non targeting control sirna  (Santa Cruz Biotechnology)

 
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    Name:
    Control siRNA I
    Description:
    resuspend to 66 µl 10 µM sufficient for 10 20 transfections suitable as a negative control for experiments using targeted siRNA transfection consists of a scrambled sequence that will not lead to the specific degradation of any cellular message aliquot and store at 20°C
    Catalog Number:
    SC-44237
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    None
    Category:
    Gene Editing Control siRNA I
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    Structured Review

    Santa Cruz Biotechnology non targeting control sirna
    MiR-205 modulates <t>LRRK2</t> protein expression in vitro . ( A , B ) Western blots show LRRK2 protein expression levels in pre-miR-205 oligo- or LRRK2 <t>siRNA-transfected</t> HEK293T cells (A) and primary cultured mouse cortical neurons (B). A scrambled microRNA oligo (Ctrl oligo), a scrambled siRNA (Ctrl siRNA) and a vehicle (Vcl) only were used as negative controls. β-Actin or β-III tubulin was used as a loading control. Bar graphs depict the signal intensities of LRRK2-positive bands in the vehicle, microRNA and siRNA-treated samples. ** P
    resuspend to 66 µl 10 µM sufficient for 10 20 transfections suitable as a negative control for experiments using targeted siRNA transfection consists of a scrambled sequence that will not lead to the specific degradation of any cellular message aliquot and store at 20°C
    https://www.bioz.com/result/non targeting control sirna/product/Santa Cruz Biotechnology
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    Images

    1) Product Images from "MicroRNA-205 regulates the expression of Parkinson's disease-related leucine-rich repeat kinase 2 protein"

    Article Title: MicroRNA-205 regulates the expression of Parkinson's disease-related leucine-rich repeat kinase 2 protein

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/dds470

    MiR-205 modulates LRRK2 protein expression in vitro . ( A , B ) Western blots show LRRK2 protein expression levels in pre-miR-205 oligo- or LRRK2 siRNA-transfected HEK293T cells (A) and primary cultured mouse cortical neurons (B). A scrambled microRNA oligo (Ctrl oligo), a scrambled siRNA (Ctrl siRNA) and a vehicle (Vcl) only were used as negative controls. β-Actin or β-III tubulin was used as a loading control. Bar graphs depict the signal intensities of LRRK2-positive bands in the vehicle, microRNA and siRNA-treated samples. ** P
    Figure Legend Snippet: MiR-205 modulates LRRK2 protein expression in vitro . ( A , B ) Western blots show LRRK2 protein expression levels in pre-miR-205 oligo- or LRRK2 siRNA-transfected HEK293T cells (A) and primary cultured mouse cortical neurons (B). A scrambled microRNA oligo (Ctrl oligo), a scrambled siRNA (Ctrl siRNA) and a vehicle (Vcl) only were used as negative controls. β-Actin or β-III tubulin was used as a loading control. Bar graphs depict the signal intensities of LRRK2-positive bands in the vehicle, microRNA and siRNA-treated samples. ** P

    Techniques Used: Expressing, In Vitro, Western Blot, Transfection, Cell Culture

    2) Product Images from "Calpastatin prevents NF-κB mediated hyperactivation of macrophages and attenuates colitis"

    Article Title: Calpastatin prevents NF-κB mediated hyperactivation of macrophages and attenuates colitis

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

    doi: 10.4049/jimmunol.1300972

    CAST deficiency in macrophages leads to decreased IκB and increased NF-κB translocation to the nucleus. (A) CAST deficiency in BMDM was accomplished by two different methods: left panel, pretreating WT BMDM with CAST siRNA and or non-targeting
    Figure Legend Snippet: CAST deficiency in macrophages leads to decreased IκB and increased NF-κB translocation to the nucleus. (A) CAST deficiency in BMDM was accomplished by two different methods: left panel, pretreating WT BMDM with CAST siRNA and or non-targeting

    Techniques Used: Translocation Assay

    3) Product Images from "Phosphorylation of p27Kip1 by Epstein-Barr Virus Protein Kinase Induces Its Degradation through SCFSkp2 Ubiquitin Ligase Actions during Viral Lytic Replication *"

    Article Title: Phosphorylation of p27Kip1 by Epstein-Barr Virus Protein Kinase Induces Its Degradation through SCFSkp2 Ubiquitin Ligase Actions during Viral Lytic Replication *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.015123

    The BGLF4 protein enhances p27 Kip1 degradation mediated by the ubiquitin ligase, SCF Skp2 . A , HEK293T cells transfected with the expression vector for FLAG-tagged p27 Kip1 were transfected with non-targeting/control siRNA ( si-control ) or Skp2 siRNA ( si-Skp2 ) together with expression vectors for FLAG-tagged WT BGLF4, kd BGLF4, or empty vector (−). Cells were harvested at 32 hpt, and whole cell lysates were prepared and subjected to immunoblot analysis with anti-Skp2, GAPDH antibodies, and anti-FLAG antibody to detect exogenously expressed BGLF4 ( Flag-BGLF4 ) and p27 Kip1 ( Flag-p27 ). B , 293/EBV-WT cells were subjected to transfection with the BZLF1 expression vector for induction of lytic EBV replication together with control siRNA or Skp2 siRNA and were harvested at 0, 24, and 48 hpt. Equal amounts of whole cell lysates were subjected to immunoblot analysis with the indicated antibodies.
    Figure Legend Snippet: The BGLF4 protein enhances p27 Kip1 degradation mediated by the ubiquitin ligase, SCF Skp2 . A , HEK293T cells transfected with the expression vector for FLAG-tagged p27 Kip1 were transfected with non-targeting/control siRNA ( si-control ) or Skp2 siRNA ( si-Skp2 ) together with expression vectors for FLAG-tagged WT BGLF4, kd BGLF4, or empty vector (−). Cells were harvested at 32 hpt, and whole cell lysates were prepared and subjected to immunoblot analysis with anti-Skp2, GAPDH antibodies, and anti-FLAG antibody to detect exogenously expressed BGLF4 ( Flag-BGLF4 ) and p27 Kip1 ( Flag-p27 ). B , 293/EBV-WT cells were subjected to transfection with the BZLF1 expression vector for induction of lytic EBV replication together with control siRNA or Skp2 siRNA and were harvested at 0, 24, and 48 hpt. Equal amounts of whole cell lysates were subjected to immunoblot analysis with the indicated antibodies.

    Techniques Used: Transfection, Expressing, Plasmid Preparation

    The stable retention of p27 Kip1 prevents efficient viral lytic replication. A , 293/EBV-WT cells were transfected with the BZLF1 expression vector together with non-targeting/control siRNA ( si-control ) or Skp2 siRNA ( si-Skp2 ) and harvested at 60 hpt. After freezing/thawing and centrifugation, the supernatants were co-cultured with Akata(−) cells. GFP-positive cells were counted by fluorescence-activated cell sorting. The results are the averages of three independent experiments and are shown as values relative to the virus yield of control siRNA-transfected cells (infectivity value of 1). B , the 293/EBV-WT cells were transfected with the BZLF1 expression vector and with increasing amounts of p27 Kip1 expression vector (1, 10, 50, and 100 ng) and empty vector and harvested at 60 hpt. Total amounts of transfected vectors were adjusted to 500 ng using empty vector. Samples were then assayed as described in A . The graph shows values relative to the virus yield in the absence of p27 Kip1 expression vector (infectivity value of 1).
    Figure Legend Snippet: The stable retention of p27 Kip1 prevents efficient viral lytic replication. A , 293/EBV-WT cells were transfected with the BZLF1 expression vector together with non-targeting/control siRNA ( si-control ) or Skp2 siRNA ( si-Skp2 ) and harvested at 60 hpt. After freezing/thawing and centrifugation, the supernatants were co-cultured with Akata(−) cells. GFP-positive cells were counted by fluorescence-activated cell sorting. The results are the averages of three independent experiments and are shown as values relative to the virus yield of control siRNA-transfected cells (infectivity value of 1). B , the 293/EBV-WT cells were transfected with the BZLF1 expression vector and with increasing amounts of p27 Kip1 expression vector (1, 10, 50, and 100 ng) and empty vector and harvested at 60 hpt. Total amounts of transfected vectors were adjusted to 500 ng using empty vector. Samples were then assayed as described in A . The graph shows values relative to the virus yield in the absence of p27 Kip1 expression vector (infectivity value of 1).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Centrifugation, Cell Culture, Fluorescence, FACS, Infection

    4) Product Images from "Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis"

    Article Title: Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis

    Journal: Nature Communications

    doi: 10.1038/ncomms10072

    Inhibition of TBK1 affects mitosis and enhances 4 n + population. ( a ) Cell cycle analysis of asynchronously growing H460 cells in the presence or absence of BX795 by flow cytometry. ( b , c ) Histograms summarizing cell cycle analysis of control and BX795-treated asynchronously growing H460 ( b ) and AALE cells ( c ). ( d , e ) Cell cycle analysis combined with pH3S10 staining of synchronized H1650 cells released for 9 h in the presence of BX795 by flow cytometry. ( f ) Cell cycle analysis of H1650 shcontrol cells (transduced with lentivirus containing scrambled sequence) or H1650 shTBK1 cells (transduced with lentivirus containing shRNA targeting TBK1) synchronized by double-thymidine block and released for 9 h by flow cytometry. In panels a, d and e, the y axis shows the quantification of cells stained for H3pS10 and the x axis shows DNA content (DAPI staining). ( g ) Graphical representation of the flow cytometry data showing reductions in the number of phospho histone H3 positive/mitotic cells when TBK1 is depleted in H1650 and H460 cells. (* P
    Figure Legend Snippet: Inhibition of TBK1 affects mitosis and enhances 4 n + population. ( a ) Cell cycle analysis of asynchronously growing H460 cells in the presence or absence of BX795 by flow cytometry. ( b , c ) Histograms summarizing cell cycle analysis of control and BX795-treated asynchronously growing H460 ( b ) and AALE cells ( c ). ( d , e ) Cell cycle analysis combined with pH3S10 staining of synchronized H1650 cells released for 9 h in the presence of BX795 by flow cytometry. ( f ) Cell cycle analysis of H1650 shcontrol cells (transduced with lentivirus containing scrambled sequence) or H1650 shTBK1 cells (transduced with lentivirus containing shRNA targeting TBK1) synchronized by double-thymidine block and released for 9 h by flow cytometry. In panels a, d and e, the y axis shows the quantification of cells stained for H3pS10 and the x axis shows DNA content (DAPI staining). ( g ) Graphical representation of the flow cytometry data showing reductions in the number of phospho histone H3 positive/mitotic cells when TBK1 is depleted in H1650 and H460 cells. (* P

    Techniques Used: Inhibition, Cell Cycle Assay, Flow Cytometry, Cytometry, Staining, Transduction, Sequencing, shRNA, Blocking Assay

    Inhibition or silencing of TBK1 induces mitotic defects and inhibits mitosis. ( a ) Knockdown of TBK1 by transfecting two different siRNAs in A549 and H1650 cells resulted reduces the number of mitotic cells. Control siRNA refers to transfection with a non-targeting siRNA, ( b ) Knockdown of TBK1 using lentiviral shRNA in H1650, H460 and A549 cells reduces the number of mitotic cells, For a , b , error bars represent standard deviation (s.d.) * P
    Figure Legend Snippet: Inhibition or silencing of TBK1 induces mitotic defects and inhibits mitosis. ( a ) Knockdown of TBK1 by transfecting two different siRNAs in A549 and H1650 cells resulted reduces the number of mitotic cells. Control siRNA refers to transfection with a non-targeting siRNA, ( b ) Knockdown of TBK1 using lentiviral shRNA in H1650, H460 and A549 cells reduces the number of mitotic cells, For a , b , error bars represent standard deviation (s.d.) * P

    Techniques Used: Inhibition, Transfection, shRNA, Standard Deviation

    TBK1 directly binds to and phosphorylates CEP170 and NuMA. ( a ) Detection of co-localization of TBK1 and CEP170 as well as TBK1 and NuMA using proximity ligation assay (PLA). Green fluorescent foci represent co-localization of TBK1 and CEP170 or TBK1 and NuMA. TBK1 antibody alone or TBK1 antibody along with IgG were used as negative controls. ( b ) Immunoprecipitation–western blot analysis showing the interaction of endogenous TBK1 with endogenous CEP170 and NuMA in lysates of asynchronously growing H1650 cells. ( c ) Immunoprecipitation–western blot analysis of H460 cell lysates showing the binding of TBK1 with NuMA and CEP170 when released from G1/S block. This interaction was inhibited when cells were released from double-thymidine block in the presence of BX795. ( d , e ) In vitro binding of full-length TBK1, the kinase domain of TBK1 (1–294) or the TBK1 C terminus (TBK1-284-729) with CEP170 ( d ) or NuMA ( e ). The data presented are representative of three independent experiments. Lysate/ input lane represents 20% of the input 35 S-methionine-labelled TBK1 used in the pull-down assay. ( f ) Immunoprecipitation–western blot analysis showing inhibition in serine and threonine phosphorylation of CEP170 and NuMA when TBK1 was depleted following transduction of cells with lentivirus expressing shRNA targeting TBK1. ( g ) In vitro kinase assays showing the phosphorylation of NuMA and CEP170 by TBK1. Coomassie brilliant blue (CBB) staining shows the amount of protein in each reaction.
    Figure Legend Snippet: TBK1 directly binds to and phosphorylates CEP170 and NuMA. ( a ) Detection of co-localization of TBK1 and CEP170 as well as TBK1 and NuMA using proximity ligation assay (PLA). Green fluorescent foci represent co-localization of TBK1 and CEP170 or TBK1 and NuMA. TBK1 antibody alone or TBK1 antibody along with IgG were used as negative controls. ( b ) Immunoprecipitation–western blot analysis showing the interaction of endogenous TBK1 with endogenous CEP170 and NuMA in lysates of asynchronously growing H1650 cells. ( c ) Immunoprecipitation–western blot analysis of H460 cell lysates showing the binding of TBK1 with NuMA and CEP170 when released from G1/S block. This interaction was inhibited when cells were released from double-thymidine block in the presence of BX795. ( d , e ) In vitro binding of full-length TBK1, the kinase domain of TBK1 (1–294) or the TBK1 C terminus (TBK1-284-729) with CEP170 ( d ) or NuMA ( e ). The data presented are representative of three independent experiments. Lysate/ input lane represents 20% of the input 35 S-methionine-labelled TBK1 used in the pull-down assay. ( f ) Immunoprecipitation–western blot analysis showing inhibition in serine and threonine phosphorylation of CEP170 and NuMA when TBK1 was depleted following transduction of cells with lentivirus expressing shRNA targeting TBK1. ( g ) In vitro kinase assays showing the phosphorylation of NuMA and CEP170 by TBK1. Coomassie brilliant blue (CBB) staining shows the amount of protein in each reaction.

