryonic and Naive Pluripotency Genes during Lineage Priming (A) WT, Smad2 KO, Smad3 KO, or Smad2/3 DKO ESCs (2iL) were stained for Oct4 and Nanog and counterstained with DAPI. (B) Venn diagrams showing significant changes in gene expression shared by Smad2 KO, Smad3 KO, and Smad2/3 DKO ESCs, relative to WT ESCs, as determined by microarray profiling (n = 3 or 4). Genes uniquely differentially expressed by Smad2 KO or Smad3 KO ESCs were excluded from this analysis. A summary of deregulated genes is presented in Table S1 . (C) Pie charts of alkaline phosphatase (AP)-stained WT, Smad2 KO, Smad3 KO, or Smad2/3 DKO ESCs cultured for 5 days in the presence or absence of LIF (n = 3), corresponding to pluripotent, differentiated, or mixed colonies. See also Figure S1 F. (D) Scatterplot showing significantly (p
    Figure Legend Snippet: Smad2/3 Repress Expression of Extra-Embryonic and Naive Pluripotency Genes during Lineage Priming (A) WT, Smad2 KO, Smad3 KO, or Smad2/3 DKO ESCs (2iL) were stained for Oct4 and Nanog and counterstained with DAPI. (B) Venn diagrams showing significant changes in gene expression shared by Smad2 KO, Smad3 KO, and Smad2/3 DKO ESCs, relative to WT ESCs, as determined by microarray profiling (n = 3 or 4). Genes uniquely differentially expressed by Smad2 KO or Smad3 KO ESCs were excluded from this analysis. A summary of deregulated genes is presented in Table S1 . (C) Pie charts of alkaline phosphatase (AP)-stained WT, Smad2 KO, Smad3 KO, or Smad2/3 DKO ESCs cultured for 5 days in the presence or absence of LIF (n = 3), corresponding to pluripotent, differentiated, or mixed colonies. See also Figure S1 F. (D) Scatterplot showing significantly (p

    Techniques Used: Expressing, Staining, Microarray, Cell Culture

    Smad2/3 Influences the Activity of Oct4-Occupied Distal Regulatory Enhancers during Priming (A) Heatmap of regulatory elements with differential chromatin accessibility in Smad2/3 DKO EpiLCs compared to WT EpiLCs, as measured by ATAC-seq (false discovery rate [FDR]
    Figure Legend Snippet: Smad2/3 Influences the Activity of Oct4-Occupied Distal Regulatory Enhancers during Priming (A) Heatmap of regulatory elements with differential chromatin accessibility in Smad2/3 DKO EpiLCs compared to WT EpiLCs, as measured by ATAC-seq (false discovery rate [FDR]

    Techniques Used: Activity Assay

    45) Product Images from "Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression"

    Article Title: Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression

    Journal: ACS Omega

    doi: 10.1021/acsomega.7b02073

    Effects of two chemical RNA splicing inhibitors on the APP expression. (A) Isoginkgetin (IGK) increases the APP expression. HEK293T cells were treated in triplicates with 30 μM IGK for 12 h. (B) Spliceostatin A (SSA) inhibits the APP expression. HEK293T cells were treated in triplicates with 25 nM for 6 h. As a solvent of these two chemicals, DMSO was used as the control. Protein relative abundance was derived from the blot intensities quantified by ImageJ and then normalized to the average of the controls. Data were from three independent experiments in both IGK and SSA treatments. Error bars are S.E.M., Student’s t -tests, * P
    Figure Legend Snippet: Effects of two chemical RNA splicing inhibitors on the APP expression. (A) Isoginkgetin (IGK) increases the APP expression. HEK293T cells were treated in triplicates with 30 μM IGK for 12 h. (B) Spliceostatin A (SSA) inhibits the APP expression. HEK293T cells were treated in triplicates with 25 nM for 6 h. As a solvent of these two chemicals, DMSO was used as the control. Protein relative abundance was derived from the blot intensities quantified by ImageJ and then normalized to the average of the controls. Data were from three independent experiments in both IGK and SSA treatments. Error bars are S.E.M., Student’s t -tests, * P

    Techniques Used: Expressing, Derivative Assay

    Analysis of APP RNA and protein expression induced by IGK. (A) Western blots of the APP expression in HEK293T and SK-N-SH neuroblastoma cells treated by IGK at different doses. (B) Schematic diagram of three major APP RNA transcripts (770, 751, and 695). (C) Western blot to show APP form change in IGK treatment. Samples were from HEK293T cell lysates. The loading amount of IGK samples was serially reduced so that the APP expression level is close to that of the DMSO control for clearer protein pattern comparison. Proteins on the membrane after electrotransfer during western blotting were stained by Ponceau S. (D) RT-PCR of APP mRNA in IGK treatment. Forward primer targets exon 6 and the reverse primer targets exon 9. All samples received the same RT-PCR protocol. After 25 amplification cycles, the DNA products of each sample were taken for separation and visualization on the 1% agarose gel. This was repeated every three cycles until the DNA bands started to be visible, so that the difference in their relative abundance would not diminish due to over amplification. The results shown were from PCR after 31 cycles.
    Figure Legend Snippet: Analysis of APP RNA and protein expression induced by IGK. (A) Western blots of the APP expression in HEK293T and SK-N-SH neuroblastoma cells treated by IGK at different doses. (B) Schematic diagram of three major APP RNA transcripts (770, 751, and 695). (C) Western blot to show APP form change in IGK treatment. Samples were from HEK293T cell lysates. The loading amount of IGK samples was serially reduced so that the APP expression level is close to that of the DMSO control for clearer protein pattern comparison. Proteins on the membrane after electrotransfer during western blotting were stained by Ponceau S. (D) RT-PCR of APP mRNA in IGK treatment. Forward primer targets exon 6 and the reverse primer targets exon 9. All samples received the same RT-PCR protocol. After 25 amplification cycles, the DNA products of each sample were taken for separation and visualization on the 1% agarose gel. This was repeated every three cycles until the DNA bands started to be visible, so that the difference in their relative abundance would not diminish due to over amplification. The results shown were from PCR after 31 cycles.

    Techniques Used: Expressing, Western Blot, Electrotransfer, Staining, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    46) Product Images from "Dual oxidase 2 and pancreatic adenocarcinoma: IFN-γ-mediated dual oxidase 2 overexpression results in H2O2-induced, ERK-associated up-regulation of HIF-1α and VEGF-A"

    Article Title: Dual oxidase 2 and pancreatic adenocarcinoma: IFN-γ-mediated dual oxidase 2 overexpression results in H2O2-induced, ERK-associated up-regulation of HIF-1α and VEGF-A

    Journal: Oncotarget

    doi: 10.18632/oncotarget.12032

    Expression of VEGF-A mRNA and DUOX and signaling proteins in BxPC-3 pancreatic cancer xenografts from untreated mice is comparable to expression in BxPC-3 cells exposed to IFN-γ for 24 h in vitro The fourth to sixth in vitro passages of cultured BxPC-3 cells were used to establish human tumor xenografts, as described in the Materials and Methods section; 300-mg tumor samples were split into quadrants, either for RNA extraction or for preparation of tumor lysates. ( A ) VEGF-A expression in BxPC-3 xenografts from individual mice was significantly increased when compared to VEGF-A expression in the BxPC-3 cell line immediately prior to inoculation, and was comparable to the expression level observed when the same cells were treated with 25 ng/ml IFN-γ for 24 h in vitro , as determined by quantitative RT-PCR. *** P
    Figure Legend Snippet: Expression of VEGF-A mRNA and DUOX and signaling proteins in BxPC-3 pancreatic cancer xenografts from untreated mice is comparable to expression in BxPC-3 cells exposed to IFN-γ for 24 h in vitro The fourth to sixth in vitro passages of cultured BxPC-3 cells were used to establish human tumor xenografts, as described in the Materials and Methods section; 300-mg tumor samples were split into quadrants, either for RNA extraction or for preparation of tumor lysates. ( A ) VEGF-A expression in BxPC-3 xenografts from individual mice was significantly increased when compared to VEGF-A expression in the BxPC-3 cell line immediately prior to inoculation, and was comparable to the expression level observed when the same cells were treated with 25 ng/ml IFN-γ for 24 h in vitro , as determined by quantitative RT-PCR. *** P

    Techniques Used: Expressing, Mouse Assay, In Vitro, Cell Culture, RNA Extraction, Quantitative RT-PCR

    47) Product Images from "SmgGDS is a transient nucleolar protein that protects cells from nucleolar stress and promotes the cell cycle by regulating DREAM complex gene expression"

    Article Title: SmgGDS is a transient nucleolar protein that protects cells from nucleolar stress and promotes the cell cycle by regulating DREAM complex gene expression

    Journal: Oncogene

    doi: 10.1038/onc.2017.280

    SmgGDS-558 physically interacts with UBF, and this interaction promotes the nucleolar accumulation of SmgGDS-558. ( a ) HEK293T cells were transfected with cDNAs encoding the HA vector or HA-tagged SmgGDS, followed by immunoprecipitation using HA antibody and silver staining to detect co-precipitating proteins. Mass spectrometry identified UBF as one of the co-precipitating proteins (HC and LC; heavy and light chains, respectively, of antibodies used in the immunoprecipitation). ( b ) Lysates from HEK293T cells transfected with cDNAs encoding the HA vector or HA-tagged SmgGDS were immunoprecipitated using HA antibody, followed by immunoblotting using antibodies to UBF and HA ( n =3). ( c ) HEK293T cells were transfected with cDNAs encoding the HA vector or SmgGDS-558-NES mut -HA along with non-targeting (NT) siRNA or UBF siRNA. After 72 h, the cells were immunofluorescently stained with HA antibody, UBF antibody and 4,6-diamidino-2-phenylindole (DAPI), and examined by confocal fluorescence microscopy ( n =3). ( d ) HEK293T cells expressing SmgGDS-558-NES mut -HA were treated with or without the RNA Pol I inhibitor CX-5461 (1 μ m , 2 h), followed by FuRD (2 m m , 15 min), and immunofluorescently stained using antibodies to HA and BrdU ( n =3). Images were obtained by confocal microscopy. ( e ) NCI-H1703 cells were transfected with the indicated siRNAs to deplete SmgGDS, and 72 h later quantitative PCR was conducted to examine 47S pre-rRNA levels (normalized to cellular GAPDH). Control cells were treated with CX-5461 (1 μ m , 2 h) before collecting RNA. Error bars represent ±s.e.m. of three biological replicates, and statistical significance was determined by one-way analysis of variance and Holm-Sidak multiple comparisons test (* P
    Figure Legend Snippet: SmgGDS-558 physically interacts with UBF, and this interaction promotes the nucleolar accumulation of SmgGDS-558. ( a ) HEK293T cells were transfected with cDNAs encoding the HA vector or HA-tagged SmgGDS, followed by immunoprecipitation using HA antibody and silver staining to detect co-precipitating proteins. Mass spectrometry identified UBF as one of the co-precipitating proteins (HC and LC; heavy and light chains, respectively, of antibodies used in the immunoprecipitation). ( b ) Lysates from HEK293T cells transfected with cDNAs encoding the HA vector or HA-tagged SmgGDS were immunoprecipitated using HA antibody, followed by immunoblotting using antibodies to UBF and HA ( n =3). ( c ) HEK293T cells were transfected with cDNAs encoding the HA vector or SmgGDS-558-NES mut -HA along with non-targeting (NT) siRNA or UBF siRNA. After 72 h, the cells were immunofluorescently stained with HA antibody, UBF antibody and 4,6-diamidino-2-phenylindole (DAPI), and examined by confocal fluorescence microscopy ( n =3). ( d ) HEK293T cells expressing SmgGDS-558-NES mut -HA were treated with or without the RNA Pol I inhibitor CX-5461 (1 μ m , 2 h), followed by FuRD (2 m m , 15 min), and immunofluorescently stained using antibodies to HA and BrdU ( n =3). Images were obtained by confocal microscopy. ( e ) NCI-H1703 cells were transfected with the indicated siRNAs to deplete SmgGDS, and 72 h later quantitative PCR was conducted to examine 47S pre-rRNA levels (normalized to cellular GAPDH). Control cells were treated with CX-5461 (1 μ m , 2 h) before collecting RNA. Error bars represent ±s.e.m. of three biological replicates, and statistical significance was determined by one-way analysis of variance and Holm-Sidak multiple comparisons test (* P

