individual sirna duplexes Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    Thermo Fisher 50 nm silencer select control small interfering rna
    50 Nm Silencer Select Control Small Interfering Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/50 nm silencer select control small interfering rna/product/Thermo Fisher
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    50 nm silencer select control small interfering rna - by Bioz Stars, 2020-08
    95/100 stars
      Buy from Supplier

    99
    Millipore individual sirna duplexes
    Identification and characterization of the <t>WWP1</t> E3 ligase for PTEN K27-linked polyubiquitination. ( A ) Lysates from DU145 cells transfected with hemagglutinin (HA)–PTEN were immunoprecipitated (IP) with an anti-PTEN antibody followed by mass spectrometric peptide sequencing. WWP1 and PTEN were identified. IgG, immunoglobulin G. ( B ) WWP1 interacts with PTEN endogenously. DU145 cells were immunoprecipitated with either anti-WWP1 or anti-PTEN antibody and then analyzed by Western blot (WB). Input is 5% of the total lysates used in IP. ( C ) Analysis of endogenous PTEN ubiquitination (Ub) in Wwp1 −/− MEFs with stable reconstitution of either WWP1 WT or its catalytically inactive mutant (C890A, abbreviated CA). ( D and E ) Effects of the indicated ubiquitin KR (Lys to Arg) (D) or K-only (E) ubiquitin mutants on WWP1-mediated PTEN polyubiquitination. HEK293T cells were transfected with the indicated constructs, and PTEN ubiquitination was analyzed. The ubiquitinated proteins were pulled down under denaturing conditions by nickel–nitrilotriacetic acid (Ni-NTA) agarose and analyzed by Western blot. ( F ) Analysis of PTEN K27-linked polyubiquitination in PC3 cells expressing the indicated NEDD4 family ubiquitin ligases as in (D). EV, cells transfected with empty vector plasmids. ( G ) In vitro ubiquitination of PTEN by WWP1 E3 ligase. Flag-PTEN purified from HEK293 cells was subject to in vitro ubiquitination reaction in the presence of E1, E2, E3, and ubiquitin or various ubiquitin mutants and then examined by Western blot with anti-PTEN antibody. The input control of WWP1 was determined by Western blotting and is shown at the bottom. ( H ) Analysis of endogenous PTEN ubiquitination by small interfering RNA <t>(siRNA)–mediated</t> WWP1 knock down in U2OS ubiquitin replacement cells treated with 1 μg/ml doxycycline for 48 hours. shUb, shRNAs targeting ubiquitin. ( I ) Effects of the indicated ubiquitin KR mutants on WWP1-mediated PTEN polyubiquitination. PC3 cells were transfected with indicated constructs, and PTEN ubiquitination was analyzed as in (D). In (D), (F), (H), and (I), actin is used as a loading control.
    Individual Sirna Duplexes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/individual sirna duplexes/product/Millipore
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    individual sirna duplexes - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    88
    Horizon Discovery individual sirna duplexes
    Confirmation that SF3A1 and GOLGA4 regulate LPS-induced IL-6 production using a second mouse macrophage cell line. A and B, indicated pools of <t>siRNA</t> duplexes were <t>transfected</t> into the RAW264.7 mouse macrophage cell line; cells were simulated with LPS
    Individual Sirna Duplexes, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 88/100, based on 999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/individual sirna duplexes/product/Horizon Discovery
    Average 88 stars, based on 999 article reviews
    Price from $9.99 to $1999.99
    individual sirna duplexes - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    88
    Thermo Fisher individual sirna duplexes
    The midbody positions the apical surface during cyst development. (A) Caco-2 transfected with control or <t>Cdc42</t> <t>siRNA</t> was fixed and stained for DNA (blue), tubulin (green), and aPKC (red). (top) A control cyst at the two-cell stage (note that abscission appears to have occurred symmetrically). (middle) A larger control cyst, with the midbody in the center of the developing structure (apical region of dividing cell) reflecting asymmetric abscission. (bottom) Cdc42 siRNA structure with one midbody positioned normally at the center and another midbody (located in a different z section) abnormally positioned. (B) Quantitation of midbodies at the center of the cyst from three independent experiments. The total number of midbodies is indicated (N). A midbody is regarded as being in the center if it is located at a distance from the centroid that is less than one third the radius of the structure.
    Individual Sirna Duplexes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/individual sirna duplexes/product/Thermo Fisher
    Average 88 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    individual sirna duplexes - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    85
    Horizon Discovery sigenome individual duplex sirnas