    Techniques Used: Proximity Ligation Assay, Immunoprecipitation, Western Blot, Binding Assay, Blocking Assay, In Vitro, Pull Down Assay, Inhibition, Transduction, Expressing, shRNA, Staining

    Phospho-TBK1 co-localizes with CEP170 and TBK1 inhibition provokes supernumerary centrosomes. ( a ) Confocal microscopy images of control and BX795-treated H460 cells at metaphase. Cells were stained for phospho-TBK1 (green), alpha tubulin (red) and DNA (DAPI, blue). Scale bar, 10 μm. ( b ) Confocal microscopy images of control and BX795-treated AALE cells at metaphase. Cells were stained for phospho-TBK1 (green), CEP170 (red) and DNA (DAPI, blue). Scale bar, 10 μm. ( c ) H460 cells stained for phospho-TBK1 (green), CEP170 (red) and DNA (DAPI, blue) in control and TBK1 inhibitor-treated cells. CEP170 fails to co-localize to centrosomes in BX795-treated cells. ( d , e ) TBK1 depletion by shRNA resulted in multiple centrosomes in H1650 ( d ) and H460 ( e ) cells. Cells were stained for phospho-TBK1 (green), CEP170 (red) and DNA (DAPI, blue). ( f ) Inhibition of TBK1 by 2 μM MRT affected spindle pole localization of NuMA. H460 and HeLa cells were stained for NuMA (green), Alpha Tubulin (red), DAPI (Blue). Scale bar, 10 μm. ( g , h ) Immunofluorescence staining for Dynein intermediate chain (red) and NuMA (green) in shcontrol and shTBK1-transfected H1650 ( g ) and A549 ( h ) cells. Scale bar, 10 μm.
    Figure Legend Snippet: Phospho-TBK1 co-localizes with CEP170 and TBK1 inhibition provokes supernumerary centrosomes. ( a ) Confocal microscopy images of control and BX795-treated H460 cells at metaphase. Cells were stained for phospho-TBK1 (green), alpha tubulin (red) and DNA (DAPI, blue). Scale bar, 10 μm. ( b ) Confocal microscopy images of control and BX795-treated AALE cells at metaphase. Cells were stained for phospho-TBK1 (green), CEP170 (red) and DNA (DAPI, blue). Scale bar, 10 μm. ( c ) H460 cells stained for phospho-TBK1 (green), CEP170 (red) and DNA (DAPI, blue) in control and TBK1 inhibitor-treated cells. CEP170 fails to co-localize to centrosomes in BX795-treated cells. ( d , e ) TBK1 depletion by shRNA resulted in multiple centrosomes in H1650 ( d ) and H460 ( e ) cells. Cells were stained for phospho-TBK1 (green), CEP170 (red) and DNA (DAPI, blue). ( f ) Inhibition of TBK1 by 2 μM MRT affected spindle pole localization of NuMA. H460 and HeLa cells were stained for NuMA (green), Alpha Tubulin (red), DAPI (Blue). Scale bar, 10 μm. ( g , h ) Immunofluorescence staining for Dynein intermediate chain (red) and NuMA (green) in shcontrol and shTBK1-transfected H1650 ( g ) and A549 ( h ) cells. Scale bar, 10 μm.

    Techniques Used: Inhibition, Confocal Microscopy, Staining, shRNA, Immunofluorescence, Transfection

    Phospho-S172 TBK1 localizes to centrosomes and mitotic spindles. ( a ) Confocal microscopy images of A549, H1650 and H460 cells progressing through different mitotic stages. Cells were stained for phospho-TBK1 (green), alpha tubulin (red) and DNA (DAPI, Blue). Scale bar, 10 μm. ( b ) Confocal microscopy images from H460 and A549 cells stained for phospho-TBK1 (green), alpha tubulin (red) and DNA (DAPI, Blue) after depleting IKKɛ using siRNAs ( c ) PLK1 using the inhibitor BI2536 in H460 and A549 cells. A non-targeting control siRNA was used as control. Depletion of IKKɛ or inhibition of PLK1 does not affect the localization of pTBK1 and alpha tubulin. ( d , e ) Western blots for pTBK1, TBK1, pPLK1, PLK1, CEP170, γ-tubulin and alpha tubulin from different fractions collected during centrosome isolation from H460 and A549 cells. pTBK1 and TBK1 are enriched in the centrosomal fraction (Fraction-4). Purity of the centrosomal fraction is indicated by high levels of γ-Tubulin in Fraction-4. CF indicates the centrosomal fraction. Lysate lane represents whole cell lysate prepared from asynchronously growing H460 or A549 cells. Experiments were repeated three times and representative images are presented.
    Figure Legend Snippet: Phospho-S172 TBK1 localizes to centrosomes and mitotic spindles. ( a ) Confocal microscopy images of A549, H1650 and H460 cells progressing through different mitotic stages. Cells were stained for phospho-TBK1 (green), alpha tubulin (red) and DNA (DAPI, Blue). Scale bar, 10 μm. ( b ) Confocal microscopy images from H460 and A549 cells stained for phospho-TBK1 (green), alpha tubulin (red) and DNA (DAPI, Blue) after depleting IKKɛ using siRNAs ( c ) PLK1 using the inhibitor BI2536 in H460 and A549 cells. A non-targeting control siRNA was used as control. Depletion of IKKɛ or inhibition of PLK1 does not affect the localization of pTBK1 and alpha tubulin. ( d , e ) Western blots for pTBK1, TBK1, pPLK1, PLK1, CEP170, γ-tubulin and alpha tubulin from different fractions collected during centrosome isolation from H460 and A549 cells. pTBK1 and TBK1 are enriched in the centrosomal fraction (Fraction-4). Purity of the centrosomal fraction is indicated by high levels of γ-Tubulin in Fraction-4. CF indicates the centrosomal fraction. Lysate lane represents whole cell lysate prepared from asynchronously growing H460 or A549 cells. Experiments were repeated three times and representative images are presented.

    Techniques Used: Confocal Microscopy, Staining, Inhibition, Western Blot, Isolation

    5) Product Images from "MACROPHAGES PRODUCE TGFBI (BIGH3) FOLLOWING INGESTION OF APOPTOTIC CELLS AND REGULATE MMP14 LEVELS AND COLLAGEN TURNOVER IN FIBROBLASTS"

    Article Title: MACROPHAGES PRODUCE TGFBI (BIGH3) FOLLOWING INGESTION OF APOPTOTIC CELLS AND REGULATE MMP14 LEVELS AND COLLAGEN TURNOVER IN FIBROBLASTS

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

    doi:

    DNA binding by p53 (A) and effects of MMP14 or p53 overexpression, or MMP14 or PU.1 siRNA inhibition on the expression levels of MMP14 and collagen (B,C). In Panel A , electromobility shift assays were used, on two independent occasions, to validate the results of the protein-DNA array experiments. The p53-specific 32 P-labeled probe was incubated with nuclear lysates from non-stimulated control or TGFBI-activated fibroblasts, as indicated, or in the presence of 10-fold excess of the mutant (scrambled) or unlabeled (cold) specific probe, as indicated. The cold but not the scrambled probe inhibited p53 binding (arrows), suggesting that the binding is specific. In Panels B and C , fibroblast cultures were transfected with MMP14-encoding or p53-encoding plasmid, or with MMP14 or PU.1 siRNA as indicated. Western blotting assays were performed using antibodies against MMP14 or collagen. At least three independent experiments were conducted for each panel shown, with consistent results.
    Figure Legend Snippet: DNA binding by p53 (A) and effects of MMP14 or p53 overexpression, or MMP14 or PU.1 siRNA inhibition on the expression levels of MMP14 and collagen (B,C). In Panel A , electromobility shift assays were used, on two independent occasions, to validate the results of the protein-DNA array experiments. The p53-specific 32 P-labeled probe was incubated with nuclear lysates from non-stimulated control or TGFBI-activated fibroblasts, as indicated, or in the presence of 10-fold excess of the mutant (scrambled) or unlabeled (cold) specific probe, as indicated. The cold but not the scrambled probe inhibited p53 binding (arrows), suggesting that the binding is specific. In Panels B and C , fibroblast cultures were transfected with MMP14-encoding or p53-encoding plasmid, or with MMP14 or PU.1 siRNA as indicated. Western blotting assays were performed using antibodies against MMP14 or collagen. At least three independent experiments were conducted for each panel shown, with consistent results.

    Techniques Used: Binding Assay, Over Expression, Inhibition, Expressing, DNA Array, Labeling, Incubation, Mutagenesis, Transfection, Plasmid Preparation, Western Blot

    6) Product Images from "Nuclear Smad7 Overexpressed in Mesenchymal Cells Acts as a Transcriptional Corepressor by Interacting with HDAC-1 and E2F to Regulate Cell Cycle"

    Article Title: Nuclear Smad7 Overexpressed in Mesenchymal Cells Acts as a Transcriptional Corepressor by Interacting with HDAC-1 and E2F to Regulate Cell Cycle

    Journal: Biology Open

    doi: 10.1242/bio.2012463

    Endogenous Smad7 regulating an E2F-responsive promoter. (A) Association of endogenous Smad7 and HDAC-1 proteins in A549 human lung cells. Nuclear extract from untransfected A549 cells were incubated with an α-HDAC-1 antibody or control IgG. Immune complexes were examined by Western blotting using α-Smad7 and α-HDAC-1 antibodies. (B) Association of endogenous Smad7 and E2F-1 proteins. Immune complexes prepared using α-E2F-1 or control IgG as in (A) were examined by Western blotting using α-Smad7 and α-E2F-1 antibodies. (C) Effect of Smad7 knock-down on the transcriptional activation of E2F-responsive promoter. (Upper panel) In combination with a vector for either Smad7-targeting or control siRNA, luciferase reporter (pGVB2-E2F×4) driven by an E2F-responsive promoter was transfected into A549 cells. Cells were serum-starved for 40 h, stimulated in medium containing 10% FBS for 18 h and then lysed for assays. Luciferase activity in the lysates is shown as means with S.D. from triplicate experiments. (Lower panel) Level of endogenous Smad7 in the lysates was examined by Western blotting using α-Smad7.
    Figure Legend Snippet: Endogenous Smad7 regulating an E2F-responsive promoter. (A) Association of endogenous Smad7 and HDAC-1 proteins in A549 human lung cells. Nuclear extract from untransfected A549 cells were incubated with an α-HDAC-1 antibody or control IgG. Immune complexes were examined by Western blotting using α-Smad7 and α-HDAC-1 antibodies. (B) Association of endogenous Smad7 and E2F-1 proteins. Immune complexes prepared using α-E2F-1 or control IgG as in (A) were examined by Western blotting using α-Smad7 and α-E2F-1 antibodies. (C) Effect of Smad7 knock-down on the transcriptional activation of E2F-responsive promoter. (Upper panel) In combination with a vector for either Smad7-targeting or control siRNA, luciferase reporter (pGVB2-E2F×4) driven by an E2F-responsive promoter was transfected into A549 cells. Cells were serum-starved for 40 h, stimulated in medium containing 10% FBS for 18 h and then lysed for assays. Luciferase activity in the lysates is shown as means with S.D. from triplicate experiments. (Lower panel) Level of endogenous Smad7 in the lysates was examined by Western blotting using α-Smad7.

    Techniques Used: Incubation, Western Blot, Activation Assay, Plasmid Preparation, Luciferase, Transfection, Activity Assay

    7) Product Images from "Nucleosomal dsDNA Stimulates APOL1 Expression in Human Cultured Podocytes by Activating the cGAS/IFI16-STING Signaling Pathway"

    Article Title: Nucleosomal dsDNA Stimulates APOL1 Expression in Human Cultured Podocytes by Activating the cGAS/IFI16-STING Signaling Pathway

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-51998-w

    STING knockdown inhibits nsDNA-induced expression of APOL1 and IFI16 but does not affect expression induced by exogenous IFNβ. ( a ) AB8/13 podocytes were transfected for 48 h with a non-targeting control siRNA or siRNA pool targeting STING. Subsequently, sets of the transfected cells were transfected for 18 h with 1 μg ml −1 nsDNA, treated for 18 h with IFNβ (10 ng ml −1 ), or pretreated with 5 μM Ruxo for 2 h followed by IFNβ stimulation for 18 h. Expression of indicated proteins was analyzed by immunoblotting. The blot images were obtained from different gels. The blot probed with IFI16 was re-probed with IRF3. Other blot images were cropped from individually probed blots. Full images of the blots are shown in Supplementary Fig. S8 . ( b , c ) STING knockdown inhibited expression of APOL1 ( b ) and IFNβ mRNA ( c ) in response to nsDNA. AB8/13 podocytes were transfected for 48 h with a non-targeting control siRNA or siRNA pool targeting STING, followed by transfection with 1 μg ml −1 nsDNA for 18 h. Expression of APOL1 and IFNβ mRNA was normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).
    Figure Legend Snippet: STING knockdown inhibits nsDNA-induced expression of APOL1 and IFI16 but does not affect expression induced by exogenous IFNβ. ( a ) AB8/13 podocytes were transfected for 48 h with a non-targeting control siRNA or siRNA pool targeting STING. Subsequently, sets of the transfected cells were transfected for 18 h with 1 μg ml −1 nsDNA, treated for 18 h with IFNβ (10 ng ml −1 ), or pretreated with 5 μM Ruxo for 2 h followed by IFNβ stimulation for 18 h. Expression of indicated proteins was analyzed by immunoblotting. The blot images were obtained from different gels. The blot probed with IFI16 was re-probed with IRF3. Other blot images were cropped from individually probed blots. Full images of the blots are shown in Supplementary Fig. S8 . ( b , c ) STING knockdown inhibited expression of APOL1 ( b ) and IFNβ mRNA ( c ) in response to nsDNA. AB8/13 podocytes were transfected for 48 h with a non-targeting control siRNA or siRNA pool targeting STING, followed by transfection with 1 μg ml −1 nsDNA for 18 h. Expression of APOL1 and IFNβ mRNA was normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).