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Silver Staining, Mass Spectrometry, Staining, Fluorescence, Microscopy, Expressing, Confocal Microscopy, Real-time Polymerase Chain Reaction

    The RNAi-mediated depletion of SmgGDS-558 disrupts nucleolar morphology. ( a ) NCI-H1703 cells were transfected with the indicated siRNAs to deplete SmgGDS isoforms, and 72 h later the cells were immunofluorescently stained with nucleolin antibody, UBF antibody and 4,6-diamidino-2-phenylindole (DAPI), and examined by confocal fluorescence microscopy. Arrows indicate circular redistribution of nucleolin, and asterisks indicate UBF cap formation ( n =3). ( b ) NCI-H1703 cells treated the same as in a were scored for disruption of nucleolar morphology according to the key described in Supplementary Figure S3 . Scoring was conducted without knowledge of the siRNAs used to treat the cells, and values represent the mean±s.e.m. from 100 cells scored in three independent experiments. Statistical significance was determined by one-way repeated measures analysis of variance and Dunnett’s multiple comparison test (* P
    Figure Legend Snippet: The RNAi-mediated depletion of SmgGDS-558 disrupts nucleolar morphology. ( a ) NCI-H1703 cells were transfected with the indicated siRNAs to deplete SmgGDS isoforms, and 72 h later the cells were immunofluorescently stained with nucleolin antibody, UBF antibody and 4,6-diamidino-2-phenylindole (DAPI), and examined by confocal fluorescence microscopy. Arrows indicate circular redistribution of nucleolin, and asterisks indicate UBF cap formation ( n =3). ( b ) NCI-H1703 cells treated the same as in a were scored for disruption of nucleolar morphology according to the key described in Supplementary Figure S3 . Scoring was conducted without knowledge of the siRNAs used to treat the cells, and values represent the mean±s.e.m. from 100 cells scored in three independent experiments. Statistical significance was determined by one-way repeated measures analysis of variance and Dunnett’s multiple comparison test (* P

    Techniques Used: Transfection, Staining, Fluorescence, Microscopy

    Depletion of SmgGDS diminishes expression of DREAM complex target genes required for cell cycle progression. ( a ) Ingenuity Pathway Analysis of microarray data from NCI-H1703 cells depleted of both SmgGDS-558 and SmgGDS-607 using siRNA I1 shows significant effects on gene expression within the indicated pathways. ( b , c ) Heatmaps display changes in expression of the 200 DREAM target genes that were most significantly altered in NCI-H1703 cells treated with siRNA I1, generated in two technical replicates (T1 and T2) from three independent experiments (Exp 1, 2 and 3). ( d ) Protein complexes containing E2F1, B-MYB, FOXM1 and Lin family members promote cell cycle progression, and yellow highlights identify specific gene products that exhibit reduced expression in NCI-H1703 cells following depletion of SmgGDS with siRNA I1. ( e ) The 200 DREAM target genes that were most significantly altered in SmgGDS-depleted cells (shown in c ) were analyzed using the targetgenereg.org 24 database to predict which of these target genes are regulated by Rb-E2F1 or MMB-FOXM1 complexes. ( f ) Immunoblotting was used to examine E2F1 protein expression in NCI-H1703 cells following depletion of different SmgGDS isoforms using specific siRNAs ( n =3). Mean normalized densitometry values are shown in Supplementary Figure S1b .
    Figure Legend Snippet: Depletion of SmgGDS diminishes expression of DREAM complex target genes required for cell cycle progression. ( a ) Ingenuity Pathway Analysis of microarray data from NCI-H1703 cells depleted of both SmgGDS-558 and SmgGDS-607 using siRNA I1 shows significant effects on gene expression within the indicated pathways. ( b , c ) Heatmaps display changes in expression of the 200 DREAM target genes that were most significantly altered in NCI-H1703 cells treated with siRNA I1, generated in two technical replicates (T1 and T2) from three independent experiments (Exp 1, 2 and 3). ( d ) Protein complexes containing E2F1, B-MYB, FOXM1 and Lin family members promote cell cycle progression, and yellow highlights identify specific gene products that exhibit reduced expression in NCI-H1703 cells following depletion of SmgGDS with siRNA I1. ( e ) The 200 DREAM target genes that were most significantly altered in SmgGDS-depleted cells (shown in c ) were analyzed using the targetgenereg.org 24 database to predict which of these target genes are regulated by Rb-E2F1 or MMB-FOXM1 complexes. ( f ) Immunoblotting was used to examine E2F1 protein expression in NCI-H1703 cells following depletion of different SmgGDS isoforms using specific siRNAs ( n =3). Mean normalized densitometry values are shown in Supplementary Figure S1b .

    Techniques Used: Expressing, Microarray, Generated

    The UBF-dependent nucleolar sequestration of SmgGDS-558 protects SmgGDS-558 from proteasome-mediated degradation in the nucleoplasm. ( a ) Lysates from HEK293T cells expressing SmgGDS-558-HA and SmgGDS-558-NES mut -HA were immunoblotted using HA antibody. Two exposures of the same immunoblot are shown; SmgGDS-558-NES mut -HA was detected only in the long exposure. Immunoblotting with GAPDH antibody was used as a loading control ( n =3). ( b ) HEK293T cells were transfected with SmgGDS-558-HA or SmgGDS-558-NES mut -HA, and 56 h later the cells were incubated with the indicated concentrations of MG-132 or lactacystin. After 16 h, cell lysates were immunoblotted using HA and GAPDH antibodies ( n =3). Long and short exposures of the immunoblots are shown. Mean normalized densitometry values are shown in Supplementary Figure S6 . ( c ) HEK293T cells expressing SmgGDS-558-HA or SmgGDS-558-NES mut -HA were incubated with cycloheximide (CHX; 1 μg/ml) for the indicated times, and cell lysates were immunoblotted using HA and GAPDH antibodies ( n =3). ( d ) Mean densitometry values obtained from three independent experiments shown in c were used to fit exponential regression curves and determine the half-life of SmgGDS-558-HA and SmgGDS-558-NES mut -HA. ( e , f ) HEK293T cells transfected with SmgGDS-558-NES mut -HA ( e ) or SmgGDS-558-HA ( f ) were co-transfected with non-targeting (NT) siRNA or UBF siRNAs, and 56 h later the cells were treated with or without MG-132 (5 μ m , 16 h; n =3). Cell lysates were immunoblotted using HA, UBF and GAPDH antibodies. Mean normalized densitometry values are shown in Supplementary Figure S6 .
    Figure Legend Snippet: The UBF-dependent nucleolar sequestration of SmgGDS-558 protects SmgGDS-558 from proteasome-mediated degradation in the nucleoplasm. ( a ) Lysates from HEK293T cells expressing SmgGDS-558-HA and SmgGDS-558-NES mut -HA were immunoblotted using HA antibody. Two exposures of the same immunoblot are shown; SmgGDS-558-NES mut -HA was detected only in the long exposure. Immunoblotting with GAPDH antibody was used as a loading control ( n =3). ( b ) HEK293T cells were transfected with SmgGDS-558-HA or SmgGDS-558-NES mut -HA, and 56 h later the cells were incubated with the indicated concentrations of MG-132 or lactacystin. After 16 h, cell lysates were immunoblotted using HA and GAPDH antibodies ( n =3). Long and short exposures of the immunoblots are shown. Mean normalized densitometry values are shown in Supplementary Figure S6 . ( c ) HEK293T cells expressing SmgGDS-558-HA or SmgGDS-558-NES mut -HA were incubated with cycloheximide (CHX; 1 μg/ml) for the indicated times, and cell lysates were immunoblotted using HA and GAPDH antibodies ( n =3). ( d ) Mean densitometry values obtained from three independent experiments shown in c were used to fit exponential regression curves and determine the half-life of SmgGDS-558-HA and SmgGDS-558-NES mut -HA. ( e , f ) HEK293T cells transfected with SmgGDS-558-NES mut -HA ( e ) or SmgGDS-558-HA ( f ) were co-transfected with non-targeting (NT) siRNA or UBF siRNAs, and 56 h later the cells were treated with or without MG-132 (5 μ m , 16 h; n =3). Cell lysates were immunoblotted using HA, UBF and GAPDH antibodies. Mean normalized densitometry values are shown in Supplementary Figure S6 .

    Techniques Used: Expressing, Transfection, Incubation, Western Blot

    48) Product Images from "Dysregulation of STAT3 signaling is associated with endplate-oriented herniations of the intervertebral disc in Adgrg6 mutant mice"

    Article Title: Dysregulation of STAT3 signaling is associated with endplate-oriented herniations of the intervertebral disc in Adgrg6 mutant mice

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008096

    Adgrg6 regulates gene expression profiles and STAT3 signaling in ATDC5 cell culture. (A) Schematic of a 17-bp deletion of Adgrg6 from a stable single cell clone of ATDC5 cell line ( Adgrg6 KO). (B) qPCR analyses of gene expression in ATDC5 cells at 5 and 15 days of maturation demonstrates decreased expression of markers of healthy disc, Col2a1 and Sox9 , and increased expression of the hypertrophic marker, Col10a1 and the extracellular matrix modifying enzyme, Mmp13 in Adgrg6 KO cells. (C) qPCR analyses revealed increased expression of Socs3 in Adrgr6 KO cells after 15 days of maturation. (D) qPCR analysis of Il1a , Il1b , Il6 and Tnf in ATDC5 cells maturated for 15 days. (E) qPCR analysis of Il6 expression in 1.5-month-old primary mouse IVDs (induced from E0.5-P20). (B-E, n = 3 biological replicates and representative result is shown. Bars represent mean ± SD. *p≤0.05, **p≤0.01, two-tailed Student's t Test). (F) Representative Western blot of wild type and Adgrg6 KO ATDC5 cell lysates showing stimulation of pSTAT3 staining after treatment with recombinant IL-6 (rIL-6) protein in both cell lines, while Adgrg6 KO cells show a mild constitutive stimulation of pSTAT3 without addition of rIL-6 ( n = 4 biological replicates and representative result is shown). (G) Densitometry of the Western blot of wild type and Adgrg6 KO ATDC5 cell with or without IL-6 (rIL-6) treatment. (Each dot represents one biological replicate. Bars plot with mean ± SD. *p≤0.05, two-tailed Student's t Test.)
    Figure Legend Snippet: Adgrg6 regulates gene expression profiles and STAT3 signaling in ATDC5 cell culture. (A) Schematic of a 17-bp deletion of Adgrg6 from a stable single cell clone of ATDC5 cell line ( Adgrg6 KO). (B) qPCR analyses of gene expression in ATDC5 cells at 5 and 15 days of maturation demonstrates decreased expression of markers of healthy disc, Col2a1 and Sox9 , and increased expression of the hypertrophic marker, Col10a1 and the extracellular matrix modifying enzyme, Mmp13 in Adgrg6 KO cells. (C) qPCR analyses revealed increased expression of Socs3 in Adrgr6 KO cells after 15 days of maturation. (D) qPCR analysis of Il1a , Il1b , Il6 and Tnf in ATDC5 cells maturated for 15 days. (E) qPCR analysis of Il6 expression in 1.5-month-old primary mouse IVDs (induced from E0.5-P20). (B-E, n = 3 biological replicates and representative result is shown. Bars represent mean ± SD. *p≤0.05, **p≤0.01, two-tailed Student's t Test). (F) Representative Western blot of wild type and Adgrg6 KO ATDC5 cell lysates showing stimulation of pSTAT3 staining after treatment with recombinant IL-6 (rIL-6) protein in both cell lines, while Adgrg6 KO cells show a mild constitutive stimulation of pSTAT3 without addition of rIL-6 ( n = 4 biological replicates and representative result is shown). (G) Densitometry of the Western blot of wild type and Adgrg6 KO ATDC5 cell with or without IL-6 (rIL-6) treatment. (Each dot represents one biological replicate. Bars plot with mean ± SD. *p≤0.05, two-tailed Student's t Test.)