    The midbody positions the apical surface during cyst development. (A) Caco-2 transfected with control or <t>Cdc42</t> <t>siRNA</t> was fixed and stained for DNA (blue), tubulin (green), and aPKC (red). (top) A control cyst at the two-cell stage (note that abscission appears to have occurred symmetrically). (middle) A larger control cyst, with the midbody in the center of the developing structure (apical region of dividing cell) reflecting asymmetric abscission. (bottom) Cdc42 siRNA structure with one midbody positioned normally at the center and another midbody (located in a different z section) abnormally positioned. (B) Quantitation of midbodies at the center of the cyst from three independent experiments. The total number of midbodies is indicated (N). A midbody is regarded as being in the center if it is located at a distance from the centroid that is less than one third the radius of the structure.
    Sigenome Individual Duplex Sirnas, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sigenome individual duplex sirnas/product/Horizon Discovery
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    sigenome individual duplex sirnas - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    85
    Horizon Discovery individual sirnas duplex dharmacon
    The midbody positions the apical surface during cyst development. (A) Caco-2 transfected with control or <t>Cdc42</t> <t>siRNA</t> was fixed and stained for DNA (blue), tubulin (green), and aPKC (red). (top) A control cyst at the two-cell stage (note that abscission appears to have occurred symmetrically). (middle) A larger control cyst, with the midbody in the center of the developing structure (apical region of dividing cell) reflecting asymmetric abscission. (bottom) Cdc42 siRNA structure with one midbody positioned normally at the center and another midbody (located in a different z section) abnormally positioned. (B) Quantitation of midbodies at the center of the cyst from three independent experiments. The total number of midbodies is indicated (N). A midbody is regarded as being in the center if it is located at a distance from the centroid that is less than one third the radius of the structure.
    Individual Sirnas Duplex Dharmacon, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/individual sirnas duplex dharmacon/product/Horizon Discovery
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    individual sirnas duplex dharmacon - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    85
    Horizon Discovery rna interference individual sirna duplexes
    Increased NF-κB luciferase activity in neuronal cells with knockdown of RNF11. (A) Stable SH-SY5Y cell lines were generated using lentiviruses containing a control scramble small hairpin <t>RNA</t> <t>(shRNA)</t> sequence (shRNA-Scramble cells) or shRNA targeted against RNF11 (shRNA-RNF11 cells). Quantitative RT-PCR was used to measure relative RNF11 mRNA levels with glucuronidase β as an internal control. Values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. (B) Stable SH-SY5Y cell lines, as well as untransduced cells, were transiently cotransfected with luciferase and Renilla plasmids and stimulated with 10 ng/ml TNF-α or PBS for 6 hours. Values represent the fold change in NF-κB-dependent activity as measured by the ratio of luciferase to Renilla luminescence in stimulated over unstimulated samples ± SEM in triplicate experiments. * P
    Rna Interference Individual Sirna Duplexes, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna interference individual sirna duplexes/product/Horizon Discovery
    Average 85 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    rna interference individual sirna duplexes - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    85
    Thermo Fisher sirnas individual duplex sirnas
    Increased NF-κB luciferase activity in neuronal cells with knockdown of RNF11. (A) Stable SH-SY5Y cell lines were generated using lentiviruses containing a control scramble small hairpin <t>RNA</t> <t>(shRNA)</t> sequence (shRNA-Scramble cells) or shRNA targeted against RNF11 (shRNA-RNF11 cells). Quantitative RT-PCR was used to measure relative RNF11 mRNA levels with glucuronidase β as an internal control. Values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. (B) Stable SH-SY5Y cell lines, as well as untransduced cells, were transiently cotransfected with luciferase and Renilla plasmids and stimulated with 10 ng/ml TNF-α or PBS for 6 hours. Values represent the fold change in NF-κB-dependent activity as measured by the ratio of luciferase to Renilla luminescence in stimulated over unstimulated samples ± SEM in triplicate experiments. * P
    Sirnas Individual Duplex Sirnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirnas individual duplex sirnas/product/Thermo Fisher
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    sirnas individual duplex sirnas - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    85
    Horizon Discovery individual gene specific sirna duplexes