    Techniques Used: Expressing, Transfection

    Knockdown of STING, TBK1, or IRF3 inhibits nsDNA-mediated APOL1 expression in human immortalized AB8/13 podocytes. AB8/13 podocytes were transfected with a non-targeting control siRNA (Co) or siRNA pool targeting STING ( a – c ), TBK1 ( d – f ), or IRF3 ( g - i ) for 48 h, and subsequently transfected with 1 μg ml −1 nsDNA for 18 h. Expression of indicated proteins ( a , d , g ) was analyzed by immunoblotting. Protein size markers (kDa) are shown. The blot images in ( a ) were obtained from different gels. One blot was probed for IRF3 and re-probed for GAPDH. Blot images in ( d ) were obtained from different gels. One blot was probed with TBK1 and re-probed with GAPDH, while two other blots were probed for APOL1 and TBK1. The blot images in ( g ) were obtained from different gels. The blot probed for TBK1 was re-probed for GAPDH. Two other blots were probed individually for IRF3 and APOL1. Full images of the blots are shown in Supplementary Fig. S3 . Expression of STING ( b ), TBK1 ( e ), IRF3 ( h ), and APOL1 ( c , f , i ) mRNA was analyzed by qRT-PCR and normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).
    Figure Legend Snippet: Knockdown of STING, TBK1, or IRF3 inhibits nsDNA-mediated APOL1 expression in human immortalized AB8/13 podocytes. AB8/13 podocytes were transfected with a non-targeting control siRNA (Co) or siRNA pool targeting STING ( a – c ), TBK1 ( d – f ), or IRF3 ( g - i ) for 48 h, and subsequently transfected with 1 μg ml −1 nsDNA for 18 h. Expression of indicated proteins ( a , d , g ) was analyzed by immunoblotting. Protein size markers (kDa) are shown. The blot images in ( a ) were obtained from different gels. One blot was probed for IRF3 and re-probed for GAPDH. Blot images in ( d ) were obtained from different gels. One blot was probed with TBK1 and re-probed with GAPDH, while two other blots were probed for APOL1 and TBK1. The blot images in ( g ) were obtained from different gels. The blot probed for TBK1 was re-probed for GAPDH. Two other blots were probed individually for IRF3 and APOL1. Full images of the blots are shown in Supplementary Fig. S3 . Expression of STING ( b ), TBK1 ( e ), IRF3 ( h ), and APOL1 ( c , f , i ) mRNA was analyzed by qRT-PCR and normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    Knockdown of cGAS or IFI16 partly inhibits nsDNA-mediated APOL1 expression in human immortalized AB8/13 podocytes. The cells were transfected for 48 h with control siRNA (Co) or siRNA pool targeting cGAS ( a – c ) or IFI16 ( d – f ) and subsequently transfected with 1 μg ml −1 nsDNA for 18 h. ( a , d ) Expression of indicated proteins was analyzed by immunoblotting. Protein size markers (kDa) are shown. Intensities of cGAS, STING, IFI16, APOL1, and GAPDH protein bands were quantified by densitometric scanning. ( a ) Expression levels of cGAS, STING, and APOL1 were normalized against GAPDH levels and presented as cGAS/GAPDH, STING/GAPDH, and APOL1/GAPDH ratios. In cells transfected with control siRNA only, the cGAS/GAPDH and STING/GAPDH ratios were both set as 1.0. The APOL1/GAPDH ratio in cells transfected with control siRNA and nsDNA was set as 1.0. The blot images were obtained from different individually probed gels. ( d ) Expression levels of IFI16, STING, and APOL1 are presented as IFI16/GAPDH, STING/GAPDH, and APOL1/GAPDH ratios. In cells transfected with control siRNA only, the IFI16/GAPDH and STING/GAPDH ratios were both set as 1.0. The APOL1/GAPDH ratio in cells transfected with control siRNA and nsDNA was set as 1.0. The blot images were obtained from different gels. The blot probed for APOL1 was re-probed for IRF3. Full images of the blots in (a) and (d) are shown in Supplementary Fig. S5 . Expression of cGAS ( b ), IFI16 ( e ), and APOL1 ( c , f ) mRNA was analyzed by qRT-PCR and normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).
    Figure Legend Snippet: Knockdown of cGAS or IFI16 partly inhibits nsDNA-mediated APOL1 expression in human immortalized AB8/13 podocytes. The cells were transfected for 48 h with control siRNA (Co) or siRNA pool targeting cGAS ( a – c ) or IFI16 ( d – f ) and subsequently transfected with 1 μg ml −1 nsDNA for 18 h. ( a , d ) Expression of indicated proteins was analyzed by immunoblotting. Protein size markers (kDa) are shown. Intensities of cGAS, STING, IFI16, APOL1, and GAPDH protein bands were quantified by densitometric scanning. ( a ) Expression levels of cGAS, STING, and APOL1 were normalized against GAPDH levels and presented as cGAS/GAPDH, STING/GAPDH, and APOL1/GAPDH ratios. In cells transfected with control siRNA only, the cGAS/GAPDH and STING/GAPDH ratios were both set as 1.0. The APOL1/GAPDH ratio in cells transfected with control siRNA and nsDNA was set as 1.0. The blot images were obtained from different individually probed gels. ( d ) Expression levels of IFI16, STING, and APOL1 are presented as IFI16/GAPDH, STING/GAPDH, and APOL1/GAPDH ratios. In cells transfected with control siRNA only, the IFI16/GAPDH and STING/GAPDH ratios were both set as 1.0. The APOL1/GAPDH ratio in cells transfected with control siRNA and nsDNA was set as 1.0. The blot images were obtained from different gels. The blot probed for APOL1 was re-probed for IRF3. Full images of the blots in (a) and (d) are shown in Supplementary Fig. S5 . Expression of cGAS ( b ), IFI16 ( e ), and APOL1 ( c , f ) mRNA was analyzed by qRT-PCR and normalized to GAPDH mRNA levels. Data are expressed as means ± SEM from three biological replicates (one-way ANOVA with post-hoc Tukey test).

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    cGAS and IFI16 are required for maximal APOL1 expression in human immortalized AB8/13 podocytes in response to nsDNA. ( a ) AB8/13 podocytes were transfected for 48 h with control siRNA (Co) or siRNA pool targeting either cGAS or IFI16 or both. The cells were subsequently transfected with 1 μg ml −1 nsDNA for the times indicated. The blot images were obtained from different gels. Left panel: The blot probed for cGAS was re-probed for GAPDH. The blot probed for APOL1 was re-probed for TBK1. Right panel: The blot probed for cGAS was re-probed for GAPDH. The blot probed for APOL1 was re-probed for TBK1. Expression levels of IFI16, STING, and APOL1 are presented as IFI16/GAPDH, STING/GAPDH, and APOL1/GAPDH ratios. The IFI16/GAPDH and STING/GAPDH ratios in cells transfected with control siRNA only were both set as 1.0. The APOL1/GAPDH ratio in cells transfected with control siRNA and nsDNA (18 h) was set as 1.0. ( b ) cGAS −/− cells generated from parental AB8/13 podocytes (cGAS +/+ ) were transfected for 48 h with control siRNA (Co) or siRNA pool targeting IFI16 and subsequently transfected with 1 μg ml −1 nsDNA for the times indicated. Expression of indicated proteins was analyzed by immunoblotting. Protein size markers (kDa) are shown. The blot images were obtained from different gels. The blot probed for cGAS was re-probed for GAPDH. The other blot images were cropped from individually probed blots. The IFI16/GAPDH and STING/GAPDH ratios in cGAS +/+ cells exposed to transfection reagent only were both set as 1.0. The APOL1/GAPDH ratio in cGAS +/+ cells transfected with nsDNA (18 h) was set as 1.0. Full images of the blots are shown in Supplementary Fig. S6 .
    Figure Legend Snippet: cGAS and IFI16 are required for maximal APOL1 expression in human immortalized AB8/13 podocytes in response to nsDNA. ( a ) AB8/13 podocytes were transfected for 48 h with control siRNA (Co) or siRNA pool targeting either cGAS or IFI16 or both. The cells were subsequently transfected with 1 μg ml −1 nsDNA for the times indicated. The blot images were obtained from different gels. Left panel: The blot probed for cGAS was re-probed for GAPDH. The blot probed for APOL1 was re-probed for TBK1. Right panel: The blot probed for cGAS was re-probed for GAPDH. The blot probed for APOL1 was re-probed for TBK1. Expression levels of IFI16, STING, and APOL1 are presented as IFI16/GAPDH, STING/GAPDH, and APOL1/GAPDH ratios. The IFI16/GAPDH and STING/GAPDH ratios in cells transfected with control siRNA only were both set as 1.0. The APOL1/GAPDH ratio in cells transfected with control siRNA and nsDNA (18 h) was set as 1.0. ( b ) cGAS −/− cells generated from parental AB8/13 podocytes (cGAS +/+ ) were transfected for 48 h with control siRNA (Co) or siRNA pool targeting IFI16 and subsequently transfected with 1 μg ml −1 nsDNA for the times indicated. Expression of indicated proteins was analyzed by immunoblotting. Protein size markers (kDa) are shown. The blot images were obtained from different gels. The blot probed for cGAS was re-probed for GAPDH. The other blot images were cropped from individually probed blots. The IFI16/GAPDH and STING/GAPDH ratios in cGAS +/+ cells exposed to transfection reagent only were both set as 1.0. The APOL1/GAPDH ratio in cGAS +/+ cells transfected with nsDNA (18 h) was set as 1.0. Full images of the blots are shown in Supplementary Fig. S6 .

    Techniques Used: Expressing, Transfection, Generated

    8) Product Images from "Species-Specific Inhibition of RIG-I Ubiquitination and IFN Induction by the Influenza A Virus NS1 Protein"

    Article Title: Species-Specific Inhibition of RIG-I Ubiquitination and IFN Induction by the Influenza A Virus NS1 Protein

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003059

    NS1 inhibits the Riplet-dependent RIG-I ubiquitination and IFN induction in murine cells. ( A ) Mouse Hepa 1.6 cells were transfected with vector or Flag-tagged mRiplet with or without NS1-PR8. WCLs were subjected to IP with RIG-I antibody, followed by IB with anti-Ub or anti-RIG-I antibody. Expression of mRiplet, NS1, and ubiquitin (Ub) was determined in the WCLs by IB with anti-Flag, anti-NS1 or anti-Ub antibody. ( B ) Mouse Hepa1.6 cells were transfected with IFN-β luciferase reporter plasmid together with empty vector or mRIG-I together with or without mRiplet and NS1-PR8. At 24 h posttransfection, cells were lysed and subjected to luciferase assay. Data shown is representative of 3 independent experiments and depicted is the mean ± SD (n = 3). ( C ) Influenza NS1 protein specifically inhibits the Riplet-dependent ubiquitination of mouse RIG-I. Hepa1.6 cells were transiently transfected with non-silencing control siRNA (si.C), or with a siRNA specific for mouse Riplet (si.Riplet) together with empty vector or NS1-PR8. At 24 h posttransfection, cells were infected with SeV (50 HA units/ml) for 22 h. WCLs were used for IP with anti-RIG-I antibody, followed by IB with anti-Ub or anti-RIG-I antibody. ( D–F ) Knockdown of endogenous Riplet in mouse embryonic fibroblasts enhances influenza A virus replication. WT or TRIM25 −/− MEFs were transfected with non-silencing control siRNA (si.C), or with a siRNA specific for mouse Riplet (si.Riplet). At 30 h posttransfection, cells were infected with recombinant A/PR/8/34 WT virus (MOI 0.1). Knockdown of endogenous Riplet was confirmed by RT-PCR ( D ). Supernatants were assayed for progeny virus yields 24 h postinfection in standard plaque titrations ( E ). Virus yields are depicted in Pfu/ml. The results of three independent experiments are shown. Furthermore, viral NS1 protein expression was determined in the WCLs of infected cells ( F ).
    Figure Legend Snippet: NS1 inhibits the Riplet-dependent RIG-I ubiquitination and IFN induction in murine cells. ( A ) Mouse Hepa 1.6 cells were transfected with vector or Flag-tagged mRiplet with or without NS1-PR8. WCLs were subjected to IP with RIG-I antibody, followed by IB with anti-Ub or anti-RIG-I antibody. Expression of mRiplet, NS1, and ubiquitin (Ub) was determined in the WCLs by IB with anti-Flag, anti-NS1 or anti-Ub antibody. ( B ) Mouse Hepa1.6 cells were transfected with IFN-β luciferase reporter plasmid together with empty vector or mRIG-I together with or without mRiplet and NS1-PR8. At 24 h posttransfection, cells were lysed and subjected to luciferase assay. Data shown is representative of 3 independent experiments and depicted is the mean ± SD (n = 3). ( C ) Influenza NS1 protein specifically inhibits the Riplet-dependent ubiquitination of mouse RIG-I. Hepa1.6 cells were transiently transfected with non-silencing control siRNA (si.C), or with a siRNA specific for mouse Riplet (si.Riplet) together with empty vector or NS1-PR8. At 24 h posttransfection, cells were infected with SeV (50 HA units/ml) for 22 h. WCLs were used for IP with anti-RIG-I antibody, followed by IB with anti-Ub or anti-RIG-I antibody. ( D–F ) Knockdown of endogenous Riplet in mouse embryonic fibroblasts enhances influenza A virus replication. WT or TRIM25 −/− MEFs were transfected with non-silencing control siRNA (si.C), or with a siRNA specific for mouse Riplet (si.Riplet). At 30 h posttransfection, cells were infected with recombinant A/PR/8/34 WT virus (MOI 0.1). Knockdown of endogenous Riplet was confirmed by RT-PCR ( D ). Supernatants were assayed for progeny virus yields 24 h postinfection in standard plaque titrations ( E ). Virus yields are depicted in Pfu/ml. The results of three independent experiments are shown. Furthermore, viral NS1 protein expression was determined in the WCLs of infected cells ( F ).

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Luciferase, Infection, Recombinant, Reverse Transcription Polymerase Chain Reaction

    Influenza A Virus NS1 interacts with TRIM25 in a species-specific manner. ( A ) HEK293T cells were transfected with V5-tagged mouse TRIM25 (V5-mTRIM25), human TRIM25 (V5-hTRIM25) or chicken TRIM25 (V5-chTRIM25) together with NS1-A/California/04/09 (Cal04) (left), or NS1-PR8, NS1-A/Hong Kong/156 (HK156), NS1-A/Swine Texas/98 (SwTx98), or NS1-Cal04 (right panel). Empty vector and V5-tagged human TRIM21 were used as negative controls. Whole-cell lysates (WCLs) of HEK293T cells were subjected to immunoprecipitation (IP) using anti-V5 antibody, followed by immunoblotting (IB) with anti-NS1. ( B–E ) TRIM25 is important for virus-induced IFN-β production in chicken cells. Chicken LMH cells were transiently transfected with non-silencing control siRNA (si.C), or with a siRNA specific for chicken TRIM25 (si.chTRIM25). At 40 h posttransfection, cells were infected with recombinant influenza viruses expressing the NS1 RNA-binding mutant R38A/K41A ( C ), or NS1 from PR8, SwTx98, or HK156 ( D ) at an MOI of 0.1 for 24 h. Knockdown was confirmed by determining chTRIM25 mRNA levels ( B ). Furthermore, the IFN-β ( C and D ) and viral M1 ( E ) mRNA levels were assessed by qPCR and the values were normalized to chicken GAPDH. ( F ) IFN induction and viral replication in murine (MEF) cells. 12 hours postinfection, IFN-β (far left panel) and viral M1 (right panel) mRNA levels were assessed by qPCR from MEFs infected with the different NS1 recombinant viruses at an MOI of 2. Values were normalized to mouse actin. IFN protein was quantified by VSV-GFP bioassay on L929 cells treated with 2-fold dilutions of post-influenza virus supernatants from MEF cells (left panel). Relative fluorescence values reported on the y-axis represent the levels of VSV-GFP replication. High IFN concentrations in the supernatants correspond to low levels of VSV-GFP replication (low fluorescence values). Each sample was assayed in triplicate, and results are representative of two independent experiments. Supernatants were assayed for progeny virus yields 12 h postinfection in standard plaque titrations (far right panel). Virus yields are depicted in PFU/ml. *p
    Figure Legend Snippet: Influenza A Virus NS1 interacts with TRIM25 in a species-specific manner. ( A ) HEK293T cells were transfected with V5-tagged mouse TRIM25 (V5-mTRIM25), human TRIM25 (V5-hTRIM25) or chicken TRIM25 (V5-chTRIM25) together with NS1-A/California/04/09 (Cal04) (left), or NS1-PR8, NS1-A/Hong Kong/156 (HK156), NS1-A/Swine Texas/98 (SwTx98), or NS1-Cal04 (right panel). Empty vector and V5-tagged human TRIM21 were used as negative controls. Whole-cell lysates (WCLs) of HEK293T cells were subjected to immunoprecipitation (IP) using anti-V5 antibody, followed by immunoblotting (IB) with anti-NS1. ( B–E ) TRIM25 is important for virus-induced IFN-β production in chicken cells. Chicken LMH cells were transiently transfected with non-silencing control siRNA (si.C), or with a siRNA specific for chicken TRIM25 (si.chTRIM25). At 40 h posttransfection, cells were infected with recombinant influenza viruses expressing the NS1 RNA-binding mutant R38A/K41A ( C ), or NS1 from PR8, SwTx98, or HK156 ( D ) at an MOI of 0.1 for 24 h. Knockdown was confirmed by determining chTRIM25 mRNA levels ( B ). Furthermore, the IFN-β ( C and D ) and viral M1 ( E ) mRNA levels were assessed by qPCR and the values were normalized to chicken GAPDH. ( F ) IFN induction and viral replication in murine (MEF) cells. 12 hours postinfection, IFN-β (far left panel) and viral M1 (right panel) mRNA levels were assessed by qPCR from MEFs infected with the different NS1 recombinant viruses at an MOI of 2. Values were normalized to mouse actin. IFN protein was quantified by VSV-GFP bioassay on L929 cells treated with 2-fold dilutions of post-influenza virus supernatants from MEF cells (left panel). Relative fluorescence values reported on the y-axis represent the levels of VSV-GFP replication. High IFN concentrations in the supernatants correspond to low levels of VSV-GFP replication (low fluorescence values). Each sample was assayed in triplicate, and results are representative of two independent experiments. Supernatants were assayed for progeny virus yields 12 h postinfection in standard plaque titrations (far right panel). Virus yields are depicted in PFU/ml. *p