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Marker, Two Tailed Test, Western Blot, Staining, Recombinant

    49) Product Images from "Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus"

    Article Title: Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20154734

    Gel electrophoresis of total RNA that was obtained from sessile cells with sheared whole-cell lysis coupled to the RNeasy Mini Kit. Lanes 1 , 2 , and 3 represent three independent experiments using the methicillin-resistant S. aureus (MRSA) isolate BMB9393 ( A ) and the methicillin-susceptible S. aureus (MSSA) isolate HC474 ( B ).
    Figure Legend Snippet: Gel electrophoresis of total RNA that was obtained from sessile cells with sheared whole-cell lysis coupled to the RNeasy Mini Kit. Lanes 1 , 2 , and 3 represent three independent experiments using the methicillin-resistant S. aureus (MRSA) isolate BMB9393 ( A ) and the methicillin-susceptible S. aureus (MSSA) isolate HC474 ( B ).

    Techniques Used: Nucleic Acid Electrophoresis, Lysis

    50) Product Images from "Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus"

    Article Title: Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20154734

    Gel electrophoresis of total RNA that was obtained from sessile cells with sheared whole-cell lysis coupled to the RNeasy Mini Kit. Lanes 1 , 2 , and 3 represent three independent experiments using the methicillin-resistant S. aureus (MRSA) isolate BMB9393 ( A ) and the methicillin-susceptible S. aureus (MSSA) isolate HC474 ( B ).
    Figure Legend Snippet: Gel electrophoresis of total RNA that was obtained from sessile cells with sheared whole-cell lysis coupled to the RNeasy Mini Kit. Lanes 1 , 2 , and 3 represent three independent experiments using the methicillin-resistant S. aureus (MRSA) isolate BMB9393 ( A ) and the methicillin-susceptible S. aureus (MSSA) isolate HC474 ( B ).

    Techniques Used: Nucleic Acid Electrophoresis, Lysis

    51) Product Images from "RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells"

    Article Title: RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197517

    A) Concentration of RNA extracted from HEK293 cells encapsulated in the four peptide hydrogels and cell-only controls using the three methods (see text for details). B Representative electrophoresis traces of total RNA extracted from cells encapsulated in the four peptide hydrogels and cell-only controls using the Tri Reagent and RNeasy MK methods and corresponding RIN values: cell-only controls (A+E), PGD-Alpha1 (B+F), PGD-Alpha2 (G), PGD-AlphaProB (C+H) and PGD-AlphaProC (D+I). C D) Representative UV spectra for RNA samples extracted using the TRI Reagent (C) and the RNeasy MK (D) methods from the four peptide hydrogels and cell-only control.
    Figure Legend Snippet: A) Concentration of RNA extracted from HEK293 cells encapsulated in the four peptide hydrogels and cell-only controls using the three methods (see text for details). B Representative electrophoresis traces of total RNA extracted from cells encapsulated in the four peptide hydrogels and cell-only controls using the Tri Reagent and RNeasy MK methods and corresponding RIN values: cell-only controls (A+E), PGD-Alpha1 (B+F), PGD-Alpha2 (G), PGD-AlphaProB (C+H) and PGD-AlphaProC (D+I). C D) Representative UV spectra for RNA samples extracted using the TRI Reagent (C) and the RNeasy MK (D) methods from the four peptide hydrogels and cell-only control.

    Techniques Used: Concentration Assay, Electrophoresis

    Ct values obtained for RT-qPCR performed using RNA extracted from the four peptide hydrogels as a template. RNA isolated from cells in suspension was used as a control. The RNA extracted using either the TRI Reagent method (A-B) or RNeasy MK method (C+D) was used as templates for the amplification of two housekeeping genes: GAPDH (A+C) and RPL13A (B+D). The cycle threshold (Ct) value was determined for three independent samples measured in triplicate (or two independent samples for PGD-Alpha1 using the RNeasy method). Data is shown as mean ± SEM. The mean values were compared to the non-encapsulated cell controls using a t-test; *, P ≤ 0.05.
    Figure Legend Snippet: Ct values obtained for RT-qPCR performed using RNA extracted from the four peptide hydrogels as a template. RNA isolated from cells in suspension was used as a control. The RNA extracted using either the TRI Reagent method (A-B) or RNeasy MK method (C+D) was used as templates for the amplification of two housekeeping genes: GAPDH (A+C) and RPL13A (B+D). The cycle threshold (Ct) value was determined for three independent samples measured in triplicate (or two independent samples for PGD-Alpha1 using the RNeasy method). Data is shown as mean ± SEM. The mean values were compared to the non-encapsulated cell controls using a t-test; *, P ≤ 0.05.

    Techniques Used: Quantitative RT-PCR, Isolation, Amplification

    52) Product Images from "Quantification of Small Non-Coding RNAs Allows an Accurate Comparison of miRNA Expression Profiles"

    Article Title: Quantification of Small Non-Coding RNAs Allows an Accurate Comparison of miRNA Expression Profiles

    Journal: Journal of Biomedicine and Biotechnology

    doi: 10.1155/2009/659028

    (a) Gel electrophoresis (polyacrylamide 15% stained with ethidium bromide) of LMW RNA samples (LCL) extracted with TRIzol reagent (lane 1), RNEasy kit (lane 2), and small RNA fraction enriched with MirVana kit (lane 3). LMW profile obtained with MirVana kit extraction is similar to that obtained with TRIzol reagent which is not shown for clarity. (b) Agilent 2100 Bioanalyzer electropherogram profile of LMW RNAs (LCL) extracted with TRIzol (black) superimposed on 5.8S (red), 5S (green), and tRNA (blue) bands eluted from polyacrylamide gel. (c) Lymphoblastoid (LCL) LMW RNA profile obtained after plotting the exported raw data from Agilent electropherogram ( ) together with the PeakFit fitted curve (solid line) and the component peak functions. Seven peaks below the LMW RNA profile were fitted by the software ( r 2 = 0.998693).
    Figure Legend Snippet: (a) Gel electrophoresis (polyacrylamide 15% stained with ethidium bromide) of LMW RNA samples (LCL) extracted with TRIzol reagent (lane 1), RNEasy kit (lane 2), and small RNA fraction enriched with MirVana kit (lane 3). LMW profile obtained with MirVana kit extraction is similar to that obtained with TRIzol reagent which is not shown for clarity. (b) Agilent 2100 Bioanalyzer electropherogram profile of LMW RNAs (LCL) extracted with TRIzol (black) superimposed on 5.8S (red), 5S (green), and tRNA (blue) bands eluted from polyacrylamide gel. (c) Lymphoblastoid (LCL) LMW RNA profile obtained after plotting the exported raw data from Agilent electropherogram ( ) together with the PeakFit fitted curve (solid line) and the component peak functions. Seven peaks below the LMW RNA profile were fitted by the software ( r 2 = 0.998693).

    Techniques Used: Nucleic Acid Electrophoresis, Staining, Software

    53) Product Images from "NF-κB promotes the stem-like properties of leukemia cells by activation of LIN28B"

    Article Title: NF-κB promotes the stem-like properties of leukemia cells by activation of LIN28B

    Journal: World Journal of Stem Cells

    doi: 10.4252/wjsc.v10.i4.34

    Effects of nuclear factor kappa B (NF-κB) inhibitors on acute myeloid leukemia cell viability and LIN28 mRNA and protein expression. TF-1a cells were treated with DMSO control, different doses of Bortezomib as indicated, followed by CellTiter-Glo ® Luminescent Cell Viability Assay (CTG assays) (A) or RNA extraction for qRT-PCR (B) and protein extraction for Western blot analysis (C), TF-1 cells were treated with DMSO control, different doses of MG-132 as indicated, followed by CTG assays (D) or RNA extraction for qRT-PCR (E) and protein extraction for Western blot analysis (F). For CTG assays, luminescence of each drug concentration and their controls were quantified. The relative inhibition induced by drug treatment was calculated relative to DMSO controls. For qRT-PCR analysis, the LIN28B mRNA level in DMSO control samples was set as 1 and the relative fold changes of LIN28B in drug treated samples were normalized to DMSO control samples. The experiments were triplicated ( n = 3, mean ± SD). For Western blot analysis, β-actin was used as loading control.
    Figure Legend Snippet: Effects of nuclear factor kappa B (NF-κB) inhibitors on acute myeloid leukemia cell viability and LIN28 mRNA and protein expression. TF-1a cells were treated with DMSO control, different doses of Bortezomib as indicated, followed by CellTiter-Glo ® Luminescent Cell Viability Assay (CTG assays) (A) or RNA extraction for qRT-PCR (B) and protein extraction for Western blot analysis (C), TF-1 cells were treated with DMSO control, different doses of MG-132 as indicated, followed by CTG assays (D) or RNA extraction for qRT-PCR (E) and protein extraction for Western blot analysis (F). For CTG assays, luminescence of each drug concentration and their controls were quantified. The relative inhibition induced by drug treatment was calculated relative to DMSO controls. For qRT-PCR analysis, the LIN28B mRNA level in DMSO control samples was set as 1 and the relative fold changes of LIN28B in drug treated samples were normalized to DMSO control samples. The experiments were triplicated ( n = 3, mean ± SD). For Western blot analysis, β-actin was used as loading control.

    Techniques Used: Expressing, Cell Viability Assay, CTG Assay, RNA Extraction, Quantitative RT-PCR, Protein Extraction, Western Blot, Concentration Assay, Inhibition

    Role of LIN28B in nuclear factor kappa B (NF-B)-mediated leukemic stem cell-like properties. A: RNAs of pEGFP and pEGFP-LIN28B transfected TF-1a cells, followed by qRT-PCR comparing LIN28B transcription level. The expression of LIN28B in each sample was normalized with GAPDH respectively ( n = 3, mean ± SD); B: A total of 2000 cells from TF-1a or LIN28B overexpression cells were initially plated in methylcellulose medium containing either DMSO as control or MG-132 at 50 nmol/L, followed by another two rounds of serial replating assays. Quantification of colonies of indicated cells over 3 rounds of replating in methylcellulose medium. Bar figures represented 3 separated experiments ( n = 3, mean ± SD). a P
    Figure Legend Snippet: Role of LIN28B in nuclear factor kappa B (NF-B)-mediated leukemic stem cell-like properties. A: RNAs of pEGFP and pEGFP-LIN28B transfected TF-1a cells, followed by qRT-PCR comparing LIN28B transcription level. The expression of LIN28B in each sample was normalized with GAPDH respectively ( n = 3, mean ± SD); B: A total of 2000 cells from TF-1a or LIN28B overexpression cells were initially plated in methylcellulose medium containing either DMSO as control or MG-132 at 50 nmol/L, followed by another two rounds of serial replating assays. Quantification of colonies of indicated cells over 3 rounds of replating in methylcellulose medium. Bar figures represented 3 separated experiments ( n = 3, mean ± SD). a P

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing, Over Expression

    54) Product Images from "Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis"