    Increased NF-κB luciferase activity in neuronal cells with knockdown of RNF11. (A) Stable SH-SY5Y cell lines were generated using lentiviruses containing a control scramble small hairpin <t>RNA</t> <t>(shRNA)</t> sequence (shRNA-Scramble cells) or shRNA targeted against RNF11 (shRNA-RNF11 cells). Quantitative RT-PCR was used to measure relative RNF11 mRNA levels with glucuronidase β as an internal control. Values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. (B) Stable SH-SY5Y cell lines, as well as untransduced cells, were transiently cotransfected with luciferase and Renilla plasmids and stimulated with 10 ng/ml TNF-α or PBS for 6 hours. Values represent the fold change in NF-κB-dependent activity as measured by the ratio of luciferase to Renilla luminescence in stimulated over unstimulated samples ± SEM in triplicate experiments. * P
    Individual Gene Specific Sirna Duplexes, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/individual gene specific sirna duplexes/product/Horizon Discovery
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    individual gene specific sirna duplexes - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    85
    Horizon Discovery pak3 3 individual sigenome duplex
    Increased NF-κB luciferase activity in neuronal cells with knockdown of RNF11. (A) Stable SH-SY5Y cell lines were generated using lentiviruses containing a control scramble small hairpin <t>RNA</t> <t>(shRNA)</t> sequence (shRNA-Scramble cells) or shRNA targeted against RNF11 (shRNA-RNF11 cells). Quantitative RT-PCR was used to measure relative RNF11 mRNA levels with glucuronidase β as an internal control. Values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. (B) Stable SH-SY5Y cell lines, as well as untransduced cells, were transiently cotransfected with luciferase and Renilla plasmids and stimulated with 10 ng/ml TNF-α or PBS for 6 hours. Values represent the fold change in NF-κB-dependent activity as measured by the ratio of luciferase to Renilla luminescence in stimulated over unstimulated samples ± SEM in triplicate experiments. * P
    Pak3 3 Individual Sigenome Duplex, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pak3 3 individual sigenome duplex/product/Horizon Discovery
    Average 85 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    pak3 3 individual sigenome duplex - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    85
    Horizon Discovery rnf11 sirna duplex
    <t>RNF11</t> is essential for the termination of NF-κB signalling. ( A ) THP-1 cells were transfected on consecutive days with control scrambled or RNF11 siRNAs. At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK and RNF11. ( B ) THP-1 cells were transfected on consecutive days with control scrambled, RNF11 or A20 siRNAs. At 2 days after the second transfection, cells were treated with LPS (1 μg/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK, A20, RNF11 and β-actin. ( C ) THP-1 cells were transfected with control scrambled or RNF11 <t>siRNA</t> as described in (A). At 2 days after the second transfection, cells were stimulated with TNF (10 ng/ml) for various times and RNA was subjected to real-time PCR for IκBα and A20 expression. This experiment was repeated twice with similar results. ( D ) THP-1 cells were transfected with siRNAs as described in (A). Supernatants were subjected to an IL-6 ELISA. Error bars indicate s.e.m. of triplicate samples. Statistical analysis was performed by one-way ANOVA, followed by the Tukey–Kramer test for multiple comparisons. * P
    Rnf11 Sirna Duplex, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnf11 sirna duplex/product/Horizon Discovery
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rnf11 sirna duplex - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    92
    Corning Life Sciences sirna duplexes
    Drug specificity and final phenotypes. A: Drug specificity tests. <t>HeLa</t> H2B-GFP cells were transfected with <t>siRNA</t> pools as indicated, or “Mock” transfected in the absence of siRNA. 24 hours after transfection, medium containing either 150 nM of taxol or 300 nM of nocadazole was added to all of the samples. Cells were then incubated for an additional 24 hours and imaged for phenotypic changes. The pools were scored visually for cells in mitotic arrest, and this value was quantified for the pool by counting > 100 cells. (B) Phenotypic categorization of hits on the basis of data in Figure 7 and 8A . Category I) failure to enter mitosis, II) brief mitotic arrest without cytokinesis, and III) brief mitotic arrest with cytokinesis (true suppressors). Shown in bold are 5 expected genes, 4 known SAC genes and KifC1, which is a known true suppressor of loss of Kinesin-5 function [28] .
    Sirna Duplexes, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna duplexes/product/Corning Life Sciences
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    sirna duplexes - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    88
    Horizon Discovery sigenome sirna oligonucleotide duplexes