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Infection, Recombinant, Expressing, RNA Binding Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Fluorescence

    NS1 proteins from human influenza strains bind and inhibit human Riplet. ( A ) HEK293T cells were transfected with empty vector or HA-tagged human Riplet (HA-hRiplet). At 30 h posttransfection, cells were either mock-treated, or infected with the indicated recombinant A/PR/8/34 viruses at an MOI of 2. 18 h later, WCLs were subjected to IP with anti-HA antibody, followed by immunoblotting using the indicated antibodies. ( B ) Tx91 recombinant virus suppresses the endogenous RIG-I ubiquitination more potently than PR8 virus. HEK293T cells, that had been transfected with HA-tagged ubiquitin, were either mock-treated, or infected with ΔNS1 PR8, PR8 WT, or Tx91-NS1 recombinant virus at an MOI of 2 for 18 h. WCLs were subjected to IP with anti-RIG-I antibody, followed by IB with anti-HA or anti-RIG-I antibody. Expression of HA-ubiquitin, viral NS1, and Actin was further determined in the WCLs. ( C ) A549 cells were infected with PR8-NS1 or Tx91-NS1 recombinant virus at an MOI of 0.1. Cells were collected at the indicated time points and IFN-β mRNA was measured by qPCR. ( D and E ) A549 cells were transiently transfected with non-silencing control siRNA (si.C), or with siRNA specific for TRIM25 (si.TRIM25), Riplet (si.Riplet), or both. At 40 h posttransfection, cells were infected with PR8 WT or Tx91 recombinant virus at an MOI of 2 for 30 h. The mRNA levels of TRIM25 and Riplet were measured by qPCR for analyzing their knockdown efficiency ( D ). Furthermore, IFN-β mRNA levels were assessed by qPCR ( E ). Results are triplicates from 3 independent experiments. NS; statistically non-significant.
    Figure Legend Snippet: NS1 proteins from human influenza strains bind and inhibit human Riplet. ( A ) HEK293T cells were transfected with empty vector or HA-tagged human Riplet (HA-hRiplet). At 30 h posttransfection, cells were either mock-treated, or infected with the indicated recombinant A/PR/8/34 viruses at an MOI of 2. 18 h later, WCLs were subjected to IP with anti-HA antibody, followed by immunoblotting using the indicated antibodies. ( B ) Tx91 recombinant virus suppresses the endogenous RIG-I ubiquitination more potently than PR8 virus. HEK293T cells, that had been transfected with HA-tagged ubiquitin, were either mock-treated, or infected with ΔNS1 PR8, PR8 WT, or Tx91-NS1 recombinant virus at an MOI of 2 for 18 h. WCLs were subjected to IP with anti-RIG-I antibody, followed by IB with anti-HA or anti-RIG-I antibody. Expression of HA-ubiquitin, viral NS1, and Actin was further determined in the WCLs. ( C ) A549 cells were infected with PR8-NS1 or Tx91-NS1 recombinant virus at an MOI of 0.1. Cells were collected at the indicated time points and IFN-β mRNA was measured by qPCR. ( D and E ) A549 cells were transiently transfected with non-silencing control siRNA (si.C), or with siRNA specific for TRIM25 (si.TRIM25), Riplet (si.Riplet), or both. At 40 h posttransfection, cells were infected with PR8 WT or Tx91 recombinant virus at an MOI of 2 for 30 h. The mRNA levels of TRIM25 and Riplet were measured by qPCR for analyzing their knockdown efficiency ( D ). Furthermore, IFN-β mRNA levels were assessed by qPCR ( E ). Results are triplicates from 3 independent experiments. NS; statistically non-significant.

    Techniques Used: Transfection, Plasmid Preparation, Infection, Recombinant, Expressing, Real-time Polymerase Chain Reaction

    9) Product Images from "Transient receptor potential ion channel Trpm7 regulates exocrine pancreatic epithelial proliferation by Mg2+-sensitive Socs3a signaling in development and cancer"

    Article Title: Transient receptor potential ion channel Trpm7 regulates exocrine pancreatic epithelial proliferation by Mg2+-sensitive Socs3a signaling in development and cancer

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.004564

    TRPM7 is overexpressed in human pancreatic adenocarcinoma and required for cellular proliferation. (A) Expression of TRPM7 protein in human pancreatic adenocarcinoma (upper panel) and normal pancreatic tissue (lower panel) by immunohistochemistry using anti-TRPM7 antibodies. The images shown are representative of five tumor specimens and five normal control samples. Scale bars: 100 μm. (B) Relative TRPM7 mRNA levels in human pancreatic adenocarcinoma cell lines by quantitative real-time PCR. Each value represents the mean ratio of each pancreatic cancer cell line to that of H6c7 from triplicate samples, and reproducible results were obtained from two independent experiments. (C) Cell morphology at 24 hours post-transfection with siRNA was examined under an inverted microscope with phase contrast. Red arrows point to multinucleated cells with cytoplasmic vacuoles.
    Figure Legend Snippet: TRPM7 is overexpressed in human pancreatic adenocarcinoma and required for cellular proliferation. (A) Expression of TRPM7 protein in human pancreatic adenocarcinoma (upper panel) and normal pancreatic tissue (lower panel) by immunohistochemistry using anti-TRPM7 antibodies. The images shown are representative of five tumor specimens and five normal control samples. Scale bars: 100 μm. (B) Relative TRPM7 mRNA levels in human pancreatic adenocarcinoma cell lines by quantitative real-time PCR. Each value represents the mean ratio of each pancreatic cancer cell line to that of H6c7 from triplicate samples, and reproducible results were obtained from two independent experiments. (C) Cell morphology at 24 hours post-transfection with siRNA was examined under an inverted microscope with phase contrast. Red arrows point to multinucleated cells with cytoplasmic vacuoles.

    Techniques Used: Expressing, Immunohistochemistry, Real-time Polymerase Chain Reaction, Transfection, Inverted Microscopy

    TRPM7 controls cellular proliferation by regulating cell-cycle progression and by modulating p21 CDKN1A , cyclin G1 and cyclin B1 expression and Mg 2+ level. (A) PANC-1 cells were treated with siRNA directed against TRPM7 , non-targeting control siRNA, or without siRNA, and then analyzed by MTS assay and counting cells. Proliferation in the anti- TRPM7- siRNA-treated cells is represented as percent of that in the cells treated with non-targeting control siRNA. Each column represents the mean from triplicate samples. Bars represent s.d. * P
    Figure Legend Snippet: TRPM7 controls cellular proliferation by regulating cell-cycle progression and by modulating p21 CDKN1A , cyclin G1 and cyclin B1 expression and Mg 2+ level. (A) PANC-1 cells were treated with siRNA directed against TRPM7 , non-targeting control siRNA, or without siRNA, and then analyzed by MTS assay and counting cells. Proliferation in the anti- TRPM7- siRNA-treated cells is represented as percent of that in the cells treated with non-targeting control siRNA. Each column represents the mean from triplicate samples. Bars represent s.d. * P

    Techniques Used: Expressing, MTS Assay

    10) Product Images from "Gadd45b is an epigenetic regulator of juvenile social behavior and alters local pro-inflammatory cytokine production in the rodent amygdala"

    Article Title: Gadd45b is an epigenetic regulator of juvenile social behavior and alters local pro-inflammatory cytokine production in the rodent amygdala

    Journal: Brain, behavior, and immunity

    doi: 10.1016/j.bbi.2015.02.018

    Neonatal Gadd45b siRNA treatment epigenetically alters Adra2a promoter DNA methylation near a CREB binding site at P2 (control: 0.175 ± 0.0175, N = 5 vs. Gadd45b: 0.371 ± 0.0764, N = 6; p = 0.0489) and P33 (control: 0.249 ± 0.0310,
    Figure Legend Snippet: Neonatal Gadd45b siRNA treatment epigenetically alters Adra2a promoter DNA methylation near a CREB binding site at P2 (control: 0.175 ± 0.0175, N = 5 vs. Gadd45b: 0.371 ± 0.0764, N = 6; p = 0.0489) and P33 (control: 0.249 ± 0.0310,

    Techniques Used: DNA Methylation Assay, Binding Assay

    Gadd45b siRNA specificity in the amygdala. Two days of Gadd45b siRNA gives approximately 20% knockdown of mRNA (control: 0.4861 ± 0.0276, N = 5; Gadd45b: 0.3967 ± 0.0130, N = 5); * p = 0.0191. Gadd45b siRNA does not significantly decrease
    Figure Legend Snippet: Gadd45b siRNA specificity in the amygdala. Two days of Gadd45b siRNA gives approximately 20% knockdown of mRNA (control: 0.4861 ± 0.0276, N = 5; Gadd45b: 0.3967 ± 0.0130, N = 5); * p = 0.0191. Gadd45b siRNA does not significantly decrease

    Techniques Used:

    Neonatal Gadd45b siRNA infusion increases the initiation of play fighting. (A) Transient neonatal disruption of Gadd45b expression in the amygdala more than doubles the frequency of juvenile social play (control siRNA males: 2.064 ± 0.5007, N
    Figure Legend Snippet: Neonatal Gadd45b siRNA infusion increases the initiation of play fighting. (A) Transient neonatal disruption of Gadd45b expression in the amygdala more than doubles the frequency of juvenile social play (control siRNA males: 2.064 ± 0.5007, N

    Techniques Used: Expressing

    Gadd45b siRNA infused males exhibit abnormal social behavior, with no preference for the social chamber. Repeated measures ANOVA analysis reveals a strong preference for the social chamber in control siRNA males ( F = 15.23, p
    Figure Legend Snippet: Gadd45b siRNA infused males exhibit abnormal social behavior, with no preference for the social chamber. Repeated measures ANOVA analysis reveals a strong preference for the social chamber in control siRNA males ( F = 15.23, p

    Techniques Used:

    3.7. Gadd45b siRNA epigenetically alters methylation of the Adra2a promoter in the amygdala
    Figure Legend Snippet: 3.7. Gadd45b siRNA epigenetically alters methylation of the Adra2a promoter in the amygdala

    Techniques Used: Methylation

    11) Product Images from "Hydroxysafflor Yellow A Suppresses MRC-5 Cell Activation Induced by TGF-β1 by Blocking TGF-β1 Binding to TβRII"

    Article Title: Hydroxysafflor Yellow A Suppresses MRC-5 Cell Activation Induced by TGF-β1 by Blocking TGF-β1 Binding to TβRII

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2017.00264

    Effects of HSYA on TGF-β1/Smad and ERK/MAPK signaling pathway in TGF-β1-treated MRC-5 cells with TβRII knockdown. MRC-5 cells were pretreated with TβRII-non-targeting control siRNA or TβRII siRNA for 24 h followed by 45 μmol/L HSYA for 30 min before stimulation with 10 ng/mL TGF-β1 for 1 h. Protein levels of phosphorylated Smad2, Smad3, and ERK were analyzed by western blotting (A) . Densitometric analyses of phosphorylation statuses of Smad2 (B) , Smad3 (C) , and ERK (D) . Data are presented as mean ± SD, n = 3 per group. ΔΔ p
    Figure Legend Snippet: Effects of HSYA on TGF-β1/Smad and ERK/MAPK signaling pathway in TGF-β1-treated MRC-5 cells with TβRII knockdown. MRC-5 cells were pretreated with TβRII-non-targeting control siRNA or TβRII siRNA for 24 h followed by 45 μmol/L HSYA for 30 min before stimulation with 10 ng/mL TGF-β1 for 1 h. Protein levels of phosphorylated Smad2, Smad3, and ERK were analyzed by western blotting (A) . Densitometric analyses of phosphorylation statuses of Smad2 (B) , Smad3 (C) , and ERK (D) . Data are presented as mean ± SD, n = 3 per group. ΔΔ p

    Techniques Used: Western Blot

    Effects of HSYA on α-SMA, COL1A1, and FN protein expression in TGF-β1-treated MRC-5 cells with TβRII knockdown. MRC-5 cells were pretreated with TβRII-non-targeting control siRNA or TβRII siRNA for 24 h followed by 45 μmol/L HSYA for 30 min before stimulation with 10 ng/mL TGF-β1 for 48 h. Protein expression levels of α-SMA, COL1A1, and FN were measured by western blotting (A) . Densitometric analyses of α-SMA (B) , COL1A1 (C) , and FN (D) . Data are presented as mean ± SD, n = 3 per group. ΔΔ p
    Figure Legend Snippet: Effects of HSYA on α-SMA, COL1A1, and FN protein expression in TGF-β1-treated MRC-5 cells with TβRII knockdown. MRC-5 cells were pretreated with TβRII-non-targeting control siRNA or TβRII siRNA for 24 h followed by 45 μmol/L HSYA for 30 min before stimulation with 10 ng/mL TGF-β1 for 48 h. Protein expression levels of α-SMA, COL1A1, and FN were measured by western blotting (A) . Densitometric analyses of α-SMA (B) , COL1A1 (C) , and FN (D) . Data are presented as mean ± SD, n = 3 per group. ΔΔ p

    Techniques Used: Expressing, Western Blot

    12) Product Images from "Decreased expression of the NF-κB family member RelB in lung fibroblasts from Smokers with and without COPD potentiates cigarette smoke-induced COX-2 expression"

    Article Title: Decreased expression of the NF-κB family member RelB in lung fibroblasts from Smokers with and without COPD potentiates cigarette smoke-induced COX-2 expression