    Article Title: Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070714

    Fixation has strong influence on RNA fragment size and impairs qRT-PCR results. Electropherograms show different RNA molecule size distribution in cryopreserved and FFPE/TFPE liver tissue samples. (A) In contrast to CRYO, ribosomal RNA peaks are absent and fragmentation of RNA has occurred in all fixed tissues. (B) RNA cleanup by Qiagen RNeasy Kit removes small (
    Figure Legend Snippet: Fixation has strong influence on RNA fragment size and impairs qRT-PCR results. Electropherograms show different RNA molecule size distribution in cryopreserved and FFPE/TFPE liver tissue samples. (A) In contrast to CRYO, ribosomal RNA peaks are absent and fragmentation of RNA has occurred in all fixed tissues. (B) RNA cleanup by Qiagen RNeasy Kit removes small (

    Techniques Used: Quantitative RT-PCR, Formalin-fixed Paraffin-Embedded

    55) Product Images from "PTK787/ZK22258 attenuates stellate cell activation and hepatic fibrosis in vivo by inhibiting VEGF signaling"

    Article Title: PTK787/ZK22258 attenuates stellate cell activation and hepatic fibrosis in vivo by inhibiting VEGF signaling

    Journal:

    doi: 10.1038/labinvest.2008.127

    mRNA expression of ( a ) procollagen, ( b ) TIMP-1, ( c ) MMP-9 and ( d ) CD31 in the livers of control and PTK/ZK-treated groups measured by real-time PCR. Total RNA was isolated from liver tissues using RNeasy Mini kit, and mRNA was quantitated by real-time
    Figure Legend Snippet: mRNA expression of ( a ) procollagen, ( b ) TIMP-1, ( c ) MMP-9 and ( d ) CD31 in the livers of control and PTK/ZK-treated groups measured by real-time PCR. Total RNA was isolated from liver tissues using RNeasy Mini kit, and mRNA was quantitated by real-time

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

    56) Product Images from "Receptor for advanced glycation end products involved in circulating endothelial cells release from human coronary endothelial cells induced by C-reactive protein"

    Article Title: Receptor for advanced glycation end products involved in circulating endothelial cells release from human coronary endothelial cells induced by C-reactive protein

    Journal: Iranian Journal of Basic Medical Sciences

    doi:

    Small interfering RNA attenuated the release of circulating endothelial cells induced by C-reactive protein. HCAECs were pretreated with siRNA and then incubated with 25 μg/ml purified human CRP for 24 hr. The number of CECs was assessed by flow cytometry. * P
    Figure Legend Snippet: Small interfering RNA attenuated the release of circulating endothelial cells induced by C-reactive protein. HCAECs were pretreated with siRNA and then incubated with 25 μg/ml purified human CRP for 24 hr. The number of CECs was assessed by flow cytometry. * P

    Techniques Used: Small Interfering RNA, Incubation, Purification, Flow Cytometry, Cytometry

    57) Product Images from "Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey"

    Article Title: Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey

    Journal: Journal of Clinical Laboratory Analysis

    doi: 10.1002/jcla.20439

    Reverse transcription polymerase chain reaction amplification of RNA extracted from VSV‐IND and VSV‐NJ spiked bovine lymph nodes by Qiagen RNeasy mini kit. Lane 1: 100 bp marker; lanes 2–6: IND and NJ RNA with NJ Primer set; lanes 7–11: IND and NJ RNA with IND Primer set; lane 12–16: IND and NJ RNA with both IND and NJ primer sets; lane 17: Negative extraction control; lane 18: Negative PCR control; lane 19: 100 bp marker.
    Figure Legend Snippet: Reverse transcription polymerase chain reaction amplification of RNA extracted from VSV‐IND and VSV‐NJ spiked bovine lymph nodes by Qiagen RNeasy mini kit. Lane 1: 100 bp marker; lanes 2–6: IND and NJ RNA with NJ Primer set; lanes 7–11: IND and NJ RNA with IND Primer set; lane 12–16: IND and NJ RNA with both IND and NJ primer sets; lane 17: Negative extraction control; lane 18: Negative PCR control; lane 19: 100 bp marker.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Marker, Polymerase Chain Reaction

    Multiplex RT‐PCR sensitivity and specificity assay for VSV‐IND and VSV‐NJ detection. RNA extracted from serial dilutions of both types of VSV with titers of 10 3.625 TCID 50 /25 µl and 10 4.375 TCID 50 /25 µl, for IND and NJ, respectively, using Qiagen RNeasy mini kit. Lanes 2–5: typical positive VSV‐IND reactions; lane 6: negative VSV‐IND reaction; lane 7–10: positive VSV‐NJ reactions; lane 11: negative VSV‐NJ reaction; lanes 12–15: VSV‐IND and VSV‐NJ positive reactions utilizing both primer sets; lane 16: negative for VSV‐IND NJ reaction; CE: negative extraction control; CP: negative PCR control; M: DNA marker.
    Figure Legend Snippet: Multiplex RT‐PCR sensitivity and specificity assay for VSV‐IND and VSV‐NJ detection. RNA extracted from serial dilutions of both types of VSV with titers of 10 3.625 TCID 50 /25 µl and 10 4.375 TCID 50 /25 µl, for IND and NJ, respectively, using Qiagen RNeasy mini kit. Lanes 2–5: typical positive VSV‐IND reactions; lane 6: negative VSV‐IND reaction; lane 7–10: positive VSV‐NJ reactions; lane 11: negative VSV‐NJ reaction; lanes 12–15: VSV‐IND and VSV‐NJ positive reactions utilizing both primer sets; lane 16: negative for VSV‐IND NJ reaction; CE: negative extraction control; CP: negative PCR control; M: DNA marker.

    Techniques Used: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Marker

    Reverse transcription polymerase chain reaction amplification of RNA extracted from spiked BHK‐21 cell suspensions by using Qiagen RNeasy mini kit. Lane 1: 100 bp marker; lane 2–6: IND and NJ RNA with IND Primer set; lane 7–11: IND and NJ RNA with NJ Primer set; lane 12: Negative extraction control (IND primer set); lane 13–17: IND and NJ RNA with both IND and NJ primer sets; lane 18: Negative extraction control (NJ primer set); lane 19: Negative extraction control (IND and NJ primer sets); lane 20: Negative PCR control.
    Figure Legend Snippet: Reverse transcription polymerase chain reaction amplification of RNA extracted from spiked BHK‐21 cell suspensions by using Qiagen RNeasy mini kit. Lane 1: 100 bp marker; lane 2–6: IND and NJ RNA with IND Primer set; lane 7–11: IND and NJ RNA with NJ Primer set; lane 12: Negative extraction control (IND primer set); lane 13–17: IND and NJ RNA with both IND and NJ primer sets; lane 18: Negative extraction control (NJ primer set); lane 19: Negative extraction control (IND and NJ primer sets); lane 20: Negative PCR control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Marker, Polymerase Chain Reaction

    58) Product Images from "Amino Acids as Signaling Molecules Modulating Bone Turnover"

    Article Title: Amino Acids as Signaling Molecules Modulating Bone Turnover

    Journal: Bone

    doi: 10.1016/j.bone.2018.02.028

    Amino Acid Effects on BMSCs Panel A: (A) Effect of low amino acid levels on BMSC survival or of amino acid supplementation at a 1x (B) or 0.5x (C) level for three days followed by cell staining with Coomassie Blue. Higher magnification images are shown in the panels below. Essentially no BMSCs survived at the end of the three-day culture in M199 (A). Increasing concentrations of the amino acid mixture resulted in increased cell survival (B,C). Images representative of fifteen independent experiments. (D) Unstained cells grown in 1x amino-acid mixture. Panel B: RT-PCR for amino acid sensors: Total RNA was extracted from BMSC cells with the RNeasy mini kit (Qiagen). cDNA was synthesized using 1 μg total RNA (SuperScript III First-Strand Synthesis System, Invitrogen) and PCR performed as described in Methods. Panel C: RT-PCR for GPRC6A: To evaluate GPRC6A expression in bone cells we ran additional controls using the same conditions as in Panel B. As seen, we could detect GPRC6A in primary calvarial osteoblasts. Additional mouse tissues were used as positive and negative controls. OS= primary mouse calvarial osteoblasts; Hrt= mouse heart; Intest= mouse intestine; Kdn=mouse kidney, H 2 0 was a negative control
    Figure Legend Snippet: Amino Acid Effects on BMSCs Panel A: (A) Effect of low amino acid levels on BMSC survival or of amino acid supplementation at a 1x (B) or 0.5x (C) level for three days followed by cell staining with Coomassie Blue. Higher magnification images are shown in the panels below. Essentially no BMSCs survived at the end of the three-day culture in M199 (A). Increasing concentrations of the amino acid mixture resulted in increased cell survival (B,C). Images representative of fifteen independent experiments. (D) Unstained cells grown in 1x amino-acid mixture. Panel B: RT-PCR for amino acid sensors: Total RNA was extracted from BMSC cells with the RNeasy mini kit (Qiagen). cDNA was synthesized using 1 μg total RNA (SuperScript III First-Strand Synthesis System, Invitrogen) and PCR performed as described in Methods. Panel C: RT-PCR for GPRC6A: To evaluate GPRC6A expression in bone cells we ran additional controls using the same conditions as in Panel B. As seen, we could detect GPRC6A in primary calvarial osteoblasts. Additional mouse tissues were used as positive and negative controls. OS= primary mouse calvarial osteoblasts; Hrt= mouse heart; Intest= mouse intestine; Kdn=mouse kidney, H 2 0 was a negative control

    Techniques Used: Staining, Reverse Transcription Polymerase Chain Reaction, Synthesized, Polymerase Chain Reaction, Expressing, Negative Control

    59) Product Images from "A preliminary analysis of hepatitis C virus in pancreatic islet cells"

    Article Title: A preliminary analysis of hepatitis C virus in pancreatic islet cells

    Journal: Virology Journal

    doi: 10.1186/s12985-017-0905-3

    Total RNA from 3 different human islets donors was isolated using TRIzol reagent in combination with the RNeasy Mini kit followed by DNase treatment. 500 ng of total RNA were retrotranscribed using the Superscript III kit. The cDNAs obtained after retrotranscription were used as templates for quantitative real-time RT-PCR for mRNAs corresponding to the HCV entry factors CD81, occludin, claudin-1, and SR-B1. The relative amount of specific mRNA was normalized to Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). mRNA levels in Huh7.5 cells and human islets are relative to those in 293 T cells. Bars represent means ± SEM from three independent experiments
    Figure Legend Snippet: Total RNA from 3 different human islets donors was isolated using TRIzol reagent in combination with the RNeasy Mini kit followed by DNase treatment. 500 ng of total RNA were retrotranscribed using the Superscript III kit. The cDNAs obtained after retrotranscription were used as templates for quantitative real-time RT-PCR for mRNAs corresponding to the HCV entry factors CD81, occludin, claudin-1, and SR-B1. The relative amount of specific mRNA was normalized to Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). mRNA levels in Huh7.5 cells and human islets are relative to those in 293 T cells. Bars represent means ± SEM from three independent experiments

    Techniques Used: Isolation, Quantitative RT-PCR

    60) Product Images from "TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562. TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562"

    Article Title: TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562. TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562