    Drug specificity and final phenotypes. A: Drug specificity tests. <t>HeLa</t> H2B-GFP cells were transfected with <t>siRNA</t> pools as indicated, or “Mock” transfected in the absence of siRNA. 24 hours after transfection, medium containing either 150 nM of taxol or 300 nM of nocadazole was added to all of the samples. Cells were then incubated for an additional 24 hours and imaged for phenotypic changes. The pools were scored visually for cells in mitotic arrest, and this value was quantified for the pool by counting > 100 cells. (B) Phenotypic categorization of hits on the basis of data in Figure 7 and 8A . Category I) failure to enter mitosis, II) brief mitotic arrest without cytokinesis, and III) brief mitotic arrest with cytokinesis (true suppressors). Shown in bold are 5 expected genes, 4 known SAC genes and KifC1, which is a known true suppressor of loss of Kinesin-5 function [28] .
    Sigenome Sirna Oligonucleotide Duplexes, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sigenome sirna oligonucleotide duplexes/product/Horizon Discovery
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    sigenome sirna oligonucleotide duplexes - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    93
    Horizon Discovery sirna duplex
    Effects of <t>siRNA</t> Treatment on Cellular <t>UGT1A6</t> Activities and Expression. In , p-nitrophenol (50 µM) glucuronidation rates were measured in Caco-2 cells treated with Lipofectamine alone (control), negative control (nonsense) siRNA pool (Non-control)
    Sirna Duplex, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 93/100, based on 437 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna duplex/product/Horizon Discovery
    Average 93 stars, based on 437 article reviews
    Price from $9.99 to $1999.99
    sirna duplex - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    92
    Horizon Discovery sirna sigenome smartpools
    Effects of <t>siRNA</t> Treatment on Cellular <t>UGT1A6</t> Activities and Expression. In , p-nitrophenol (50 µM) glucuronidation rates were measured in Caco-2 cells treated with Lipofectamine alone (control), negative control (nonsense) siRNA pool (Non-control)
    Sirna Sigenome Smartpools, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna sigenome smartpools/product/Horizon Discovery
    Average 92 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    sirna sigenome smartpools - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    85
    Horizon Discovery smartpool deconvoluted sirna
    Kinome <t>siRNA</t> screen to determine genes involved in AT 1 R–EGFR transactivation. (A) Using the HMEC-LST-AT 1 R cell line, we performed a primary siRNA screen using the Dharmacon siGENOME <t>SMARTpool</t> siRNA library targeting kinase genes (720 in total,
    Smartpool Deconvoluted Sirna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/smartpool deconvoluted sirna/product/Horizon Discovery
    Average 85 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    smartpool deconvoluted sirna - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    88
    Horizon Discovery rgs12
    <t>RGS12</t> coordinates a Ras/Raf/MEK/ERK complex and enhances signaling to ERK. ( A ) CHO-K1 cells with endogenous PDGFβR were transfected with ERK2–GFP and either empty vector or increasing amounts of HA-RGS12 vector, serum-starved overnight,
    Rgs12, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rgs12/product/Horizon Discovery
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rgs12 - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    85
    Horizon Discovery app sirna duplex gcctaaagctgataagaag
    <t>RGS12</t> coordinates a Ras/Raf/MEK/ERK complex and enhances signaling to ERK. ( A ) CHO-K1 cells with endogenous PDGFβR were transfected with ERK2–GFP and either empty vector or increasing amounts of HA-RGS12 vector, serum-starved overnight,
    App Sirna Duplex Gcctaaagctgataagaag, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/app sirna duplex gcctaaagctgataagaag/product/Horizon Discovery
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    app sirna duplex gcctaaagctgataagaag - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    86
    Santa Cruz Biotechnology sgk3 sirna duplexes
    Androgen/AR-dependent <t>SGK3</t> transcription involves ER. A, LNCaP cells were transfected with the <t>siRNA</t> negative control (siNC) or ERβ siRNA (siERβ) for 72 hours. Cell extracts were subjected to Western blotting. B, After being hormone-stripped for 2 days, LNCaP cells were transfected with the siRNA negative control or ERβ siRNA for 48 hours and then were transfected with the luciferase reporters as indicated and cultured in the presence or absence of 1 nM DHT for 24 hours. Luciferase activity of cell extracts was measured and normalized to protein concentration. Data are expressed as means ± SD from 3 independent experiments. **, P
    Sgk3 Sirna Duplexes, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sgk3 sirna duplexes/product/Santa Cruz Biotechnology
    Average 86 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    sgk3 sirna duplexes - by Bioz Stars, 2020-08
    86/100 stars
      Buy from Supplier