    Journal: Respiratory Research

    doi: 10.1186/s12931-015-0214-6

    siRNA-mediated knock-down of RelB potentiates CSE-induced Cox-2 mRNA and protein expression in Normal lung fibroblasts. (A) RelB siRNA: Lung fibroblasts derived from Normal subjects were transfected with siRNA against RelB (siRelB). RelB protein levels were decreased by approximately 40% compared to Ctrl-transfected (siCtrl; relative-change: 0.59 ± 0.065). Results are expressed as the mean ± SEM, n = 4 independent experiments . (B) Cox-2 mRNA: Normal fibroblasts transfected with RelB siRNA were exposed to 2% CSE for 6 hours and Cox-2 mRNA expression evaluated by qRT-PCR. There was a significant increase in Cox-2 mRNA expression only when RelB expression is reduced (siRelB; fold-induction 15 ± 8; *p
    Figure Legend Snippet: siRNA-mediated knock-down of RelB potentiates CSE-induced Cox-2 mRNA and protein expression in Normal lung fibroblasts. (A) RelB siRNA: Lung fibroblasts derived from Normal subjects were transfected with siRNA against RelB (siRelB). RelB protein levels were decreased by approximately 40% compared to Ctrl-transfected (siCtrl; relative-change: 0.59 ± 0.065). Results are expressed as the mean ± SEM, n = 4 independent experiments . (B) Cox-2 mRNA: Normal fibroblasts transfected with RelB siRNA were exposed to 2% CSE for 6 hours and Cox-2 mRNA expression evaluated by qRT-PCR. There was a significant increase in Cox-2 mRNA expression only when RelB expression is reduced (siRelB; fold-induction 15 ± 8; *p

    Techniques Used: Expressing, Derivative Assay, Transfection, Quantitative RT-PCR

    13) Product Images from "Autophagy Promotes Intracellular Degradation of Type I Collagen Induced by Transforming Growth Factor (TGF)-?1 *"

    Article Title: Autophagy Promotes Intracellular Degradation of Type I Collagen Induced by Transforming Growth Factor (TGF)-?1 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.308460

    Reduced expression of Beclin 1 increases Col-I levels in mesangial cells. A , Western blot analysis of type I collagen (Col-I) and Beclin 1 in MMC obtained from beclin 1 +/+ and beclin 1 −/− mice. Cells were treated with TGF-β1 (2 ng/ml) for the indicated time periods. B , expression levels of Col-I and Beclin 1 in beclin 1 +/+ MMC transfected with either control siRNA or beclin 1 siRNA and treated with TGF-β1 (2 ng/ml) for the indicated time periods. Immunoblotting for β-actin was used as protein loading control ( A and B ). The levels of Col-I were quantitated as ratio to β-actin by densitometry. Data are presented as the mean ± S.E. of three independent experiments (for each time point *, p
    Figure Legend Snippet: Reduced expression of Beclin 1 increases Col-I levels in mesangial cells. A , Western blot analysis of type I collagen (Col-I) and Beclin 1 in MMC obtained from beclin 1 +/+ and beclin 1 −/− mice. Cells were treated with TGF-β1 (2 ng/ml) for the indicated time periods. B , expression levels of Col-I and Beclin 1 in beclin 1 +/+ MMC transfected with either control siRNA or beclin 1 siRNA and treated with TGF-β1 (2 ng/ml) for the indicated time periods. Immunoblotting for β-actin was used as protein loading control ( A and B ). The levels of Col-I were quantitated as ratio to β-actin by densitometry. Data are presented as the mean ± S.E. of three independent experiments (for each time point *, p

    Techniques Used: Expressing, Western Blot, Mouse Assay, Transfection

    14) Product Images from "Role of microRNAs and microRNA machinery in the pathogenesis of diffuse large B-cell lymphoma"

    Article Title: Role of microRNAs and microRNA machinery in the pathogenesis of diffuse large B-cell lymphoma

    Journal: Blood Cancer Journal

    doi: 10.1038/bcj.2013.49

    Silencing of TARBP2 reduces cell proliferation and increases apoptosis in DLBCL cell lines. Histograms show the effect of ( a ) cell viability measured by WST-1 colorimetric assay and ( b ) cell death evaluated by Caspase-3/CPP32 colorimetric assay in OCI-Ly-1 and OCI-Ly-3 cells transfected with TARBP2 siRNA as compared with si-CTR cells. Data presented represent mean of three independent experiments. Error bars represent standard deviations from the mean. All comparisons were evaluated by paired t -test. P -values
    Figure Legend Snippet: Silencing of TARBP2 reduces cell proliferation and increases apoptosis in DLBCL cell lines. Histograms show the effect of ( a ) cell viability measured by WST-1 colorimetric assay and ( b ) cell death evaluated by Caspase-3/CPP32 colorimetric assay in OCI-Ly-1 and OCI-Ly-3 cells transfected with TARBP2 siRNA as compared with si-CTR cells. Data presented represent mean of three independent experiments. Error bars represent standard deviations from the mean. All comparisons were evaluated by paired t -test. P -values

    Techniques Used: Colorimetric Assay, Transfection

    15) Product Images from "Transient receptor potential channel TRPM8 is over-expressed and required for cellular proliferation in pancreatic adenocarcinoma"

    Article Title: Transient receptor potential channel TRPM8 is over-expressed and required for cellular proliferation in pancreatic adenocarcinoma

    Journal: Cancer letters

    doi: 10.1016/j.canlet.2010.04.023

    TRPM8-deficient pancreatic adenocarcinoma cells have impaired ability to progress through the cell division cycle. Serum-deprived PANC-1 and BxPC-3 cells were transfected with anti- TRPM8 siRNA or non-targeting control siRNA. Forty-eight hours later, the cells were analyzed for DNA content by flow cytometry. The proportion of cells in each phase of the cell cycle is indicated. Non-specific sub-G 0 /G 1 events were gated out for accurate analysis of cell cycle distribution curves in the viable cells. This experiment was repeated with similar results.
    Figure Legend Snippet: TRPM8-deficient pancreatic adenocarcinoma cells have impaired ability to progress through the cell division cycle. Serum-deprived PANC-1 and BxPC-3 cells were transfected with anti- TRPM8 siRNA or non-targeting control siRNA. Forty-eight hours later, the cells were analyzed for DNA content by flow cytometry. The proportion of cells in each phase of the cell cycle is indicated. Non-specific sub-G 0 /G 1 events were gated out for accurate analysis of cell cycle distribution curves in the viable cells. This experiment was repeated with similar results.

    Techniques Used: Transfection, Flow Cytometry, Cytometry

    RNA interference-mediated silencing of TRPM8 induces alterations of cellular and nuclear morphology suggestive of proliferative arrest. PANC-1 and BxPC-3 cells transfected with siRNA directed against TR PM8 or non-targeting control siRNA. A. Phase contrast micrographs of TRPM8-deficient cells with multiple micronuclei (red arrowheads). B. Fluorescent micrographs of DAPI-stained TRPM8-deficient cells with abnormal nuclear morphology (white arrows). These experiments were repeated two times with essentially the same results.
    Figure Legend Snippet: RNA interference-mediated silencing of TRPM8 induces alterations of cellular and nuclear morphology suggestive of proliferative arrest. PANC-1 and BxPC-3 cells transfected with siRNA directed against TR PM8 or non-targeting control siRNA. A. Phase contrast micrographs of TRPM8-deficient cells with multiple micronuclei (red arrowheads). B. Fluorescent micrographs of DAPI-stained TRPM8-deficient cells with abnormal nuclear morphology (white arrows). These experiments were repeated two times with essentially the same results.

    Techniques Used: Transfection, Staining

    Anti- TRPM8 siRNA up-regulates expression of p21 CDKN1A and p27 CDKN1B . Total RNA extracted from PANC-1 and BxPC-3 cells 72 h following transfection with siRNA directed against TR PM8 or non-targeting control siRNA was analyzed for p21 CDKN1A and p27 CDKN1B mRNA levels by quantitative real-time PCR. Each value represents the relative p21 CDKN1A or p27 CDKN1B mRNA level in the anti- TRPM8 siRNA transfected cells as compared to that in control siRNA transfected cells. This experiment was independently performed twice, and the same pattern of changes in p21 CDKN1A and p27 CDKN1B mRNA was observed.
    Figure Legend Snippet: Anti- TRPM8 siRNA up-regulates expression of p21 CDKN1A and p27 CDKN1B . Total RNA extracted from PANC-1 and BxPC-3 cells 72 h following transfection with siRNA directed against TR PM8 or non-targeting control siRNA was analyzed for p21 CDKN1A and p27 CDKN1B mRNA levels by quantitative real-time PCR. Each value represents the relative p21 CDKN1A or p27 CDKN1B mRNA level in the anti- TRPM8 siRNA transfected cells as compared to that in control siRNA transfected cells. This experiment was independently performed twice, and the same pattern of changes in p21 CDKN1A and p27 CDKN1B mRNA was observed.

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

    SiRNA-induced knock down of TRPM8 reduces the ability ofpan creatic adenocarcinoma cells to proliferate. PANC-1 and BxPC-3 cells treated with siRNA anti- TRPM8 were analyzed by the MTS assay and by counting cells. Proliferation of the TRPM8-deficient cells is expressed as a percentage of that in control siRNA transfected cells. Each value represents the mean of 6 independent cell samples +/− standard deviation. * P
    Figure Legend Snippet: SiRNA-induced knock down of TRPM8 reduces the ability ofpan creatic adenocarcinoma cells to proliferate. PANC-1 and BxPC-3 cells treated with siRNA anti- TRPM8 were analyzed by the MTS assay and by counting cells. Proliferation of the TRPM8-deficient cells is expressed as a percentage of that in control siRNA transfected cells. Each value represents the mean of 6 independent cell samples +/− standard deviation. * P

    Techniques Used: MTS Assay, Transfection, Standard Deviation

    16) Product Images from "Nischarin-siRNA delivered by polyethylenimine-alginate nanoparticles accelerates motor function recovery after spinal cord injury"

    Article Title: Nischarin-siRNA delivered by polyethylenimine-alginate nanoparticles accelerates motor function recovery after spinal cord injury

    Journal: Neural Regeneration Research

    doi: 10.4103/1673-5374.217348

    Effect of Nis-siRNA delivered by PEI-ALG nanoparticles on GAP-43 protein expression in the injured spinal cord of rats on day 21 after SCI. Protein expression of GAP-43 was markedly decreased in the SCI, PEI-ALG, and PEI-ALG/Ctl-siRNA groups compared with the sham group (§ P
    Figure Legend Snippet: Effect of Nis-siRNA delivered by PEI-ALG nanoparticles on GAP-43 protein expression in the injured spinal cord of rats on day 21 after SCI. Protein expression of GAP-43 was markedly decreased in the SCI, PEI-ALG, and PEI-ALG/Ctl-siRNA groups compared with the sham group (§ P

    Techniques Used: Expressing, CTL Assay

    Nischarin protein expression in the injured spinal cord of rats. (A, B) Western blot assay shows markedly upregulated nischarin protein expression in SCI, PEI-ALG, and PEI-ALG/Ctl-siRNA groups compared with the sham group (§ P
    Figure Legend Snippet: Nischarin protein expression in the injured spinal cord of rats. (A, B) Western blot assay shows markedly upregulated nischarin protein expression in SCI, PEI-ALG, and PEI-ALG/Ctl-siRNA groups compared with the sham group (§ P

    Techniques Used: Expressing, Western Blot, CTL Assay

    17) Product Images from "Knockdown BMI1 expression inhibits proliferation and invasion in human bladder cancer T24 cells"

    Article Title: Knockdown BMI1 expression inhibits proliferation and invasion in human bladder cancer T24 cells

    Journal: Molecular and Cellular Biochemistry

    doi: 10.1007/s11010-013-1745-0

    Effect of BMI1 knockdown on the expression of epithelial marker E-cadherin and mesenchymal marker vimentin and twist, measured by western blot. The epithelial marker E-cadherin was increased, whereas the mesenchymal marker vimentin and twist decreased in BMI1 siRNA group cells than control group cells
    Figure Legend Snippet: Effect of BMI1 knockdown on the expression of epithelial marker E-cadherin and mesenchymal marker vimentin and twist, measured by western blot. The epithelial marker E-cadherin was increased, whereas the mesenchymal marker vimentin and twist decreased in BMI1 siRNA group cells than control group cells

    Techniques Used: Expressing, Marker, Western Blot

    a BMI1 knockdown enhanced the apoptosis of T24 cells. Apoptotic cells of different groups were measured by flow cytometry after 48 h transduction. The cell populations of Annexin-V +/PI− and Annexin-V +/PI+ were used to assess apoptotic events. ( a ) BMI1 siRNA group, ( b ) control group. b The apoptosis rate of BMI1 siRNA group cells was significantly higher than that of control group cells. p
    Figure Legend Snippet: a BMI1 knockdown enhanced the apoptosis of T24 cells. Apoptotic cells of different groups were measured by flow cytometry after 48 h transduction. The cell populations of Annexin-V +/PI− and Annexin-V +/PI+ were used to assess apoptotic events. ( a ) BMI1 siRNA group, ( b ) control group. b The apoptosis rate of BMI1 siRNA group cells was significantly higher than that of control group cells. p

    Techniques Used: Flow Cytometry, Cytometry, Transduction

    Morphologic changes in T24 cells after BMI1 knockdown. a BMI1 siRNA group, b control group. In the BMI1 siRNA group, cells showed cobblestone-like morphology, whereas the control group cells kept their fibroblastic, elongated like morphology. Photos were taken under ×200 magnification
    Figure Legend Snippet: Morphologic changes in T24 cells after BMI1 knockdown. a BMI1 siRNA group, b control group. In the BMI1 siRNA group, cells showed cobblestone-like morphology, whereas the control group cells kept their fibroblastic, elongated like morphology. Photos were taken under ×200 magnification

    Techniques Used:

    Effect of BMI1 knockdown on the expression of cell cycle and apoptosis related genes, measured by western blot. Cyclin D1, cdk2, BCL2 were decreased in BMI1 siRNA group cells than control cells, while apoptosis protein cleaved-caspase3 was increased
    Figure Legend Snippet: Effect of BMI1 knockdown on the expression of cell cycle and apoptosis related genes, measured by western blot. Cyclin D1, cdk2, BCL2 were decreased in BMI1 siRNA group cells than control cells, while apoptosis protein cleaved-caspase3 was increased

    Techniques Used: Expressing, Western Blot

    a Effect of BMI1 siRNA on the migration of T24 cells in vitro. A.Transwell migration assay was performed to measure T24 cell migration. Crystal violet staining of the lower surface filters showed that the cells were able to migrate to the filter (×400). The number of the invaded cells of the control group is dramatically more than that of the BMI1 siRNA group. ( a ) BMI1 siRNA group, ( b ) control group. b Quantification of the numbers of BMI1 siRNA group and control group cells migrating through the matrigel. p
    Figure Legend Snippet: a Effect of BMI1 siRNA on the migration of T24 cells in vitro. A.Transwell migration assay was performed to measure T24 cell migration. Crystal violet staining of the lower surface filters showed that the cells were able to migrate to the filter (×400). The number of the invaded cells of the control group is dramatically more than that of the BMI1 siRNA group. ( a ) BMI1 siRNA group, ( b ) control group. b Quantification of the numbers of BMI1 siRNA group and control group cells migrating through the matrigel. p

    Techniques Used: Migration, In Vitro, Transwell Migration Assay, Staining

    Effect of BMI1 knockdown on the expression of BMI1, P16, and P14 in bladder cancer T24 cells, measured by western blot. The BMI1 was knockdown by using siRNA transfected. After BMI1 knockdown, the expression of P16 and P14 increased
    Figure Legend Snippet: Effect of BMI1 knockdown on the expression of BMI1, P16, and P14 in bladder cancer T24 cells, measured by western blot. The BMI1 was knockdown by using siRNA transfected. After BMI1 knockdown, the expression of P16 and P14 increased