    Journal: Physiological Reports

    doi: 10.14814/phy2.13796

    si RNA knockdown of TRPM 7 channel abrogates Ca 2+ influx and I spont in K562 cells. (A) detection of TRPM 7 transcripts (398 bp) by conventional RT ‐ PCR . Ten μ mol/L doxycycline ( DXC ) almost completely eliminated TRPM 7 mRNA expression. (B) immunoblots of K562 protein extracts by TRPM 7 antibody. Doxycycline concentration dependently (0.1–10 μ mol/L) reduced TRPM 7 protein expression. The results shown in A and B are representative of three independent experiments. (C) fura‐2 Ca 2+ fluorescence imaging from untreated ([ DXC (−)] and 10 μ mol/L doxycycline‐treated [ DXC (+)] K562 cells stably expressing tet ‐inducible TRPM 7‐specific si RNA (si TRPM 7). n = 36 and 26, respectively. (D) density of I spont recorded by conventional whole‐cell recording from untreated ([ DXC (−)] and 10 μ mol/L doxycycline‐treated [ DXC (+)] K562 cells stably expressing si TRPM 7. To facilitate the induction of TRPM 7 currents, ATP and Mg 2+ were absent in the patch pipette. The numbers of cells tested are shown in parentheses.
    Figure Legend Snippet: si RNA knockdown of TRPM 7 channel abrogates Ca 2+ influx and I spont in K562 cells. (A) detection of TRPM 7 transcripts (398 bp) by conventional RT ‐ PCR . Ten μ mol/L doxycycline ( DXC ) almost completely eliminated TRPM 7 mRNA expression. (B) immunoblots of K562 protein extracts by TRPM 7 antibody. Doxycycline concentration dependently (0.1–10 μ mol/L) reduced TRPM 7 protein expression. The results shown in A and B are representative of three independent experiments. (C) fura‐2 Ca 2+ fluorescence imaging from untreated ([ DXC (−)] and 10 μ mol/L doxycycline‐treated [ DXC (+)] K562 cells stably expressing tet ‐inducible TRPM 7‐specific si RNA (si TRPM 7). n = 36 and 26, respectively. (D) density of I spont recorded by conventional whole‐cell recording from untreated ([ DXC (−)] and 10 μ mol/L doxycycline‐treated [ DXC (+)] K562 cells stably expressing si TRPM 7. To facilitate the induction of TRPM 7 currents, ATP and Mg 2+ were absent in the patch pipette. The numbers of cells tested are shown in parentheses.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Concentration Assay, Fluorescence, Imaging, Stable Transfection, Transferring

    K562 cell growth is dependent on Ca 2+ influx mediated by TRPM 7 channel. (A) Dependency of K562 cell growth on extracellular Ca 2+ concentration evaluated at 72 h after culture. Open and filled circles indicate K562 cells untreated [ DXC (‐)] or treated with 10 μ mol/L doxycycline [ DXC (+)], respectively. (B) time courses of K562 cell growth under various conditions, i.e. DXC (−) (no doxycycline and normal Ca 2+ in RPMI medium), DXC (−)0Ca 2+ (no doxycycline and Ca 2+ free in the medium), DXC (+) (10 μ mol/L doxycycline and normal Ca 2+ in the medium) and DXC (+)0Ca 2+ (10 μ mol/L doxycycline and Ca 2+ free in the medium). In both types of experiments (A and B), K562 cells stably expressing tet ‐inducible TRPM 7‐si RNA (si TRPM 7) were used. n = 5 for each condition. * P
    Figure Legend Snippet: K562 cell growth is dependent on Ca 2+ influx mediated by TRPM 7 channel. (A) Dependency of K562 cell growth on extracellular Ca 2+ concentration evaluated at 72 h after culture. Open and filled circles indicate K562 cells untreated [ DXC (‐)] or treated with 10 μ mol/L doxycycline [ DXC (+)], respectively. (B) time courses of K562 cell growth under various conditions, i.e. DXC (−) (no doxycycline and normal Ca 2+ in RPMI medium), DXC (−)0Ca 2+ (no doxycycline and Ca 2+ free in the medium), DXC (+) (10 μ mol/L doxycycline and normal Ca 2+ in the medium) and DXC (+)0Ca 2+ (10 μ mol/L doxycycline and Ca 2+ free in the medium). In both types of experiments (A and B), K562 cells stably expressing tet ‐inducible TRPM 7‐si RNA (si TRPM 7) were used. n = 5 for each condition. * P

    Techniques Used: Concentration Assay, Stable Transfection, Expressing

    Erythroid differentiation of K562 cell by hemin is regulated by Ca 2+ influx mediated by TRPM 7. (A) representative RT ‐ PCR experiments showing hemin‐induced γ ‐globin synthesis in K562 cells untreated (control) and treated with 10 μ mol/L FTY 720 (+ FTY ), 1 mmol/L EGTA (+ EGTA ) or 10 μ mol/L doxycycline (+dxc). In all conditions, K562 cells stably expressing tet ‐inducible TRPM 7‐si RNA (si TRPM 7) were used. The expression of γ ‐globin mRNA was greatly decreased by these procedues, while that of GAPDH stayed almost constant. (B and C) statistical evaluation of pooled data from experiments as shown in A: 10 μ mol/L FTY 720 and 1 mmol/L EGTA (B): si TRPM 7 (C). (D and E) effects of 10 μ mol/L FTY 720 ( FTY ), 1 mmol/L EGTA ( EGTA ) or 10 μ mol/L doxycycline (+dxc) on hemoglobin synthesis at rest or 3 days after hemin treatment in K562‐cells stably expressing si TRPM 7. n = 5 for each condition. * P
    Figure Legend Snippet: Erythroid differentiation of K562 cell by hemin is regulated by Ca 2+ influx mediated by TRPM 7. (A) representative RT ‐ PCR experiments showing hemin‐induced γ ‐globin synthesis in K562 cells untreated (control) and treated with 10 μ mol/L FTY 720 (+ FTY ), 1 mmol/L EGTA (+ EGTA ) or 10 μ mol/L doxycycline (+dxc). In all conditions, K562 cells stably expressing tet ‐inducible TRPM 7‐si RNA (si TRPM 7) were used. The expression of γ ‐globin mRNA was greatly decreased by these procedues, while that of GAPDH stayed almost constant. (B and C) statistical evaluation of pooled data from experiments as shown in A: 10 μ mol/L FTY 720 and 1 mmol/L EGTA (B): si TRPM 7 (C). (D and E) effects of 10 μ mol/L FTY 720 ( FTY ), 1 mmol/L EGTA ( EGTA ) or 10 μ mol/L doxycycline (+dxc) on hemoglobin synthesis at rest or 3 days after hemin treatment in K562‐cells stably expressing si TRPM 7. n = 5 for each condition. * P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Expressing

    61) Product Images from "Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens"

    Article Title: Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0196913

    Comparison of sera exosomal RNA using four different RNA extraction methods. (A) Total RNA yield from ultracentrifugation (UC) and ExoQuick (EQ) treated samples using the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and AllPrep DNA/RNA Mini Kit. (B) Demonstration of RNA quality measured by OD 260 /OD 280 in EQ and UC treated samples. Data are shown as the mean ± SD from six independent patient samples. *** P
    Figure Legend Snippet: Comparison of sera exosomal RNA using four different RNA extraction methods. (A) Total RNA yield from ultracentrifugation (UC) and ExoQuick (EQ) treated samples using the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and AllPrep DNA/RNA Mini Kit. (B) Demonstration of RNA quality measured by OD 260 /OD 280 in EQ and UC treated samples. Data are shown as the mean ± SD from six independent patient samples. *** P

    Techniques Used: RNA Extraction

    Exosomal RNAs are stable over two decades. Dot plots of total RNA yield and OD 260 /OD 280 from 105 EQ-treated archival patient samples using the RNeasy Mini Kit combined with TRIzol LS. Storage time for each sample is indicated on the x-axis. The Spearman’s rank correlations for RNA yield versus storing time and OD 260 /OD 280 versus storage time are 0.185 ( P = 0.06) and 0.04 ( P = 0.70), respectively.
    Figure Legend Snippet: Exosomal RNAs are stable over two decades. Dot plots of total RNA yield and OD 260 /OD 280 from 105 EQ-treated archival patient samples using the RNeasy Mini Kit combined with TRIzol LS. Storage time for each sample is indicated on the x-axis. The Spearman’s rank correlations for RNA yield versus storing time and OD 260 /OD 280 versus storage time are 0.185 ( P = 0.06) and 0.04 ( P = 0.70), respectively.

    Techniques Used:

    62) Product Images from "Comparison of RNA Isolation Methods From Insect Larvae"

    Article Title: Comparison of RNA Isolation Methods From Insect Larvae

    Journal: Journal of Insect Science

    doi: 10.1093/jisesa/ieu130

    Agilent 2100 Bioanalyser electropherogram graphs showing RNA extracted using the RNeasy Mini Kit, a CTAB-based protocol, TRIzol reagent and the SV Total RNA kit Markers can be seen at 25 nt, 18S RNA subunit at 2,000 nt, the 28S large subunit at 3,800 nt, and a combination of 5S, 5.8S, and tRNAs at ± 180 nt.
    Figure Legend Snippet: Agilent 2100 Bioanalyser electropherogram graphs showing RNA extracted using the RNeasy Mini Kit, a CTAB-based protocol, TRIzol reagent and the SV Total RNA kit Markers can be seen at 25 nt, 18S RNA subunit at 2,000 nt, the 28S large subunit at 3,800 nt, and a combination of 5S, 5.8S, and tRNAs at ± 180 nt.

    Techniques Used:

    Pseudo-gel image produced using an Agilent 2100 Bioanalyser, showing the results of RNA extracted from T. leucotreta using the RNeasy Mini Kit, a CTAB-based protocol, TRIzol reagent, and the SV Total RNA kit. The 18S RNA subunit is visible at 2,000 nt, the 28S large subunit at 3,800 nt, and a combination of 5S, 5.8S, and tRNAs at ± 180 nt.
    Figure Legend Snippet: Pseudo-gel image produced using an Agilent 2100 Bioanalyser, showing the results of RNA extracted from T. leucotreta using the RNeasy Mini Kit, a CTAB-based protocol, TRIzol reagent, and the SV Total RNA kit. The 18S RNA subunit is visible at 2,000 nt, the 28S large subunit at 3,800 nt, and a combination of 5S, 5.8S, and tRNAs at ± 180 nt.

    Techniques Used: Produced

    63) Product Images from "Bias in recent miRBase annotations potentially associated with RNA quality issues"

    Article Title: Bias in recent miRBase annotations potentially associated with RNA quality issues

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-05070-0

    Experimental design. ( a ) Liver, heart and brain of male mice were harvested immediately after death, divided into 8 parts of about equal size, and stored at either 4 °C or at room temperature (RT) for the indicated time periods before RNA isolation. Experiments were performed in biological triplicates. RNA integrity was measured with Bioanalyzer. Gel-like image of brain tissue is given as example. MiRNA expression profiles of one replicate were measured using microarrays. ( b ) Liver tissue of 3 male mice was harvested immediately after death and divided into 5 parts of about equal size. Three parts were immediately transferred into RNAlater (0 h), two parts were stored for 96 h at room temperature (96 h). Two samples (0 h and 96 h) were isolated using standard procedure with miRNeasy Kit without DNase digestion. Two samples (0 h and 96 h) were isolated with optional DNase digestion to exclude DNA background. From the remaining undegraded sample (0 h), total RNA without small RNAs was isolated using RNeasy Kit with optional DNase digestion. Isolated RNA was further treated with 0 U, 0.026 U and 0.67 U RNase for 30 min to generate artificial RNA degradation. RNA integrity was measured with Bioanalyzer. MiRNA expression profiles of all replicates were measured using microarrays. The schematic drawings were prepared using the Biomedical-PPT-Toolkit-Suite from Motifolio Inc., USA.
    Figure Legend Snippet: Experimental design. ( a ) Liver, heart and brain of male mice were harvested immediately after death, divided into 8 parts of about equal size, and stored at either 4 °C or at room temperature (RT) for the indicated time periods before RNA isolation. Experiments were performed in biological triplicates. RNA integrity was measured with Bioanalyzer. Gel-like image of brain tissue is given as example. MiRNA expression profiles of one replicate were measured using microarrays. ( b ) Liver tissue of 3 male mice was harvested immediately after death and divided into 5 parts of about equal size. Three parts were immediately transferred into RNAlater (0 h), two parts were stored for 96 h at room temperature (96 h). Two samples (0 h and 96 h) were isolated using standard procedure with miRNeasy Kit without DNase digestion. Two samples (0 h and 96 h) were isolated with optional DNase digestion to exclude DNA background. From the remaining undegraded sample (0 h), total RNA without small RNAs was isolated using RNeasy Kit with optional DNase digestion. Isolated RNA was further treated with 0 U, 0.026 U and 0.67 U RNase for 30 min to generate artificial RNA degradation. RNA integrity was measured with Bioanalyzer. MiRNA expression profiles of all replicates were measured using microarrays. The schematic drawings were prepared using the Biomedical-PPT-Toolkit-Suite from Motifolio Inc., USA.