    86
    Thermo Fisher human gnrh r sirna
    Validation of the detection of <t>GnRH-R</t> in immunoblots. A. Representative immunoblots of GnRH-R detection (upper panel) after 48 h transfection with the Human cDNA clone pCMV6-XL5/GNRH-R in 16HBE14o − (1) and CFBE41o − (2) cells. pCMV6-XL5 empty plasmid was used as a control. B. The densitometric analysis after normalization by G3PDH expression and comparison with the controls, indicate that the GnRH-R expression in significantly increased, (n = 5). C. Representative immunoblots of GnRH-R detection after 72 h transfection with a siGENOME individual duplex targeting GnRH-R in 16HBE14o − (1) and CFBE41o − (2) cells. siGENOME Non-Targeting was used as control. A decreased expression of GnRH-R is observed in both cell types. D. The densitometric analysis after normalization by G3PDH expression and comparison with the controls, indicate that the GnRH-R expression is significantly decreased in 16HBE14o − (1) and CFBE41o − (2) cells (n = 7) in the presence of <t>siRNA.</t>
    Human Gnrh R Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human gnrh r sirna/product/Thermo Fisher
    Average 86 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    human gnrh r sirna - by Bioz Stars, 2020-08
    86/100 stars
      Buy from Supplier

    Image Search Results


    Identification and characterization of the WWP1 E3 ligase for PTEN K27-linked polyubiquitination. ( A ) Lysates from DU145 cells transfected with hemagglutinin (HA)–PTEN were immunoprecipitated (IP) with an anti-PTEN antibody followed by mass spectrometric peptide sequencing. WWP1 and PTEN were identified. IgG, immunoglobulin G. ( B ) WWP1 interacts with PTEN endogenously. DU145 cells were immunoprecipitated with either anti-WWP1 or anti-PTEN antibody and then analyzed by Western blot (WB). Input is 5% of the total lysates used in IP. ( C ) Analysis of endogenous PTEN ubiquitination (Ub) in Wwp1 −/− MEFs with stable reconstitution of either WWP1 WT or its catalytically inactive mutant (C890A, abbreviated CA). ( D and E ) Effects of the indicated ubiquitin KR (Lys to Arg) (D) or K-only (E) ubiquitin mutants on WWP1-mediated PTEN polyubiquitination. HEK293T cells were transfected with the indicated constructs, and PTEN ubiquitination was analyzed. The ubiquitinated proteins were pulled down under denaturing conditions by nickel–nitrilotriacetic acid (Ni-NTA) agarose and analyzed by Western blot. ( F ) Analysis of PTEN K27-linked polyubiquitination in PC3 cells expressing the indicated NEDD4 family ubiquitin ligases as in (D). EV, cells transfected with empty vector plasmids. ( G ) In vitro ubiquitination of PTEN by WWP1 E3 ligase. Flag-PTEN purified from HEK293 cells was subject to in vitro ubiquitination reaction in the presence of E1, E2, E3, and ubiquitin or various ubiquitin mutants and then examined by Western blot with anti-PTEN antibody. The input control of WWP1 was determined by Western blotting and is shown at the bottom. ( H ) Analysis of endogenous PTEN ubiquitination by small interfering RNA (siRNA)–mediated WWP1 knock down in U2OS ubiquitin replacement cells treated with 1 μg/ml doxycycline for 48 hours. shUb, shRNAs targeting ubiquitin. ( I ) Effects of the indicated ubiquitin KR mutants on WWP1-mediated PTEN polyubiquitination. PC3 cells were transfected with indicated constructs, and PTEN ubiquitination was analyzed as in (D). In (D), (F), (H), and (I), actin is used as a loading control.