    Techniques Used: Expressing, Western Blot, Transfection

    a Soft agar colony formation assay showed that BMI1 knockdown reduced T24 cell colony formation. Colonies larger than 1 mm in diameter were counted. The number of T24 colonies was significantly reduced by BMI1 knockdown. ( a ) BMI1 siRNA group ( b ) control group. b The bar graph showed the mean number of colonies in 10 low power fields for each group. p
    Figure Legend Snippet: a Soft agar colony formation assay showed that BMI1 knockdown reduced T24 cell colony formation. Colonies larger than 1 mm in diameter were counted. The number of T24 colonies was significantly reduced by BMI1 knockdown. ( a ) BMI1 siRNA group ( b ) control group. b The bar graph showed the mean number of colonies in 10 low power fields for each group. p

    Techniques Used: Soft Agar Assay

    18) Product Images from "Transient receptor potential ion channel Trpm7 regulates exocrine pancreatic epithelial proliferation by Mg2+-sensitive Socs3a signaling in development and cancer"

    Article Title: Transient receptor potential ion channel Trpm7 regulates exocrine pancreatic epithelial proliferation by Mg2+-sensitive Socs3a signaling in development and cancer

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.004564

    TRPM7 is overexpressed in human pancreatic adenocarcinoma and required for cellular proliferation. (A) Expression of TRPM7 protein in human pancreatic adenocarcinoma (upper panel) and normal pancreatic tissue (lower panel) by immunohistochemistry using anti-TRPM7 antibodies. The images shown are representative of five tumor specimens and five normal control samples. Scale bars: 100 μm. (B) Relative TRPM7 mRNA levels in human pancreatic adenocarcinoma cell lines by quantitative real-time PCR. Each value represents the mean ratio of each pancreatic cancer cell line to that of H6c7 from triplicate samples, and reproducible results were obtained from two independent experiments. (C) Cell morphology at 24 hours post-transfection with siRNA was examined under an inverted microscope with phase contrast. Red arrows point to multinucleated cells with cytoplasmic vacuoles.
    Figure Legend Snippet: TRPM7 is overexpressed in human pancreatic adenocarcinoma and required for cellular proliferation. (A) Expression of TRPM7 protein in human pancreatic adenocarcinoma (upper panel) and normal pancreatic tissue (lower panel) by immunohistochemistry using anti-TRPM7 antibodies. The images shown are representative of five tumor specimens and five normal control samples. Scale bars: 100 μm. (B) Relative TRPM7 mRNA levels in human pancreatic adenocarcinoma cell lines by quantitative real-time PCR. Each value represents the mean ratio of each pancreatic cancer cell line to that of H6c7 from triplicate samples, and reproducible results were obtained from two independent experiments. (C) Cell morphology at 24 hours post-transfection with siRNA was examined under an inverted microscope with phase contrast. Red arrows point to multinucleated cells with cytoplasmic vacuoles.

    Techniques Used: Expressing, Immunohistochemistry, Real-time Polymerase Chain Reaction, Transfection, Inverted Microscopy

    TRPM7 controls cellular proliferation by regulating cell-cycle progression and by modulating p21 CDKN1A , cyclin G1 and cyclin B1 expression and Mg 2+ level. (A) PANC-1 cells were treated with siRNA directed against TRPM7 , non-targeting control siRNA, or without siRNA, and then analyzed by MTS assay and counting cells. Proliferation in the anti- TRPM7- siRNA-treated cells is represented as percent of that in the cells treated with non-targeting control siRNA. Each column represents the mean from triplicate samples. Bars represent s.d. * P
    Figure Legend Snippet: TRPM7 controls cellular proliferation by regulating cell-cycle progression and by modulating p21 CDKN1A , cyclin G1 and cyclin B1 expression and Mg 2+ level. (A) PANC-1 cells were treated with siRNA directed against TRPM7 , non-targeting control siRNA, or without siRNA, and then analyzed by MTS assay and counting cells. Proliferation in the anti- TRPM7- siRNA-treated cells is represented as percent of that in the cells treated with non-targeting control siRNA. Each column represents the mean from triplicate samples. Bars represent s.d. * P

    Techniques Used: Expressing, MTS Assay

    19) Product Images from "Suxiao Jiuxin pill promotes exosome secretion from mouse cardiac mesenchymal stem cells in vitro"

    Article Title: Suxiao Jiuxin pill promotes exosome secretion from mouse cardiac mesenchymal stem cells in vitro

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2018.19

    Knockdown of Rab27a or Rab27b inhibits SJP-mediated exosome release in C-MSCs. (A, B) qRT-PCR quantification of Rab27a and Rab27b mRNA levels (normalized to GADPH levels) in C-MSCs transfected with Rab27a siRNA, Rab27b siRNA, or non-targeting (NT) siRNA ( * P
    Figure Legend Snippet: Knockdown of Rab27a or Rab27b inhibits SJP-mediated exosome release in C-MSCs. (A, B) qRT-PCR quantification of Rab27a and Rab27b mRNA levels (normalized to GADPH levels) in C-MSCs transfected with Rab27a siRNA, Rab27b siRNA, or non-targeting (NT) siRNA ( * P

    Techniques Used: Quantitative RT-PCR, Transfection

    20) Product Images from "Bacterial lipoproteins induce distinct IL-8 inductions relying on their lipid moieties in pulmonary epithelial cells"

    Article Title: Bacterial lipoproteins induce distinct IL-8 inductions relying on their lipid moieties in pulmonary epithelial cells

    Journal: bioRxiv

    doi: 10.1101/2020.04.03.024471

    Lipopeptides induce IL-8 expression via intracellular TLR2 in A549 cells. (A) TLR2 knockdown effect on IL-8 production induced by lipopeptides in A549 cells. siRNA-transfected cells were treated with either pam2CSK4 or pam3CSK4 for additional 24 h. (B) Blocking antibodies for TLR2, TLR4, and CD14 had no effect on lipopeptide-induced IL-8 production. Cells were treated with blocking antibodies for 1 h prior to the lipopeptide stimulation for 24 h. Mouse IgG2aκ (for TLR2 and TLR4 antibodies) and IgG1aκ (for CD14 antibody) were used as isotype controls. Bars indicate means ± S.D. (n=3). (C) Intracellular localization of TLR2 in A549 cells was determined by immunostaining and FACS. Grey areas indicate isotype control. Dotted lines indicate fixed cells for surface staining and closed lines indicate permeabilized cells for intracellular staining of TLR.
    Figure Legend Snippet: Lipopeptides induce IL-8 expression via intracellular TLR2 in A549 cells. (A) TLR2 knockdown effect on IL-8 production induced by lipopeptides in A549 cells. siRNA-transfected cells were treated with either pam2CSK4 or pam3CSK4 for additional 24 h. (B) Blocking antibodies for TLR2, TLR4, and CD14 had no effect on lipopeptide-induced IL-8 production. Cells were treated with blocking antibodies for 1 h prior to the lipopeptide stimulation for 24 h. Mouse IgG2aκ (for TLR2 and TLR4 antibodies) and IgG1aκ (for CD14 antibody) were used as isotype controls. Bars indicate means ± S.D. (n=3). (C) Intracellular localization of TLR2 in A549 cells was determined by immunostaining and FACS. Grey areas indicate isotype control. Dotted lines indicate fixed cells for surface staining and closed lines indicate permeabilized cells for intracellular staining of TLR.

    Techniques Used: Expressing, Transfection, Blocking Assay, Immunostaining, FACS, Staining

    TLR2 knockdown efficiency. A549 cells were transfected with either control or TLR2 siRNA for 6 h and then treated with either pam2CSK4 or pam3CSK4 for additional 24 h to determine IL-8 secretion. The cells were collected and TLR2 mRNA expression was determined by quantitative RT-PCR from total RNA. Bars indicate means ± S.D. (n=3).
    Figure Legend Snippet: TLR2 knockdown efficiency. A549 cells were transfected with either control or TLR2 siRNA for 6 h and then treated with either pam2CSK4 or pam3CSK4 for additional 24 h to determine IL-8 secretion. The cells were collected and TLR2 mRNA expression was determined by quantitative RT-PCR from total RNA. Bars indicate means ± S.D. (n=3).

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR

    21) Product Images from "Transient receptor potential ion channel Trpm7 regulates exocrine pancreatic epithelial proliferation by Mg2+-sensitive Socs3a signaling in development and cancer"

    Article Title: Transient receptor potential ion channel Trpm7 regulates exocrine pancreatic epithelial proliferation by Mg2+-sensitive Socs3a signaling in development and cancer

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.004564

    TRPM7 is overexpressed in human pancreatic adenocarcinoma and required for cellular proliferation. (A) Expression of TRPM7 protein in human pancreatic adenocarcinoma (upper panel) and normal pancreatic tissue (lower panel) by immunohistochemistry using anti-TRPM7 antibodies. The images shown are representative of five tumor specimens and five normal control samples. Scale bars: 100 μm. (B) Relative TRPM7 mRNA levels in human pancreatic adenocarcinoma cell lines by quantitative real-time PCR. Each value represents the mean ratio of each pancreatic cancer cell line to that of H6c7 from triplicate samples, and reproducible results were obtained from two independent experiments. (C) Cell morphology at 24 hours post-transfection with siRNA was examined under an inverted microscope with phase contrast. Red arrows point to multinucleated cells with cytoplasmic vacuoles.
    Figure Legend Snippet: TRPM7 is overexpressed in human pancreatic adenocarcinoma and required for cellular proliferation. (A) Expression of TRPM7 protein in human pancreatic adenocarcinoma (upper panel) and normal pancreatic tissue (lower panel) by immunohistochemistry using anti-TRPM7 antibodies. The images shown are representative of five tumor specimens and five normal control samples. Scale bars: 100 μm. (B) Relative TRPM7 mRNA levels in human pancreatic adenocarcinoma cell lines by quantitative real-time PCR. Each value represents the mean ratio of each pancreatic cancer cell line to that of H6c7 from triplicate samples, and reproducible results were obtained from two independent experiments. (C) Cell morphology at 24 hours post-transfection with siRNA was examined under an inverted microscope with phase contrast. Red arrows point to multinucleated cells with cytoplasmic vacuoles.

    Techniques Used: Expressing, Immunohistochemistry, Real-time Polymerase Chain Reaction, Transfection, Inverted Microscopy

    TRPM7 controls cellular proliferation by regulating cell-cycle progression and by modulating p21 CDKN1A , cyclin G1 and cyclin B1 expression and Mg 2+ level. (A) PANC-1 cells were treated with siRNA directed against TRPM7 , non-targeting control siRNA, or without siRNA, and then analyzed by MTS assay and counting cells. Proliferation in the anti- TRPM7- siRNA-treated cells is represented as percent of that in the cells treated with non-targeting control siRNA. Each column represents the mean from triplicate samples. Bars represent s.d. * P
    Figure Legend Snippet: TRPM7 controls cellular proliferation by regulating cell-cycle progression and by modulating p21 CDKN1A , cyclin G1 and cyclin B1 expression and Mg 2+ level. (A) PANC-1 cells were treated with siRNA directed against TRPM7 , non-targeting control siRNA, or without siRNA, and then analyzed by MTS assay and counting cells. Proliferation in the anti- TRPM7- siRNA-treated cells is represented as percent of that in the cells treated with non-targeting control siRNA. Each column represents the mean from triplicate samples. Bars represent s.d. * P

    Techniques Used: Expressing, MTS Assay

    22) Product Images from "Targeted silencing of TRPM7 ion channel induces replicative senescence and produces enhanced cytotoxicity with gemcitabine in pancreatic adenocarcinoma"

    Article Title: Targeted silencing of TRPM7 ion channel induces replicative senescence and produces enhanced cytotoxicity with gemcitabine in pancreatic adenocarcinoma

    Journal: Cancer Letters

    doi: 10.1016/j.canlet.2011.12.007

    Combination of anti- TRPM7 siRNA with gemcitabine produced enhanced cytotoxicity. PANC-1 cells transfected with anti- TRPM7 siRNA or non-targeting control siRNA were incubated in the presence or absence of gemcitabine at 1 µM or 5 µM and
    Figure Legend Snippet: Combination of anti- TRPM7 siRNA with gemcitabine produced enhanced cytotoxicity. PANC-1 cells transfected with anti- TRPM7 siRNA or non-targeting control siRNA were incubated in the presence or absence of gemcitabine at 1 µM or 5 µM and

    Techniques Used: Produced, Transfection, Incubation

    RNA interference-mediated silencing of TRPM7 did not induce apoptotic cell death. BxPC-3 and PANC-1 cells transfected with anti- TRPM7 siRNA or non-targeting control siRNA were incubated with FITC-conjugated annexin V and PI, and then analyzed for apoptosis
    Figure Legend Snippet: RNA interference-mediated silencing of TRPM7 did not induce apoptotic cell death. BxPC-3 and PANC-1 cells transfected with anti- TRPM7 siRNA or non-targeting control siRNA were incubated with FITC-conjugated annexin V and PI, and then analyzed for apoptosis

    Techniques Used: Transfection, Incubation

    SiRNA-mediated repression of TRPM7 induced cellular morphology indicative of a senescent phenotype. BxPC-3 and PANC-1 cells transfected with anti- TRPM7 siRNA or non-targeting control siRNA were stained with hematoxylin and eosin, and examined under a
    Figure Legend Snippet: SiRNA-mediated repression of TRPM7 induced cellular morphology indicative of a senescent phenotype. BxPC-3 and PANC-1 cells transfected with anti- TRPM7 siRNA or non-targeting control siRNA were stained with hematoxylin and eosin, and examined under a

    Techniques Used: Transfection, Staining

    TRPM7-deficient cells contained multiple nuclei suggesting mitotic arrest. BxPC-3 and PANC-1 cells transfected with anti- TRPM7 siRNA or non-targeting control siRNA were stained with DAPI, and examined under a compound light microscope with fluorescence.
    Figure Legend Snippet: TRPM7-deficient cells contained multiple nuclei suggesting mitotic arrest. BxPC-3 and PANC-1 cells transfected with anti- TRPM7 siRNA or non-targeting control siRNA were stained with DAPI, and examined under a compound light microscope with fluorescence.