    Techniques Used: Mouse Assay, Isolation, Expressing

    64) Product Images from "Glycosylation is an Androgen-Regulated Process Essential for Prostate Cancer Cell Viability"

    Article Title: Glycosylation is an Androgen-Regulated Process Essential for Prostate Cancer Cell Viability

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2016.04.018

    Reciprocal gene expression signatures identify a core set of clinically relevant androgen-regulated target genes. (A) RNA-Seq expression analysis of LNCaP cells treated with or without 10 nM R1881 (a synthetic androgen) for 24 h in triplicate, and of 7 PCa patient prostate biopsies before and after androgen ablation therapy (ADT). Venn diagrams show the number of genes with significant differential expression (q
    Figure Legend Snippet: Reciprocal gene expression signatures identify a core set of clinically relevant androgen-regulated target genes. (A) RNA-Seq expression analysis of LNCaP cells treated with or without 10 nM R1881 (a synthetic androgen) for 24 h in triplicate, and of 7 PCa patient prostate biopsies before and after androgen ablation therapy (ADT). Venn diagrams show the number of genes with significant differential expression (q

    Techniques Used: Expressing, RNA Sequencing Assay

    65) Product Images from "Knockdown of LMP1-induced miR-155 sensitizes nasopharyngeal carcinoma cells to radiotherapy in vitro"

    Article Title: Knockdown of LMP1-induced miR-155 sensitizes nasopharyngeal carcinoma cells to radiotherapy in vitro

    Journal: Oncology Letters

    doi: 10.3892/ol.2016.4400

    LMP1 of Epstein-Barr virus promotes miR-155 expression in CNE-2 cells. CNE-2 cells were separated into three groups: Non-transfected CNE-2 cells (Blank), CNE-2 cells transfected with empty pcDNA3.1 as negative control (Con) and LMP1-pcDNA3.1-transfected CNE-2 cells (LMP1). (A) Relative mRNA expression levels of LMP1 in the three groups of CNE-2 cells, compared with the levels of GAPDH. (B) Percentage of LMP1 expression at the protein level in the three CNE-2 cell groups, as revealed by western blot analysis. (C) Relative mRNA levels of LMP1 vs. GAPDH in the three groups of CNE-2 cells upon a number of serial passages, as determined by reverse transcription-quantitative polymerase chain reaction. (D) Overexpression of LMP1 protein in CNE-2 cells following a number of serial passages. (E) Relative miR-155 levels in the three groups of CNE-2 cells, compared with the levels of U6 snRNA. (F) Relative miR-155 levels in CNE-2 cells transfected with various concentrations of LMP1-pcDNA3.1, compared with the levels of U6 snRNA. *P
    Figure Legend Snippet: LMP1 of Epstein-Barr virus promotes miR-155 expression in CNE-2 cells. CNE-2 cells were separated into three groups: Non-transfected CNE-2 cells (Blank), CNE-2 cells transfected with empty pcDNA3.1 as negative control (Con) and LMP1-pcDNA3.1-transfected CNE-2 cells (LMP1). (A) Relative mRNA expression levels of LMP1 in the three groups of CNE-2 cells, compared with the levels of GAPDH. (B) Percentage of LMP1 expression at the protein level in the three CNE-2 cell groups, as revealed by western blot analysis. (C) Relative mRNA levels of LMP1 vs. GAPDH in the three groups of CNE-2 cells upon a number of serial passages, as determined by reverse transcription-quantitative polymerase chain reaction. (D) Overexpression of LMP1 protein in CNE-2 cells following a number of serial passages. (E) Relative miR-155 levels in the three groups of CNE-2 cells, compared with the levels of U6 snRNA. (F) Relative miR-155 levels in CNE-2 cells transfected with various concentrations of LMP1-pcDNA3.1, compared with the levels of U6 snRNA. *P

    Techniques Used: Expressing, Transfection, Negative Control, Western Blot, Real-time Polymerase Chain Reaction, Over Expression

    66) Product Images from "Differential association of microRNAs with polysomes reflects distinct strengths of interactions with their mRNA targets"

    Article Title: Differential association of microRNAs with polysomes reflects distinct strengths of interactions with their mRNA targets

    Journal: RNA

    doi: 10.1261/rna.033142.112

    High-throughput method for measuring polysome occupancy of microRNAs. ( A ) Schematics of the approach. MicroRNA occupancies were computed based on the levels measured in the “polysome-bound” and “ribosome-free” fractions
    Figure Legend Snippet: High-throughput method for measuring polysome occupancy of microRNAs. ( A ) Schematics of the approach. MicroRNA occupancies were computed based on the levels measured in the “polysome-bound” and “ribosome-free” fractions

    Techniques Used: High Throughput Screening Assay

    The mature form of the microRNA, but not its abundance, specifies its preference for low or high occupancy. Polysome occupancy and expression of let-7b ( A ) and miR-598 ( B ) measured in (1) hESCs transfected with the mature form of the microRNAs (overexpression
    Figure Legend Snippet: The mature form of the microRNA, but not its abundance, specifies its preference for low or high occupancy. Polysome occupancy and expression of let-7b ( A ) and miR-598 ( B ) measured in (1) hESCs transfected with the mature form of the microRNAs (overexpression

    Techniques Used: Expressing, Transfection, Over Expression

    Dependence of microRNA occupancy on cellular context. ( A ) Distributions of microRNA occupancies evaluated in undifferentiated human embryonic stem cells (hESCs) and human foreskin fibroblasts (hFFs). ( B ) Color-coded heat map displaying side-by-side measurements
    Figure Legend Snippet: Dependence of microRNA occupancy on cellular context. ( A ) Distributions of microRNA occupancies evaluated in undifferentiated human embryonic stem cells (hESCs) and human foreskin fibroblasts (hFFs). ( B ) Color-coded heat map displaying side-by-side measurements

    Techniques Used:

    MicroRNA occupancy is functionally dependent on the choice of seed. ( A ) Schematics of chimeric microRNAs designed for testing relative contributions of the microRNA seed and body to the degree of its association with polysomes. ( B ) Polysome occupancies
    Figure Legend Snippet: MicroRNA occupancy is functionally dependent on the choice of seed. ( A ) Schematics of chimeric microRNAs designed for testing relative contributions of the microRNA seed and body to the degree of its association with polysomes. ( B ) Polysome occupancies

    Techniques Used:

    67) Product Images from "Characterization of macroautophagic flux in vivo using a leupeptin-based assay"

    Article Title: Characterization of macroautophagic flux in vivo using a leupeptin-based assay

    Journal: Autophagy

    doi: 10.4161/auto.7.6.15100

    Analysis of basal macroautophagic flux in beclin 1 +/+ and beclin 1 +/− mice using the leupeptin assay. Mice were injected with PBS or 40 mg/kg leupeptin and sacrificed 0–180 min later (n = 3–4 mice per time point). (A) Western blot
    Figure Legend Snippet: Analysis of basal macroautophagic flux in beclin 1 +/+ and beclin 1 +/− mice using the leupeptin assay. Mice were injected with PBS or 40 mg/kg leupeptin and sacrificed 0–180 min later (n = 3–4 mice per time point). (A) Western blot

    Techniques Used: Mouse Assay, Injection, Western Blot

    68) Product Images from "Role of the Phosphatidylinositol-3-Kinase and Extracellular Regulated Kinase Pathways in the Induction of Hypoxia-Inducible Factor (HIF)-1 Activity and the HIF-1 Target Vascular Endothelial Growth Factor in Ovarian Granulosa Cells in Response to Follicle-Stimulating Hormone"

    Article Title: Role of the Phosphatidylinositol-3-Kinase and Extracellular Regulated Kinase Pathways in the Induction of Hypoxia-Inducible Factor (HIF)-1 Activity and the HIF-1 Target Vascular Endothelial Growth Factor in Ovarian Granulosa Cells in Response to Follicle-Stimulating Hormone

    Journal: Endocrinology

    doi: 10.1210/en.2008-0850

    Interaction of HIF-1 with the VEGF promoter is abrogated by PD98059. ChIP assays were performed as described in Materials and Methods . GCs were pretreated with DMSO vehicle or 50 μ m PD98059 for 1 h and then left untreated (CON) and treated with
    Figure Legend Snippet: Interaction of HIF-1 with the VEGF promoter is abrogated by PD98059. ChIP assays were performed as described in Materials and Methods . GCs were pretreated with DMSO vehicle or 50 μ m PD98059 for 1 h and then left untreated (CON) and treated with

    Techniques Used: Chromatin Immunoprecipitation

    The PI3-kinase/mTOR inhibitor LY294002 inhibits FSH-stimulated HIF-1α accumulation more than the mTOR inhibitor rapamycin. GCs were pretreated with DMSO vehicle (Veh) or 12.5 μ m LY294002 for 1 h, 50 μ m PD98059 for 1 h, or 100 n
    Figure Legend Snippet: The PI3-kinase/mTOR inhibitor LY294002 inhibits FSH-stimulated HIF-1α accumulation more than the mTOR inhibitor rapamycin. GCs were pretreated with DMSO vehicle (Veh) or 12.5 μ m LY294002 for 1 h, 50 μ m PD98059 for 1 h, or 100 n

    Techniques Used:

    69) Product Images from "MicroRNA-375 Suppresses Extracellular Matrix Degradation and Invadopodial Activity in Head and Neck Squamous Cell Carcinoma"

    Article Title: MicroRNA-375 Suppresses Extracellular Matrix Degradation and Invadopodial Activity in Head and Neck Squamous Cell Carcinoma

    Journal: Archives of pathology & laboratory medicine

    doi: 10.5858/arpa.2014-0471-OA

    Elevated expression of microRNA-375 in HNSCC cells significantly reduces kallikrein 6, kallikrein 10, and MMP-9 messenger RNA (mRNA) expression. The mRNA expression levels in the transductant lines were measured by TaqMan quantitative real-time polymerase
    Figure Legend Snippet: Elevated expression of microRNA-375 in HNSCC cells significantly reduces kallikrein 6, kallikrein 10, and MMP-9 messenger RNA (mRNA) expression. The mRNA expression levels in the transductant lines were measured by TaqMan quantitative real-time polymerase

    Techniques Used: Expressing

    70) Product Images from "Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity"

    Article Title: Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2015-0095OC

    Immunophenotyping of PDGFRα + and PDGFRα ⁻ fibroblasts in adult mouse lungs. Lin ⁻ (CD45 ⁻ CD326 ⁻ CD31 ⁻ ) stromal cells were gated for CD140α + ( green ) or CD140α ( red ) subpopulations, and
    Figure Legend Snippet: Immunophenotyping of PDGFRα + and PDGFRα ⁻ fibroblasts in adult mouse lungs. Lin ⁻ (CD45 ⁻ CD326 ⁻ CD31 ⁻ ) stromal cells were gated for CD140α + ( green ) or CD140α ( red ) subpopulations, and

    Techniques Used:

    71) Product Images from "Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation"