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

    Article Title: Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway

    doi: 10.1126/science.aau0159

    Figure Lengend Snippet: Identification and characterization of the WWP1 E3 ligase for PTEN K27-linked polyubiquitination. ( A ) Lysates from DU145 cells transfected with hemagglutinin (HA)–PTEN were immunoprecipitated (IP) with an anti-PTEN antibody followed by mass spectrometric peptide sequencing. WWP1 and PTEN were identified. IgG, immunoglobulin G. ( B ) WWP1 interacts with PTEN endogenously. DU145 cells were immunoprecipitated with either anti-WWP1 or anti-PTEN antibody and then analyzed by Western blot (WB). Input is 5% of the total lysates used in IP. ( C ) Analysis of endogenous PTEN ubiquitination (Ub) in Wwp1 −/− MEFs with stable reconstitution of either WWP1 WT or its catalytically inactive mutant (C890A, abbreviated CA). ( D and E ) Effects of the indicated ubiquitin KR (Lys to Arg) (D) or K-only (E) ubiquitin mutants on WWP1-mediated PTEN polyubiquitination. HEK293T cells were transfected with the indicated constructs, and PTEN ubiquitination was analyzed. The ubiquitinated proteins were pulled down under denaturing conditions by nickel–nitrilotriacetic acid (Ni-NTA) agarose and analyzed by Western blot. ( F ) Analysis of PTEN K27-linked polyubiquitination in PC3 cells expressing the indicated NEDD4 family ubiquitin ligases as in (D). EV, cells transfected with empty vector plasmids. ( G ) In vitro ubiquitination of PTEN by WWP1 E3 ligase. Flag-PTEN purified from HEK293 cells was subject to in vitro ubiquitination reaction in the presence of E1, E2, E3, and ubiquitin or various ubiquitin mutants and then examined by Western blot with anti-PTEN antibody. The input control of WWP1 was determined by Western blotting and is shown at the bottom. ( H ) Analysis of endogenous PTEN ubiquitination by small interfering RNA (siRNA)–mediated WWP1 knock down in U2OS ubiquitin replacement cells treated with 1 μg/ml doxycycline for 48 hours. shUb, shRNAs targeting ubiquitin. ( I ) Effects of the indicated ubiquitin KR mutants on WWP1-mediated PTEN polyubiquitination. PC3 cells were transfected with indicated constructs, and PTEN ubiquitination was analyzed as in (D). In (D), (F), (H), and (I), actin is used as a loading control.

    Article Snippet: The two individual siRNA duplexes targeted to WWP1 and control nontarget siRNA were purchased from Sigma Aldrich, whereas siRNA SMARTpool targeted to MYC was purchased from Dharmacon.

    Techniques: Transfection, Immunoprecipitation, Sequencing, Western Blot, Mutagenesis, Construct, Expressing, Plasmid Preparation, In Vitro, Purification, Small Interfering RNA

    mRNA expression of NMIIA, NMIIB, and NMIIC in two different samples of WJ-MSCs, as analyzed by semiquantitative RT-PCR using isoform-specific primers. 18s rRNA was used as an internal control (A) . Protein expression of NMIIC was analyzed in WJ-MSCs. The MCF-7 cell line was used as positive control. The same blot was reprobed with the anti-GAPDH antibody to demonstrate equal loading (B) . Localization of NMIIA and NMIIB in cultured WJ-MSCs. Representative confocal images of cultured WJ-MSCs immunostained for NMII A and NMIIB ( C , F , respectively ) , phalloidin-stained F-actin ( D , G) , merged (E , H) and mesenchymal marker vimentin ( I) are presented. Nuclei are stained with DAPI. A negative control with the omission of incubation with primary antibody is shown (J) . Scale bar: 50 μm. Results are representative of at least two independent biological or donor samples. MSCs, mesenchymal stem cells; NMII, nonmuscle myosin II; RT-PCR, reverse transcription-polymerase chain reaction; WJ, Wharton's jelly; WJ-MSCs, WJ-derived MSCs.

    Journal: Stem Cells and Development

    Article Title: Role of Nonmuscle Myosin II in Migration of Wharton's Jelly-Derived Mesenchymal Stem Cells

    doi: 10.1089/scd.2015.0095

    Figure Lengend Snippet: mRNA expression of NMIIA, NMIIB, and NMIIC in two different samples of WJ-MSCs, as analyzed by semiquantitative RT-PCR using isoform-specific primers. 18s rRNA was used as an internal control (A) . Protein expression of NMIIC was analyzed in WJ-MSCs. The MCF-7 cell line was used as positive control. The same blot was reprobed with the anti-GAPDH antibody to demonstrate equal loading (B) . Localization of NMIIA and NMIIB in cultured WJ-MSCs. Representative confocal images of cultured WJ-MSCs immunostained for NMII A and NMIIB ( C , F , respectively ) , phalloidin-stained F-actin ( D , G) , merged (E , H) and mesenchymal marker vimentin ( I) are presented. Nuclei are stained with DAPI. A negative control with the omission of incubation with primary antibody is shown (J) . Scale bar: 50 μm. Results are representative of at least two independent biological or donor samples. MSCs, mesenchymal stem cells; NMII, nonmuscle myosin II; RT-PCR, reverse transcription-polymerase chain reaction; WJ, Wharton's jelly; WJ-MSCs, WJ-derived MSCs.