    Techniques Used: Transfection, Staining, Light Microscopy, Fluorescence

    TRPM7-deficient cells underwent replicative senescence. BxPC-3 and PANC-1 cells transfected with anti- TRPM7 siRNA or non-targeting control siRNA were analyzed for SA β-gal activity. Images were acquired under an inverted light microscope with
    Figure Legend Snippet: TRPM7-deficient cells underwent replicative senescence. BxPC-3 and PANC-1 cells transfected with anti- TRPM7 siRNA or non-targeting control siRNA were analyzed for SA β-gal activity. Images were acquired under an inverted light microscope with

    Techniques Used: Transfection, Activity Assay, Light Microscopy

    No enhanced cytotoxicity by anti- TRPM7 siRNA in combination with SAHA. PANC-1 cells transfected with anti- TRPM7 siRNA or non-targeting control siRNA were incubated in the presence or absence of SAHA at 1 µM or 5 µM and then analyzed for
    Figure Legend Snippet: No enhanced cytotoxicity by anti- TRPM7 siRNA in combination with SAHA. PANC-1 cells transfected with anti- TRPM7 siRNA or non-targeting control siRNA were incubated in the presence or absence of SAHA at 1 µM or 5 µM and then analyzed for

    Techniques Used: Transfection, Incubation

    23) Product Images from "Activation of HuR downstream of p38 MAPK promotes cardiomyocyte hypertrophy"

    Article Title: Activation of HuR downstream of p38 MAPK promotes cardiomyocyte hypertrophy

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2016.08.005

    HuR knockdown or inhibition inhibits NFAT transcriptional activation and downstream gene expression ( A ) Expression level of the NFAT-dependent gene RCAN was assessed via qRT-PCR in control or PE-treated NRVMs with and without siRNA-mediated HuR knockdown. N ≥ 3. * P ≤ 0.05. ( B ) NFAT transcriptional activation was directly assessed via transient transfection of NRVMs with an NFAT-luciferase reporter. Consistent with other assessed timepoints of NRVM hypertrophy, luciferase activity was measured 24 hours following treatment with PE and HuR pharmacological inhibition (via CMLD1 and CMLD2). N ≥ 8. * P ≤ 0.05.
    Figure Legend Snippet: HuR knockdown or inhibition inhibits NFAT transcriptional activation and downstream gene expression ( A ) Expression level of the NFAT-dependent gene RCAN was assessed via qRT-PCR in control or PE-treated NRVMs with and without siRNA-mediated HuR knockdown. N ≥ 3. * P ≤ 0.05. ( B ) NFAT transcriptional activation was directly assessed via transient transfection of NRVMs with an NFAT-luciferase reporter. Consistent with other assessed timepoints of NRVM hypertrophy, luciferase activity was measured 24 hours following treatment with PE and HuR pharmacological inhibition (via CMLD1 and CMLD2). N ≥ 8. * P ≤ 0.05.

    Techniques Used: Inhibition, Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay

    24) Product Images from "AhR and Arnt differentially regulate NF-κB signaling and chemokine responses in human bronchial epithelial cells"

    Article Title: AhR and Arnt differentially regulate NF-κB signaling and chemokine responses in human bronchial epithelial cells

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-014-0048-8

    AhR and Arnt differentially regulate CXCL8 and CCL5 responses as well as p65 phosphorylation in Poly I: C- exposed BEAS- 2B cells. Cells were transfected with siRNA against p65 (siP65), AhR (siAHR), Arnt (siARNT) or non-targeting control siRNA (siNT), and exposed to 10 μg/ml Poly I:C. Intracellular protein levels of total and phospho-p65 (Ser536) as well as AhR and Arnt were detected by Western blotting after 2 and 4 h exposure, as described under “Materials and methods”. The figure displays representative blots (A) , as well as relative changes in phospho-p65 compared to total p65 quantified by densitometric analysis of the Western blots (B) . CXCL8 (C and E) and CCL5 (D and F) protein levels in the medium were measured by ELISA after 18 h exposure, as described under “Materials and methods”. The results are expressed as mean ± SEM (n ≥ 3). *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.
    Figure Legend Snippet: AhR and Arnt differentially regulate CXCL8 and CCL5 responses as well as p65 phosphorylation in Poly I: C- exposed BEAS- 2B cells. Cells were transfected with siRNA against p65 (siP65), AhR (siAHR), Arnt (siARNT) or non-targeting control siRNA (siNT), and exposed to 10 μg/ml Poly I:C. Intracellular protein levels of total and phospho-p65 (Ser536) as well as AhR and Arnt were detected by Western blotting after 2 and 4 h exposure, as described under “Materials and methods”. The figure displays representative blots (A) , as well as relative changes in phospho-p65 compared to total p65 quantified by densitometric analysis of the Western blots (B) . CXCL8 (C and E) and CCL5 (D and F) protein levels in the medium were measured by ELISA after 18 h exposure, as described under “Materials and methods”. The results are expressed as mean ± SEM (n ≥ 3). *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.

    Techniques Used: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

    RelB suppresses CXCL8, but not CCL5, in BEAS- 2B cells. Cells were transfected with siRNA against RelB (siRELB) or non-targeting control siRNA (siNT). Transfected cells were exposed to 20 μM 1-NP, 1-AP, or vehicle (DMSO) alone (A-D) or 10 μg/ml Poly I:C or vehicle (water) alone (E and F) . CXCL8 (A) and CCL5 (B) gene expression were measured after 6 h by real-time PCR. CXCL8 (C and E) and CCL5 (D and F) protein levels in the medium were measured by ELISA after 18 h exposure as described under “Materials and methods”. Efficiency of transfection was assessed by Western blotting (G) . The results are expressed as mean ± SEM (A/B: n = 1 (triplicate determinations); C-F : n ≥ 3; G: representative blot, n = 3) *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.
    Figure Legend Snippet: RelB suppresses CXCL8, but not CCL5, in BEAS- 2B cells. Cells were transfected with siRNA against RelB (siRELB) or non-targeting control siRNA (siNT). Transfected cells were exposed to 20 μM 1-NP, 1-AP, or vehicle (DMSO) alone (A-D) or 10 μg/ml Poly I:C or vehicle (water) alone (E and F) . CXCL8 (A) and CCL5 (B) gene expression were measured after 6 h by real-time PCR. CXCL8 (C and E) and CCL5 (D and F) protein levels in the medium were measured by ELISA after 18 h exposure as described under “Materials and methods”. Efficiency of transfection was assessed by Western blotting (G) . The results are expressed as mean ± SEM (A/B: n = 1 (triplicate determinations); C-F : n ≥ 3; G: representative blot, n = 3) *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.

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

    Depletion of AhR and Arnt increases RelB levels in Poly I: C - exposed BEAS- 2B cells. Cells were transfected with siRNA against AhR (siAHR), Arnt (siARNT) or non-targeting control siRNA (siNT), and exposed to 10 μg/ml Poly I:C for 2 and 4 h. Intracellular protein levels of RelB and β-actin were detected by Western blotting as described under “Materials and methods”. The figure displays representative blots of RelB levels in unexposed cells (A) and Poly I:C-exposed cells (B) . The graph depicts relative changes in RelB (B) compared to β-actin quantified by densitometric analysis of the Western blots. The results are expressed as mean ± SEM (n = 3). *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.
    Figure Legend Snippet: Depletion of AhR and Arnt increases RelB levels in Poly I: C - exposed BEAS- 2B cells. Cells were transfected with siRNA against AhR (siAHR), Arnt (siARNT) or non-targeting control siRNA (siNT), and exposed to 10 μg/ml Poly I:C for 2 and 4 h. Intracellular protein levels of RelB and β-actin were detected by Western blotting as described under “Materials and methods”. The figure displays representative blots of RelB levels in unexposed cells (A) and Poly I:C-exposed cells (B) . The graph depicts relative changes in RelB (B) compared to β-actin quantified by densitometric analysis of the Western blots. The results are expressed as mean ± SEM (n = 3). *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.

    Techniques Used: Transfection, Western Blot

    AhR and Arnt differentially regulate CXCL8 and CCL5 responses in 1- NP- or 1- AP- exposed BEAS- 2B cells. Cells were transfected with siRNA against AhR (siAHR) and Arnt (siARNT) or non-targeting control siRNA (siNT), and exposed to 20 μM 1-NP, 1-AP or vehicle (DMSO) alone. CXCL8 (A) and CCL5 (B) gene expression were measured after 6 h by real-time PCR. CXCL8 (C) and CCL5 (D) protein levels in the medium were measured by ELISA after 18 h exposure as described under “Materials and methods”. Efficiency of transfection was assessed by Western blotting (E) as well as expression of CYP1A1 after 6 h exposure to 20 μM B[a]P, 1-NP and 1-AP, by real-time PCR (F) . The results are expressed as mean ± SEM (A/B/F: n = 1 (triplicate determinations); C/D: n ≥ 3; E: representative blots, n ≥ 3). *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.
    Figure Legend Snippet: AhR and Arnt differentially regulate CXCL8 and CCL5 responses in 1- NP- or 1- AP- exposed BEAS- 2B cells. Cells were transfected with siRNA against AhR (siAHR) and Arnt (siARNT) or non-targeting control siRNA (siNT), and exposed to 20 μM 1-NP, 1-AP or vehicle (DMSO) alone. CXCL8 (A) and CCL5 (B) gene expression were measured after 6 h by real-time PCR. CXCL8 (C) and CCL5 (D) protein levels in the medium were measured by ELISA after 18 h exposure as described under “Materials and methods”. Efficiency of transfection was assessed by Western blotting (E) as well as expression of CYP1A1 after 6 h exposure to 20 μM B[a]P, 1-NP and 1-AP, by real-time PCR (F) . The results are expressed as mean ± SEM (A/B/F: n = 1 (triplicate determinations); C/D: n ≥ 3; E: representative blots, n ≥ 3). *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.

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

    AhR silencing increased TNF- α-, but not 1- NP- induced p50/ p65 reporter gene expression in BEAS- 2B cells. Cells were transfected with a NF-κB-luciferase (p50/p65) reporter gene, and exposed to 1-NP or TNF-α for 6 or 16 h (A) , and assayed for luciferase activity as described under “Materials and methods”. Cells transfected siRNAs against AhR (siAHR), Arnt (siARNT) or non-targeting control siRNA (siNT) were further transfected with NF-κB-luciferase promoter and assayed for luciferase activity after a 16 h exposure to 1-NP (20 μM) or TNF-α (50 ng/ml) (B) . The results are expressed as mean ± SEM (A: n = 2; B: n = 5). *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.
    Figure Legend Snippet: AhR silencing increased TNF- α-, but not 1- NP- induced p50/ p65 reporter gene expression in BEAS- 2B cells. Cells were transfected with a NF-κB-luciferase (p50/p65) reporter gene, and exposed to 1-NP or TNF-α for 6 or 16 h (A) , and assayed for luciferase activity as described under “Materials and methods”. Cells transfected siRNAs against AhR (siAHR), Arnt (siARNT) or non-targeting control siRNA (siNT) were further transfected with NF-κB-luciferase promoter and assayed for luciferase activity after a 16 h exposure to 1-NP (20 μM) or TNF-α (50 ng/ml) (B) . The results are expressed as mean ± SEM (A: n = 2; B: n = 5). *Significantly different from unexposed controls; †Significantly different from cells transfected with non-targeting siRNA.

    Techniques Used: Expressing, Transfection, Luciferase, Activity Assay

    25) Product Images from "Raloxifene Induces Autophagy-Dependent Cell Death in Breast Cancer Cells via the Activation of AMP-Activated Protein Kinase"

    Article Title: Raloxifene Induces Autophagy-Dependent Cell Death in Breast Cancer Cells via the Activation of AMP-Activated Protein Kinase

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2015.2193

    Raloxifene induces autophagy-dependent cell death. (A) MCF-7 cells were transfected with 0.17 μM non-targeting control siRNA (siCont) or BECN1 siRNA (si BECN1 ) for 48 h. Bars denote cell viability of cells treated with 10 μM raloxifene
    Figure Legend Snippet: Raloxifene induces autophagy-dependent cell death. (A) MCF-7 cells were transfected with 0.17 μM non-targeting control siRNA (siCont) or BECN1 siRNA (si BECN1 ) for 48 h. Bars denote cell viability of cells treated with 10 μM raloxifene

    Techniques Used: Transfection

    26) Product Images from "The EphB4 Receptor Tyrosine Kinase Promotes Lung Cancer Growth: A Potential Novel Therapeutic Target"

    Article Title: The EphB4 Receptor Tyrosine Kinase Promotes Lung Cancer Growth: A Potential Novel Therapeutic Target

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067668

    EphB4 protein knockdown reduces SCLC cell viability. A. EphB4 protein knockdown following siRNA transfection. GAPDH was used as a protein loading control. Scr, scrambled siRNA. B. Viability of cells following EphB4 knockdown and/or treatment with SN-38. Error bars indicate SD. **, p
    Figure Legend Snippet: EphB4 protein knockdown reduces SCLC cell viability. A. EphB4 protein knockdown following siRNA transfection. GAPDH was used as a protein loading control. Scr, scrambled siRNA. B. Viability of cells following EphB4 knockdown and/or treatment with SN-38. Error bars indicate SD. **, p

    Techniques Used: Transfection

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    Small Interfering RNA:

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    Santa Cruz Biotechnology non targeting sirna control sirna
    Endogenous immunofluorescence of LC3 after PKCε downregulation. a Cells were <t>transfected</t> with PKCε <t>siRNA</t> (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated with rapamycin (300 nM) for 24 h, as described in Autophagosome visualization section. Representative images from HeLaPKCεA/E, U-138 MG, and U-118 MG cells are shown. Scale bars equate to 10 μm. b AUTOCOUNTER analysis in imaging of U-138 MG and U-118 MG cells after siRNA silencing of PKCε (72 h) and Rapamycin (300 nM) treatment (24 h). The ratio between vesicle area and cell area (Aves/Acell), number of vesicles (#ves), and number of vesicles grouped, according to their area, into three defined classes (small: 0
    Non Targeting Sirna Control Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Transcription factor activities involved in mediating NaCl-induced expression of the c-Fos gene in RPE cells. mRNA levels were determined with real-time reverse transcription (RT)–PCR analysis in cells cultured for 2 h in iso- (control) and hyperosmotic (+ 100 mM NaCl) media, and are expressed as folds of the unstimulated control. A : The level of c-Fos mRNA was determined in cells cultured in the presence of the following inhibitory compounds: the AP-1 inhibitor SR11302 (5 µM), the cAMP response element-binding protein (CREB) inhibitor 666–15 (250 nM), a HIF inhibitor (HIF-Inh; 5 µM), the STAT3 inhibitor Stattic (1 µM), and the nuclear factor (NF)-κB inhibitor CAPE (5 µM). B, C : mRNA levels were determined in cells transfected with nuclear factor of activated T cell 5 <t>(NFAT5)</t> <t>siRNA</t> (siNFAT5; 5 nM) and non-targeted siRNA (siNon; 5 nM), respectively. B : Transfection of RPE cells with NFAT5 siRNA for 48 h resulted in a reduction of the NFAT5 mRNA level in cells cultured for 2 h in iso- and hyperosmotic media. Non-targeted siRNA had no effects. As negative control, transfection reagent (TR) without siRNA was tested. C : Knocking down NFAT5 with siRNA reduced the level of c-Fos mRNA under hyperosmotic conditions. Each bar represents data obtained in three to nine independent experiments using cell lines from different donors. Significant difference versus unstimulated control: *p
    Non Targeted Control Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Depletion of Epac1 attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. <t>HPAEC</t> were treated with Epac1 specific <t>siRNA</t> or non-targeting siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Epac1 significantly attenuated adenosine-induced endothelial barrier enhancement. n=3; mean +/− SEM; * p
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    Image Search Results


    Endogenous immunofluorescence of LC3 after PKCε downregulation. a Cells were transfected with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated with rapamycin (300 nM) for 24 h, as described in Autophagosome visualization section. Representative images from HeLaPKCεA/E, U-138 MG, and U-118 MG cells are shown. Scale bars equate to 10 μm. b AUTOCOUNTER analysis in imaging of U-138 MG and U-118 MG cells after siRNA silencing of PKCε (72 h) and Rapamycin (300 nM) treatment (24 h). The ratio between vesicle area and cell area (Aves/Acell), number of vesicles (#ves), and number of vesicles grouped, according to their area, into three defined classes (small: 0