    Article Title: Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation

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

    doi: 10.1073/pnas.1613156113

    Microtubules are not critical for activation of the Nlrp3 inflammasome. LPS-primed BMDMs were pretreated with colchicine, nocodazole, ABT-751, CA4P, or CYT997 before stimulation with nigericin. Combined supernatant and cell lysates were immunoblotted
    Figure Legend Snippet: Microtubules are not critical for activation of the Nlrp3 inflammasome. LPS-primed BMDMs were pretreated with colchicine, nocodazole, ABT-751, CA4P, or CYT997 before stimulation with nigericin. Combined supernatant and cell lysates were immunoblotted

    Techniques Used: Activation Assay

    TcdA and TcdB activate the Pyrin inflammasome. ( A and B ) LPS-primed BMDMs were stimulated with TcdB. Samples were immunoblotted for IL-1β ( A ), and cell-culture supernatants were collected and analyzed for IL-1β ( B ). ( C – F ) LPS-primed
    Figure Legend Snippet: TcdA and TcdB activate the Pyrin inflammasome. ( A and B ) LPS-primed BMDMs were stimulated with TcdB. Samples were immunoblotted for IL-1β ( A ), and cell-culture supernatants were collected and analyzed for IL-1β ( B ). ( C – F ) LPS-primed

    Techniques Used: Cell Culture

    Microtubule-depolymerizing drugs do not inhibit activation of Nlrp1b, Nlrp3, Nlrc4, and AIM2 inflammasomes. ( A – D ) LPS-primed BMDMs were pretreated with colchicine before stimulation with activators of the Nlrp1b (anthrax lethal toxin; LeTx) (
    Figure Legend Snippet: Microtubule-depolymerizing drugs do not inhibit activation of Nlrp1b, Nlrp3, Nlrc4, and AIM2 inflammasomes. ( A – D ) LPS-primed BMDMs were pretreated with colchicine before stimulation with activators of the Nlrp1b (anthrax lethal toxin; LeTx) (

    Techniques Used: Activation Assay

    Actin dynamics and microtubule stabilization are not required for activation of the Pyrin inflammasome. ( A and B ) LPS-primed BMDMs ( A ) and PBMCs from healthy donors ( n = 3) ( B ) were pretreated with STI-571, blebbistatin, Y-27632, or cytochalasin D. Stimulation
    Figure Legend Snippet: Actin dynamics and microtubule stabilization are not required for activation of the Pyrin inflammasome. ( A and B ) LPS-primed BMDMs ( A ) and PBMCs from healthy donors ( n = 3) ( B ) were pretreated with STI-571, blebbistatin, Y-27632, or cytochalasin D. Stimulation

    Techniques Used: Activation Assay

    Microtubule depolymerizing drugs specifically inhibit the Pyrin inflammasome. ( A and B ) Wild-type LPS-primed BMDMs were pretreated with lumicolchicine or colchicine before stimulation with TcdA. Samples were immunoblotted for caspase-1 and IL-1β
    Figure Legend Snippet: Microtubule depolymerizing drugs specifically inhibit the Pyrin inflammasome. ( A and B ) Wild-type LPS-primed BMDMs were pretreated with lumicolchicine or colchicine before stimulation with TcdA. Samples were immunoblotted for caspase-1 and IL-1β

    Techniques Used:

    Pyrin is up-regulated upon LPS priming. ( A – C ) Wild-type unprimed or LPS-primed BMDMs were stimulated with TcdA or TcdB. Combined supernatant and cell lysates were immunoblotted for caspase-1 and IL-1β ( A ), and supernatants were analyzed
    Figure Legend Snippet: Pyrin is up-regulated upon LPS priming. ( A – C ) Wild-type unprimed or LPS-primed BMDMs were stimulated with TcdA or TcdB. Combined supernatant and cell lysates were immunoblotted for caspase-1 and IL-1β ( A ), and supernatants were analyzed

    Techniques Used:

    72) Product Images from "Identification of a unique gene expression signature in mercury and 2,3,7,8-tetrachlorodibenzo-p-dioxin co-exposed cells"

    Article Title: Identification of a unique gene expression signature in mercury and 2,3,7,8-tetrachlorodibenzo-p-dioxin co-exposed cells

    Journal: Toxicology research

    doi: 10.1039/C6TX00432F

    Differential gene expression (≥1.5 fold up- or down-regulated compared to control) identified using RNA-Seq analysis in BEAS-2B cells treated with (i) Hg, (ii) TCDD and (iii) Hg and TCDD in combination. (A) Low-dose exposure and (B) high-dose exposure. Co-exposure to Hg and TCDD induces the differential expression of 486 (low-dose) and 190 (high-dose) genes that are not transcriptionally altered by either Hg or TCDD alone.
    Figure Legend Snippet: Differential gene expression (≥1.5 fold up- or down-regulated compared to control) identified using RNA-Seq analysis in BEAS-2B cells treated with (i) Hg, (ii) TCDD and (iii) Hg and TCDD in combination. (A) Low-dose exposure and (B) high-dose exposure. Co-exposure to Hg and TCDD induces the differential expression of 486 (low-dose) and 190 (high-dose) genes that are not transcriptionally altered by either Hg or TCDD alone.

    Techniques Used: Expressing, RNA Sequencing Assay

    73) Product Images from "IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation *"

    Article Title: IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.645960

    IPO3 deficiency is associated with defective nuclear import of NEMO. A , control or HeLa cell clones stably expressing the indicated IPO3 shRNA constructs were left untreated or treated with doxorubicin (8 μg/ml, 2 h). NF-κB activation
    Figure Legend Snippet: IPO3 deficiency is associated with defective nuclear import of NEMO. A , control or HeLa cell clones stably expressing the indicated IPO3 shRNA constructs were left untreated or treated with doxorubicin (8 μg/ml, 2 h). NF-κB activation

    Techniques Used: Clone Assay, Stable Transfection, Expressing, shRNA, Construct, Activation Assay

    74) Product Images from "Small RNAs Control Sodium Channel Expression, Nociceptor Excitability, and Pain Thresholds"

    Article Title: Small RNAs Control Sodium Channel Expression, Nociceptor Excitability, and Pain Thresholds

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1980-10.2010

    Microarray analysis. A , Summary of changed transcripts in Dicer conditional null mutant. After isolation of total RNA from DRGs of Dicer conditional null mice ( n = 6, males) and floxed littermate control ( n = 6, males), the expression of mRNA was detected with GeneChip (mouse genome 430 2.0 array). Four thousand six hundred and eighteen (4618) transcripts ( p
    Figure Legend Snippet: Microarray analysis. A , Summary of changed transcripts in Dicer conditional null mutant. After isolation of total RNA from DRGs of Dicer conditional null mice ( n = 6, males) and floxed littermate control ( n = 6, males), the expression of mRNA was detected with GeneChip (mouse genome 430 2.0 array). Four thousand six hundred and eighteen (4618) transcripts ( p

    Techniques Used: Microarray, Mutagenesis, Isolation, Mouse Assay, Expressing

    Representative qRT-PCR analysis of selected transcripts and correlation between fold decrease in Nav1.8 + ) and fold decrease in Dicer conditional null mutants. A , After isolation of DRG, total RNA was extracted from DRG. Representative mRNA and pre-mRNAs were subjected to analysis by qRT-PCR. Some of the DRG-specific gene (Edg7, TRPC6, Nav1.9, Nav1.8, Nav1.7, Runx1, CaMKIIa, and Mrgpra3) transcripts were significantly downregulated in Dicer knock-out mice. B , Representative DRG nonspecific gene (Mtap2, Gbp1, ApoD, and Rgs5) transcripts show significant upregulation in Dicer knock-out mice. C , The pre-mRNAs of DRG-specific genes (Nav1.8, Nav1.9) and Edg7 and CaMKIIa were downregulated in Dicer knock-out mice. No alteration of pre-mRNA of ApoD was detected in Dicer knock-out mice. The data are normalized with β-actin. Each bar represents n = 3 mice. All data are presented as means ± SEM; * p
    Figure Legend Snippet: Representative qRT-PCR analysis of selected transcripts and correlation between fold decrease in Nav1.8 + ) and fold decrease in Dicer conditional null mutants. A , After isolation of DRG, total RNA was extracted from DRG. Representative mRNA and pre-mRNAs were subjected to analysis by qRT-PCR. Some of the DRG-specific gene (Edg7, TRPC6, Nav1.9, Nav1.8, Nav1.7, Runx1, CaMKIIa, and Mrgpra3) transcripts were significantly downregulated in Dicer knock-out mice. B , Representative DRG nonspecific gene (Mtap2, Gbp1, ApoD, and Rgs5) transcripts show significant upregulation in Dicer knock-out mice. C , The pre-mRNAs of DRG-specific genes (Nav1.8, Nav1.9) and Edg7 and CaMKIIa were downregulated in Dicer knock-out mice. No alteration of pre-mRNA of ApoD was detected in Dicer knock-out mice. The data are normalized with β-actin. Each bar represents n = 3 mice. All data are presented as means ± SEM; * p

    Techniques Used: Quantitative RT-PCR, Isolation, Knock-Out, Mouse Assay

    75) Product Images from "Evaluation of cell damage induced by irradiated Zinc-Phthalocyanine-gold dendrimeric nanoparticles in a breast cancer cell line"

    Article Title: Evaluation of cell damage induced by irradiated Zinc-Phthalocyanine-gold dendrimeric nanoparticles in a breast cancer cell line

    Journal: Biomedical Journal

    doi: 10.1016/j.bj.2018.05.002

    The primary response of MPDC-mediated PDT on the expression of genes involved in cell death pathways was the up-regulation of Ulk-1, Bax, Casp-2 and Bcl-2 genes. The Ulk-1 protein protonates and activates the FIP200. ULK is part of a protein complex containing Atg13, Atg17 and FIP200 (autophagosome), which drives the subsequent cellular damage and death. The Bax protein directly affects the mitochondria while the Cas-2 protein is activated by reactive oxygen species (ROS) and then Casp-2 transforms a mitochondrial damaging protein into its truncated and activated form (tBid). The p53-induced death domain associated protein (PIDD) can also convert pro-Casp-2 into the active Casp-2. Apoptogenic proteins (such as Cytochrome C) released from mitochondria participate in the assemblage of the apoptosome, activation of other effectors (Casp-3/6/7) and cell death. Mitochondrial damage and depolarization induce change in cellular ATP levels, activation of the 5′ adenosine monophosphate activated protein kinase (AMPK) and AMPK-induced cell death. This cell death response stimulates Bcl-2 protein to prevent further cell damage.
    Figure Legend Snippet: The primary response of MPDC-mediated PDT on the expression of genes involved in cell death pathways was the up-regulation of Ulk-1, Bax, Casp-2 and Bcl-2 genes. The Ulk-1 protein protonates and activates the FIP200. ULK is part of a protein complex containing Atg13, Atg17 and FIP200 (autophagosome), which drives the subsequent cellular damage and death. The Bax protein directly affects the mitochondria while the Cas-2 protein is activated by reactive oxygen species (ROS) and then Casp-2 transforms a mitochondrial damaging protein into its truncated and activated form (tBid). The p53-induced death domain associated protein (PIDD) can also convert pro-Casp-2 into the active Casp-2. Apoptogenic proteins (such as Cytochrome C) released from mitochondria participate in the assemblage of the apoptosome, activation of other effectors (Casp-3/6/7) and cell death. Mitochondrial damage and depolarization induce change in cellular ATP levels, activation of the 5′ adenosine monophosphate activated protein kinase (AMPK) and AMPK-induced cell death. This cell death response stimulates Bcl-2 protein to prevent further cell damage.