    Article Snippet: Isoform-specific individual siRNA duplexes against NMIIA and NMIIB (Sigma) were used at a final concentration of 20 nM, and the MISSION siRNA Universal Negative Control (Sigma) was used at the same concentration in the control experiments.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Cell Culture, Staining, Marker, Negative Control, Incubation, Derivative Assay

    SiRNA-mediated downregulation of NMII isoforms. Western blots of WJ-MSC lysates, prepared 72 h post-transfection, showed selective downregulation of NMIIA and NMIIB protein expression by siRNA specific for NMIIA or NMIIB isoforms, respectively. The same blot was reprobed with anti-GAPDH antibody to demonstrate equal loading (A) . Morphology of WJ-MSCs 72 h after transfection with negative control siRNA, NMIIA siRNA, NMIIB SiRNA, or cotransfection with NMIIA plus NMIIB siRNA. SiRNA-mediated downregulation of NMIIA, but not NMIIB, resulted in extended tails as shown by white arrows . Boxed regions are zoomed. Scale bar: 200 μm (B) . Scratch migration assay of siRNA-transfected WJ-MSCs. WJ-MSCs were seeded in 12-well plates and transfected with negative control siRNA or siRNAs against NMIIA, NMIIB, and both NMIIA and NMIIB using Lipofectamine 3000. Seventy-two hours after siRNA transfection, confluent monolayer cultures were wounded with a sterile pipette tip. Images of the wound healing were captured at 0 h, 5, and 12 h (C) . Scale bar: 200 μm. Results are representative of at least three independent biological or donor samples.

    Journal: Stem Cells and Development

    Article Title: Role of Nonmuscle Myosin II in Migration of Wharton's Jelly-Derived Mesenchymal Stem Cells

    doi: 10.1089/scd.2015.0095

    Figure Lengend Snippet: SiRNA-mediated downregulation of NMII isoforms. Western blots of WJ-MSC lysates, prepared 72 h post-transfection, showed selective downregulation of NMIIA and NMIIB protein expression by siRNA specific for NMIIA or NMIIB isoforms, respectively. The same blot was reprobed with anti-GAPDH antibody to demonstrate equal loading (A) . Morphology of WJ-MSCs 72 h after transfection with negative control siRNA, NMIIA siRNA, NMIIB SiRNA, or cotransfection with NMIIA plus NMIIB siRNA. SiRNA-mediated downregulation of NMIIA, but not NMIIB, resulted in extended tails as shown by white arrows . Boxed regions are zoomed. Scale bar: 200 μm (B) . Scratch migration assay of siRNA-transfected WJ-MSCs. WJ-MSCs were seeded in 12-well plates and transfected with negative control siRNA or siRNAs against NMIIA, NMIIB, and both NMIIA and NMIIB using Lipofectamine 3000. Seventy-two hours after siRNA transfection, confluent monolayer cultures were wounded with a sterile pipette tip. Images of the wound healing were captured at 0 h, 5, and 12 h (C) . Scale bar: 200 μm. Results are representative of at least three independent biological or donor samples.

    Article Snippet: Isoform-specific individual siRNA duplexes against NMIIA and NMIIB (Sigma) were used at a final concentration of 20 nM, and the MISSION siRNA Universal Negative Control (Sigma) was used at the same concentration in the control experiments.

    Techniques: Western Blot, Transfection, Expressing, Negative Control, Cotransfection, Migration, Transferring

    Confirmation that SF3A1 and GOLGA4 regulate LPS-induced IL-6 production using a second mouse macrophage cell line. A and B, indicated pools of siRNA duplexes were transfected into the RAW264.7 mouse macrophage cell line; cells were simulated with LPS

    Journal: The Journal of Biological Chemistry

    Article Title: An Evolutionarily Conserved Innate Immunity Protein Interaction Network *

    doi: 10.1074/jbc.M112.407205

    Figure Lengend Snippet: Confirmation that SF3A1 and GOLGA4 regulate LPS-induced IL-6 production using a second mouse macrophage cell line. A and B, indicated pools of siRNA duplexes were transfected into the RAW264.7 mouse macrophage cell line; cells were simulated with LPS

    Article Snippet: In brief, pools of four siRNA duplexes or individual siRNA duplexes (Dharmacon) were transfected into either of two mouse macrophage cell lines (J774A.1 or RAW264.7) using the Amaxa nucleofector 96-well shuttle according to the manufacturer's instructions.