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Endogenous immunofluorescence of LC3 after PKCε downregulation. a Cells were transfected with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated with rapamycin (300 nM) for 24 h, as described in Autophagosome visualization section. Representative images from HeLaPKCεA/E, U-138 MG, and U-118 MG cells are shown. Scale bars equate to 10 μm. b AUTOCOUNTER analysis in imaging of U-138 MG and U-118 MG cells after siRNA silencing of PKCε (72 h) and Rapamycin (300 nM) treatment (24 h). The ratio between vesicle area and cell area (Aves/Acell), number of vesicles (#ves), and number of vesicles grouped, according to their area, into three defined classes (small: 0

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Immunofluorescence, Transfection, Imaging

    Knockdown of PKCɛ diminishes the level and phosphorylation of Akt in glioma cells. a U-138 MG and b U-118 MG were transfected for 72 h with PKCε siRNA and Control siRNA and then were treated for 24 h with rapamycin (300 nM) or 3-MA (5 mM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Knockdown of PKCɛ diminishes the level and phosphorylation of Akt in glioma cells. a U-138 MG and b U-118 MG were transfected for 72 h with PKCε siRNA and Control siRNA and then were treated for 24 h with rapamycin (300 nM) or 3-MA (5 mM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Transfection

    Changes in autophagy gene expression in U-138 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-138 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Changes in autophagy gene expression in U-138 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-138 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Expressing, Transfection

    Influence of PKCε downregulation and rapamycin or 3-MA treatment on protein level directly involved in autophagy activation (mTOR, PI3K, Beclin1). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. In case of mTOR the order of bands has been changing, due to a different final construct of probe layout. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Influence of PKCε downregulation and rapamycin or 3-MA treatment on protein level directly involved in autophagy activation (mTOR, PI3K, Beclin1). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. In case of mTOR the order of bands has been changing, due to a different final construct of probe layout. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Activation Assay, Transfection, Construct

    Effect of PKCε downregulation and rapamycin or 3-MA treatment on proteins engaged in the formation of a double-membrane structure known as autophagosome (Atg5, LC3-II). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation and rapamycin or 3-MA treatment on proteins engaged in the formation of a double-membrane structure known as autophagosome (Atg5, LC3-II). a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Transfection

    Effect of PKCε downregulation and rapamycin or 3-MA treatment on Bcl-2, a protein that regulates autophagy process. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation and rapamycin or 3-MA treatment on Bcl-2, a protein that regulates autophagy process. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM) (PKCε siRNA + Rap) or 3-MA (5 mM) (PKCε siRNA + 3-MA). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Transfection

    Changes in autophagy gene expression in U-118 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-118 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Changes in autophagy gene expression in U-118 MG cells after PKCε downregulation and rapamycin or 3-MA treatment. The heat map shows the relative expression of human autophagy genes in U-118 MG cells transfected for 72 h with PKCε siRNA (PKCε siRNA) and in cells transfected and then treated for 24 h with rapamycin (PKCε siRNA + Rap) or 3-MA (PKCε siRNA + 3-MA). Each row represents an average gene expression normalized to the expression of the indicated gene in cells transfected with non-targeting siRNA (Control siRNA). Red color indicates genes that were upregulated, and blue color indicates genes that were downregulated. White indicates genes whose expression is unchanged in analyzed cells as compared to Control siRNA. Experiments were performed three times in duplicates

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Expressing, Transfection

    Effect of PKCε downregulation on the adhesion of glioblastoma cells. a . U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and Control siRNA and then were seeded in culture plates coated with Matrigel (Corning Life Sciences, NY, USA). The photos represent cell adhesion under the microscope at 100 x magnification field. c Evaluation of FAK expression in U-138 MG and U-118 MG cells with knockdown PKCε. Total protein expression and FAK phosphorylation at Tyr-397/Tyr-576/577 were assessed. GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Effect of PKCε downregulation on the adhesion of glioblastoma cells. a . U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and Control siRNA and then were seeded in culture plates coated with Matrigel (Corning Life Sciences, NY, USA). The photos represent cell adhesion under the microscope at 100 x magnification field. c Evaluation of FAK expression in U-138 MG and U-118 MG cells with knockdown PKCε. Total protein expression and FAK phosphorylation at Tyr-397/Tyr-576/577 were assessed. GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Transfection, Microscopy, Expressing

    Modulation SQSTM1/p62 after PRKCE silencing and rapamycin treatment. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: Modulation SQSTM1/p62 after PRKCE silencing and rapamycin treatment. a U-138 MG and b U-118 MG cells were transfected for 72 h with PKCε siRNA (PKCε siRNA) and non-targeting siRNA (Control siRNA) and then were treated for 24 h with rapamycin (300 nM). GAPDH was used as a loading control and as an internal standard. Representative blots are shown. The densitometric analysis represents means ±SD of three independent experiments. * P

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Transfection

    The effect of PRKCE silencing on U-138 MG and U-118 MG cells. Cells were harvested 72 h after transfection with non-targeting siRNA (Control siRNA) or PKCε siRNA. The downregulation of PRKCE mRNA expression ( a ) and PKCε protein level ( b ) are shown. The bar graphs present average values of three independent experiments. ** P

    Journal: BMC Cancer

    Article Title: Impact of PKCε downregulation on autophagy in glioblastoma cells

    doi: 10.1186/s12885-018-4095-1

    Figure Lengend Snippet: The effect of PRKCE silencing on U-138 MG and U-118 MG cells. Cells were harvested 72 h after transfection with non-targeting siRNA (Control siRNA) or PKCε siRNA. The downregulation of PRKCE mRNA expression ( a ) and PKCε protein level ( b ) are shown. The bar graphs present average values of three independent experiments. ** P

    Article Snippet: siRNA transfection Two glioblastoma cell lines (U-138 MG and U-118 MG) were transfected with small interfering RNA against PKCε (PKCε siRNA) and non-targeting siRNA (Control-siRNA) (Santa Cruz Biotechnology, CA, USA).

    Techniques: Transfection, Expressing

    Transcription factor activities involved in mediating NaCl-induced expression of the c-Fos gene in RPE cells. mRNA levels were determined with real-time reverse transcription (RT)–PCR analysis in cells cultured for 2 h in iso- (control) and hyperosmotic (+ 100 mM NaCl) media, and are expressed as folds of the unstimulated control. A : The level of c-Fos mRNA was determined in cells cultured in the presence of the following inhibitory compounds: the AP-1 inhibitor SR11302 (5 µM), the cAMP response element-binding protein (CREB) inhibitor 666–15 (250 nM), a HIF inhibitor (HIF-Inh; 5 µM), the STAT3 inhibitor Stattic (1 µM), and the nuclear factor (NF)-κB inhibitor CAPE (5 µM). B, C : mRNA levels were determined in cells transfected with nuclear factor of activated T cell 5 (NFAT5) siRNA (siNFAT5; 5 nM) and non-targeted siRNA (siNon; 5 nM), respectively. B : Transfection of RPE cells with NFAT5 siRNA for 48 h resulted in a reduction of the NFAT5 mRNA level in cells cultured for 2 h in iso- and hyperosmotic media. Non-targeted siRNA had no effects. As negative control, transfection reagent (TR) without siRNA was tested. C : Knocking down NFAT5 with siRNA reduced the level of c-Fos mRNA under hyperosmotic conditions. Each bar represents data obtained in three to nine independent experiments using cell lines from different donors. Significant difference versus unstimulated control: *p

    Journal: Molecular Vision

    Article Title: Activator protein-1 contributes to the NaCl-induced expression of VEGF and PlGF in RPE cells

    doi:

    Figure Lengend Snippet: Transcription factor activities involved in mediating NaCl-induced expression of the c-Fos gene in RPE cells. mRNA levels were determined with real-time reverse transcription (RT)–PCR analysis in cells cultured for 2 h in iso- (control) and hyperosmotic (+ 100 mM NaCl) media, and are expressed as folds of the unstimulated control. A : The level of c-Fos mRNA was determined in cells cultured in the presence of the following inhibitory compounds: the AP-1 inhibitor SR11302 (5 µM), the cAMP response element-binding protein (CREB) inhibitor 666–15 (250 nM), a HIF inhibitor (HIF-Inh; 5 µM), the STAT3 inhibitor Stattic (1 µM), and the nuclear factor (NF)-κB inhibitor CAPE (5 µM). B, C : mRNA levels were determined in cells transfected with nuclear factor of activated T cell 5 (NFAT5) siRNA (siNFAT5; 5 nM) and non-targeted siRNA (siNon; 5 nM), respectively. B : Transfection of RPE cells with NFAT5 siRNA for 48 h resulted in a reduction of the NFAT5 mRNA level in cells cultured for 2 h in iso- and hyperosmotic media. Non-targeted siRNA had no effects. As negative control, transfection reagent (TR) without siRNA was tested. C : Knocking down NFAT5 with siRNA reduced the level of c-Fos mRNA under hyperosmotic conditions. Each bar represents data obtained in three to nine independent experiments using cell lines from different donors. Significant difference versus unstimulated control: *p

    Article Snippet: Human-specific siRNA against NFAT5 and non-targeted control siRNA were obtained from Santa Cruz Biotechnology (Heidelberg, Germany).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Binding Assay, Transfection, Negative Control

    MiR-205 modulates LRRK2 protein expression in vitro . ( A , B ) Western blots show LRRK2 protein expression levels in pre-miR-205 oligo- or LRRK2 siRNA-transfected HEK293T cells (A) and primary cultured mouse cortical neurons (B). A scrambled microRNA oligo (Ctrl oligo), a scrambled siRNA (Ctrl siRNA) and a vehicle (Vcl) only were used as negative controls. β-Actin or β-III tubulin was used as a loading control. Bar graphs depict the signal intensities of LRRK2-positive bands in the vehicle, microRNA and siRNA-treated samples. ** P

    Journal: Human Molecular Genetics

    Article Title: MicroRNA-205 regulates the expression of Parkinson's disease-related leucine-rich repeat kinase 2 protein

    doi: 10.1093/hmg/dds470

    Figure Lengend Snippet: MiR-205 modulates LRRK2 protein expression in vitro . ( A , B ) Western blots show LRRK2 protein expression levels in pre-miR-205 oligo- or LRRK2 siRNA-transfected HEK293T cells (A) and primary cultured mouse cortical neurons (B). A scrambled microRNA oligo (Ctrl oligo), a scrambled siRNA (Ctrl siRNA) and a vehicle (Vcl) only were used as negative controls. β-Actin or β-III tubulin was used as a loading control. Bar graphs depict the signal intensities of LRRK2-positive bands in the vehicle, microRNA and siRNA-treated samples. ** P

    Article Snippet: Small interfering RNA (siRNA) against human LRRK2 and non-targeting control siRNA were purchased from Santa Cruz Biotechnology.

    Techniques: Expressing, In Vitro, Western Blot, Transfection, Cell Culture

    Depletion of Epac1 attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Epac1 specific siRNA or non-targeting siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Epac1 significantly attenuated adenosine-induced endothelial barrier enhancement. n=3; mean +/− SEM; * p

    Journal: Journal of cellular physiology

    Article Title: Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: molecular mechanisms and barrier-protective role

    doi: 10.1002/jcp.26281

    Figure Lengend Snippet: Depletion of Epac1 attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Epac1 specific siRNA or non-targeting siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Epac1 significantly attenuated adenosine-induced endothelial barrier enhancement. n=3; mean +/− SEM; * p

    Article Snippet: Briefly, HPAEC were transfected either with control, non-targeting siRNA or with siRNAs specific for protein of interest (listed above).

    Techniques: Activation Assay

    Depletion of Vav2 attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Vav2, specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in the TER measurement assay. Depletion of Vav2 significantly attenuated adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM;*** p

    Journal: Journal of cellular physiology

    Article Title: Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: molecular mechanisms and barrier-protective role

    doi: 10.1002/jcp.26281

    Figure Lengend Snippet: Depletion of Vav2 attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Vav2, specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in the TER measurement assay. Depletion of Vav2 significantly attenuated adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM;*** p

    Article Snippet: Briefly, HPAEC were transfected either with control, non-targeting siRNA or with siRNAs specific for protein of interest (listed above).

    Techniques: Activation Assay

    Depletion of Rap1b modestly affects adenosine-induced increase in TER while substantially decreases Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Rap1b specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Rap1b modestly affected maximal adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; B. Effect of adenosine in Rac1 activation in Rap1b depleted cells. HPAEC were transfected with Rap1b specific siRNA or non-specific siRNA and then 72 hours later challenged with 50 μM adenosine at different time points in Rac1 activity assay. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured according to the manufacturer’s protocol. n=4; mean +/− SEM; **p

    Journal: Journal of cellular physiology

    Article Title: Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: molecular mechanisms and barrier-protective role

    doi: 10.1002/jcp.26281

    Figure Lengend Snippet: Depletion of Rap1b modestly affects adenosine-induced increase in TER while substantially decreases Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Rap1b specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Rap1b modestly affected maximal adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; B. Effect of adenosine in Rac1 activation in Rap1b depleted cells. HPAEC were transfected with Rap1b specific siRNA or non-specific siRNA and then 72 hours later challenged with 50 μM adenosine at different time points in Rac1 activity assay. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured according to the manufacturer’s protocol. n=4; mean +/− SEM; **p

    Article Snippet: Briefly, HPAEC were transfected either with control, non-targeting siRNA or with siRNAs specific for protein of interest (listed above).

    Techniques: Activation Assay, Transfection, Activity Assay

    Tiam1 depletion has no effect on adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Tiam1 specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Tiam1 did not affect adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; B. Effect of adenosine on Rac1 activation in Tiam1 depleted cells. HPAEC were transfected with Tiam1 specific siRNA or non-specific siRNA and then 72 hours later challenged with 50 μM adenosine at different time points in Rac1 activity assay. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured according to the manufacturer’s protocol. n=4; mean +/− SEM; **P

    Journal: Journal of cellular physiology

    Article Title: Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: molecular mechanisms and barrier-protective role

    doi: 10.1002/jcp.26281

    Figure Lengend Snippet: Tiam1 depletion has no effect on adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Tiam1 specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Tiam1 did not affect adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; B. Effect of adenosine on Rac1 activation in Tiam1 depleted cells. HPAEC were transfected with Tiam1 specific siRNA or non-specific siRNA and then 72 hours later challenged with 50 μM adenosine at different time points in Rac1 activity assay. Cells were serum starved for 2 hours before adenosine treatment and Rac1 activity was measured according to the manufacturer’s protocol. n=4; mean +/− SEM; **P

    Article Snippet: Briefly, HPAEC were transfected either with control, non-targeting siRNA or with siRNAs specific for protein of interest (listed above).

    Techniques: Activation Assay, Transfection, Activity Assay

    Depletion of Rap1a attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Rap1a specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Rap1a significantly attenuated adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; **p

    Journal: Journal of cellular physiology

    Article Title: Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: molecular mechanisms and barrier-protective role

    doi: 10.1002/jcp.26281

    Figure Lengend Snippet: Depletion of Rap1a attenuates adenosine-induced increase in TER and Rac1 activation in pulmonary endothelial cells. A. HPAEC were treated with Rap1a specific siRNA or non-specific siRNA and then challenged with 50 μM adenosine in TER measurement assay. Depletion of Rap1a significantly attenuated adenosine-induced endothelial barrier enhancement. Arrow indicates the time point when adenosine was added. n=4; mean +/− SEM; **p

    Article Snippet: Briefly, HPAEC were transfected either with control, non-targeting siRNA or with siRNAs specific for protein of interest (listed above).

    Techniques: Activation Assay