    Techniques Used: Expressing, Activation Assay

    Gene expression profiles of PDT-treated MCF-7 cells with 0.3 μM MPDC and 10 J/cm 2 was analyzed using the SABiosciences Human Cell Death Pathway Finder Profiler™ PCR Array System. PDT-induced changes in gene expression and BAX, BCL-2, CASP-2 and ULK-1 genes were significantly up-regulated as represented in the volcano plot. In the volcano plot, the horizontal line designates the target threshold ( p = 0.05) and vertical lines, the fold change (central) and target fold change threshold (peripheral) in gene expression.
    Figure Legend Snippet: Gene expression profiles of PDT-treated MCF-7 cells with 0.3 μM MPDC and 10 J/cm 2 was analyzed using the SABiosciences Human Cell Death Pathway Finder Profiler™ PCR Array System. PDT-induced changes in gene expression and BAX, BCL-2, CASP-2 and ULK-1 genes were significantly up-regulated as represented in the volcano plot. In the volcano plot, the horizontal line designates the target threshold ( p = 0.05) and vertical lines, the fold change (central) and target fold change threshold (peripheral) in gene expression.

    Techniques Used: Expressing, Polymerase Chain Reaction

    Morphology of untreated, irradiated, MPDC-treated and PDT-treated MCF-7 cells. No morphological change was noted in irradiated or MPDC treated cells when compared to untreated cells. The morphology of PDT-treated MCF-7 cells changed, include an elongation of cells, decrease in cell number, detachment and rounding off (200× magnification).
    Figure Legend Snippet: Morphology of untreated, irradiated, MPDC-treated and PDT-treated MCF-7 cells. No morphological change was noted in irradiated or MPDC treated cells when compared to untreated cells. The morphology of PDT-treated MCF-7 cells changed, include an elongation of cells, decrease in cell number, detachment and rounding off (200× magnification).

    Techniques Used: Irradiation

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    Polymerase Chain Reaction:

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    Multiplex Assay:

    Article Title: Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey
    Article Snippet: The use of this extraction method in addition to the multiplex RT‐PCR assay, allows a rapid approach for the differential detection of VSV‐IND and VSV‐NJ a single assay. .. In summary, the results of the study showed that the use of Qiagen RNeasy mini kit in addition to the one‐step RT‐PCR kit (Qiagen, Valencia, CA) as a simple, practicable, and reliable tool for rapid, highly sensitive, and specific differential diagnosis of VSV‐IND and VSV‐NJ in cell lysate and spiked samples.

    RNA Expression:

    Article Title: Receptor for advanced glycation end products involved in circulating endothelial cells release from human coronary endothelial cells induced by C-reactive protein
    Article Snippet: The effect of siRNA on the RNA expression of RAGE was assessed by real-time quantitative PCR after 48 hr of transfection. .. Total cellular RNA was extracted from HCAECs using an RNA isolation kit (RNeasy Mini kit, Qiagen, Duesseldorf, Germany).

    Agarose Gel Electrophoresis:

    Article Title: TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562. TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562
    Article Snippet: Reverse transcription polymerase reaction (RT‐PCR) and real‐time PCR Total RNA (about 1 μ g) was extracted from about a million of K562 cells with the total RNA extraction kit (RNeasy Minikit, Qiagen) and reverse transcribed (in a 10 μ L scale) by SuperScriptII (Thermo Fisher Scientific) with a randomized or an oligo‐dT primer to obtain the first strand DNA. .. The PCR amplicons were electrophoresed in a 1.2–1.5% agarose gel for 45–60 min, hybridized with ethidium bromide (Nacalai Tesque, Kyoto, Japan) or GelRed™ (Biotium, Fremont, CA), and visualized by ultraviolet or Blue/Green LED light, respectively.

    Preserving:

    Article Title: Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis
    Article Snippet: RNA Purification and Complementary DNA (cDNA) Synthesis Where indicated, the total RNA obtained by Trizol extraction was purified by processing with RNeasy Mini Kit (Qiagen) or RNeasy miRNA Mini Kit (Qiagen) according to manufactureŕs instructions. .. The Agilent 2100 Bioanalyzer (Agilent Technologies) was used to profile and compare RNA fragment length from the three different preservation methods with the RNA 6000 LabChip kit (Agilent Technologies).

    Spectrophotometry:

    Article Title: Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis
    Article Snippet: RNA Purification and Complementary DNA (cDNA) Synthesis Where indicated, the total RNA obtained by Trizol extraction was purified by processing with RNeasy Mini Kit (Qiagen) or RNeasy miRNA Mini Kit (Qiagen) according to manufactureŕs instructions. .. After extraction and purification, the RNA yield and purity was determined by measuring absorbance at 260 nm/280 nm on a Nanodrop spectrophotometer (Thermo Fisher Scientific).

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    Qiagen rneasy mini kit
    Gel electrophoresis of total <t>RNA</t> that was obtained from sessile cells with sheared whole-cell lysis coupled to the <t>RNeasy</t> Mini Kit. Lanes 1 , 2 , and 3 represent three independent experiments using the methicillin-resistant S. aureus (MRSA) isolate BMB9393 ( A ) and the methicillin-susceptible S. aureus (MSSA) isolate HC474 ( B ).
    Rneasy Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 8428 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gel electrophoresis of total RNA that was obtained from sessile cells with sheared whole-cell lysis coupled to the RNeasy Mini Kit. Lanes 1 , 2 , and 3 represent three independent experiments using the methicillin-resistant S. aureus (MRSA) isolate BMB9393 ( A ) and the methicillin-susceptible S. aureus (MSSA) isolate HC474 ( B ).

    Journal: Brazilian Journal of Medical and Biological Research

    Article Title: Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus

    doi: 10.1590/1414-431X20154734

    Figure Lengend Snippet: Gel electrophoresis of total RNA that was obtained from sessile cells with sheared whole-cell lysis coupled to the RNeasy Mini Kit. Lanes 1 , 2 , and 3 represent three independent experiments using the methicillin-resistant S. aureus (MRSA) isolate BMB9393 ( A ) and the methicillin-susceptible S. aureus (MSSA) isolate HC474 ( B ).

    Article Snippet: Therefore, we designed a simple optimized protocol using the RNeasy Mini Kit that ensures high-quality RNA preparations from planktonic and sessile cells of MRSA, compatible with experiments that require integrity and good quantity of this molecule.

    Techniques: Nucleic Acid Electrophoresis, Lysis

    A) Concentration of RNA extracted from HEK293 cells encapsulated in the four peptide hydrogels and cell-only controls using the three methods (see text for details). B Representative electrophoresis traces of total RNA extracted from cells encapsulated in the four peptide hydrogels and cell-only controls using the Tri Reagent and RNeasy MK methods and corresponding RIN values: cell-only controls (A+E), PGD-Alpha1 (B+F), PGD-Alpha2 (G), PGD-AlphaProB (C+H) and PGD-AlphaProC (D+I). C D) Representative UV spectra for RNA samples extracted using the TRI Reagent (C) and the RNeasy MK (D) methods from the four peptide hydrogels and cell-only control.

    Journal: PLoS ONE

    Article Title: RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

    doi: 10.1371/journal.pone.0197517

    Figure Lengend Snippet: A) Concentration of RNA extracted from HEK293 cells encapsulated in the four peptide hydrogels and cell-only controls using the three methods (see text for details). B Representative electrophoresis traces of total RNA extracted from cells encapsulated in the four peptide hydrogels and cell-only controls using the Tri Reagent and RNeasy MK methods and corresponding RIN values: cell-only controls (A+E), PGD-Alpha1 (B+F), PGD-Alpha2 (G), PGD-AlphaProB (C+H) and PGD-AlphaProC (D+I). C D) Representative UV spectra for RNA samples extracted using the TRI Reagent (C) and the RNeasy MK (D) methods from the four peptide hydrogels and cell-only control.

    Article Snippet: RNA extraction from peptide hydrogels Attempts were made to extract RNA from cells using two commercial kits: one solution-based, TRI Reagent® (Sigma-Aldrich), and one column-based, RNeasy Mini Kit® (Qiagen).

    Techniques: Concentration Assay, Electrophoresis

    Ct values obtained for RT-qPCR performed using RNA extracted from the four peptide hydrogels as a template. RNA isolated from cells in suspension was used as a control. The RNA extracted using either the TRI Reagent method (A-B) or RNeasy MK method (C+D) was used as templates for the amplification of two housekeeping genes: GAPDH (A+C) and RPL13A (B+D). The cycle threshold (Ct) value was determined for three independent samples measured in triplicate (or two independent samples for PGD-Alpha1 using the RNeasy method). Data is shown as mean ± SEM. The mean values were compared to the non-encapsulated cell controls using a t-test; *, P ≤ 0.05.

    Journal: PLoS ONE

    Article Title: RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

    doi: 10.1371/journal.pone.0197517

    Figure Lengend Snippet: Ct values obtained for RT-qPCR performed using RNA extracted from the four peptide hydrogels as a template. RNA isolated from cells in suspension was used as a control. The RNA extracted using either the TRI Reagent method (A-B) or RNeasy MK method (C+D) was used as templates for the amplification of two housekeeping genes: GAPDH (A+C) and RPL13A (B+D). The cycle threshold (Ct) value was determined for three independent samples measured in triplicate (or two independent samples for PGD-Alpha1 using the RNeasy method). Data is shown as mean ± SEM. The mean values were compared to the non-encapsulated cell controls using a t-test; *, P ≤ 0.05.

    Article Snippet: RNA extraction from peptide hydrogels Attempts were made to extract RNA from cells using two commercial kits: one solution-based, TRI Reagent® (Sigma-Aldrich), and one column-based, RNeasy Mini Kit® (Qiagen).

    Techniques: Quantitative RT-PCR, Isolation, Amplification

    (a) Gel electrophoresis (polyacrylamide 15% stained with ethidium bromide) of LMW RNA samples (LCL) extracted with TRIzol reagent (lane 1), RNEasy kit (lane 2), and small RNA fraction enriched with MirVana kit (lane 3). LMW profile obtained with MirVana kit extraction is similar to that obtained with TRIzol reagent which is not shown for clarity. (b) Agilent 2100 Bioanalyzer electropherogram profile of LMW RNAs (LCL) extracted with TRIzol (black) superimposed on 5.8S (red), 5S (green), and tRNA (blue) bands eluted from polyacrylamide gel. (c) Lymphoblastoid (LCL) LMW RNA profile obtained after plotting the exported raw data from Agilent electropherogram ( ) together with the PeakFit fitted curve (solid line) and the component peak functions. Seven peaks below the LMW RNA profile were fitted by the software ( r 2 = 0.998693).

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Quantification of Small Non-Coding RNAs Allows an Accurate Comparison of miRNA Expression Profiles

    doi: 10.1155/2009/659028

    Figure Lengend Snippet: (a) Gel electrophoresis (polyacrylamide 15% stained with ethidium bromide) of LMW RNA samples (LCL) extracted with TRIzol reagent (lane 1), RNEasy kit (lane 2), and small RNA fraction enriched with MirVana kit (lane 3). LMW profile obtained with MirVana kit extraction is similar to that obtained with TRIzol reagent which is not shown for clarity. (b) Agilent 2100 Bioanalyzer electropherogram profile of LMW RNAs (LCL) extracted with TRIzol (black) superimposed on 5.8S (red), 5S (green), and tRNA (blue) bands eluted from polyacrylamide gel. (c) Lymphoblastoid (LCL) LMW RNA profile obtained after plotting the exported raw data from Agilent electropherogram ( ) together with the PeakFit fitted curve (solid line) and the component peak functions. Seven peaks below the LMW RNA profile were fitted by the software ( r 2 = 0.998693).

    Article Snippet: Total RNA Extraction and Small RNAs Enrichment Protocols Total RNA was extracted using three different methods: an acid phenol/guanidine isothiocyanate solution (TRIzol Reagent, Invitrogen), a glass-fiber filtration protocol (MirVana miRNA Isolation Kit, Ambion) that provides also a procedure to isolate and enrich low molecular weight (LMW) RNAs from higher molecular weight (HMW) RNAs, and another common glass-fiber purification protocol (RNEasy Mini Kit, Qiagen).

    Techniques: Nucleic Acid Electrophoresis, Staining, Software