    Techniques: Transfection

    Most genes in the innate immunity protein interaction network affect LPS-induced IL-6 production in the J774A.1 mouse macrophage cell line. A, pools of four siRNA duplexes per gene were transfected into the mouse macrophage cell line J774A.1; cells were

    Journal: The Journal of Biological Chemistry

    Article Title: An Evolutionarily Conserved Innate Immunity Protein Interaction Network *

    doi: 10.1074/jbc.M112.407205

    Figure Lengend Snippet: Most genes in the innate immunity protein interaction network affect LPS-induced IL-6 production in the J774A.1 mouse macrophage cell line. A, pools of four siRNA duplexes per gene were transfected into the mouse macrophage cell line J774A.1; cells were

    Article Snippet: In brief, pools of four siRNA duplexes or individual siRNA duplexes (Dharmacon) were transfected into either of two mouse macrophage cell lines (J774A.1 or RAW264.7) using the Amaxa nucleofector 96-well shuttle according to the manufacturer's instructions.

    Techniques: Transfection

    MACF1 inhibits PAMP-induced cytokine production in RAW264.7 cells and in vivo . A, pool of four siRNA duplexes targeting either Macf1 or a nontargeting control siRNA duplex pool were transfected into the mouse macrophage cell line RAW264.7; cells were

    Journal: The Journal of Biological Chemistry

    Article Title: An Evolutionarily Conserved Innate Immunity Protein Interaction Network *

    doi: 10.1074/jbc.M112.407205

    Figure Lengend Snippet: MACF1 inhibits PAMP-induced cytokine production in RAW264.7 cells and in vivo . A, pool of four siRNA duplexes targeting either Macf1 or a nontargeting control siRNA duplex pool were transfected into the mouse macrophage cell line RAW264.7; cells were

    Article Snippet: In brief, pools of four siRNA duplexes or individual siRNA duplexes (Dharmacon) were transfected into either of two mouse macrophage cell lines (J774A.1 or RAW264.7) using the Amaxa nucleofector 96-well shuttle according to the manufacturer's instructions.

    Techniques: In Vivo, Transfection

    An siRNA-based screen for ISGs that inhibit VSV replication. (A) Schematic representation of the screening procedure for siRNA SMARTpools directed against ISGs. (B) VSV IND (nLuc) replication (fold increase in nLuc signal compared to the signal in the control) in cells transfected with siRNA SMARTpools ( n = 400 SMARTpools). (C) Confirmatory assays of VSV IND (nLuc) replication (luciferase activity in relative light units [RLU]) in cells transfected with the indicated siRNA SMARTpools and treated with 0, 5, 10, or 20 U/ml of IFN-α. (D) VSV IND (nLuc) replication (luciferase activity in RLU) in HT1080 cells transfected with the indicated individual siRNAs, alone or in combination, and treated with 10 U/ml of IFN-α. Mean values ± standard deviations (SD) are shown.

    Journal: Journal of Virology

    Article Title: Vesicular Stomatitis Virus Transcription Is Inhibited by TRIM69 in the Interferon-Induced Antiviral State

    doi: 10.1128/JVI.01372-19

    Figure Lengend Snippet: An siRNA-based screen for ISGs that inhibit VSV replication. (A) Schematic representation of the screening procedure for siRNA SMARTpools directed against ISGs. (B) VSV IND (nLuc) replication (fold increase in nLuc signal compared to the signal in the control) in cells transfected with siRNA SMARTpools ( n = 400 SMARTpools). (C) Confirmatory assays of VSV IND (nLuc) replication (luciferase activity in relative light units [RLU]) in cells transfected with the indicated siRNA SMARTpools and treated with 0, 5, 10, or 20 U/ml of IFN-α. (D) VSV IND (nLuc) replication (luciferase activity in RLU) in HT1080 cells transfected with the indicated individual siRNAs, alone or in combination, and treated with 10 U/ml of IFN-α. Mean values ± standard deviations (SD) are shown.

    Article Snippet: We also designed a small interfering RNA (siRNA) library containing siRNA pools (Dharmacon SMARTpools containing 4 individual siRNA duplexes) representing the 400 most strongly upregulated genes among a panel of cell lines, along with 18 siRNA controls ( ).

    Techniques: Transfection, Luciferase, Activity Assay

    Two-step validation RNAi assays identify six novel hepcidin regulators. (A) Thirteen screening hits were validated by the analysis of endogenous hepcidin mRNA levels upon knockdown with pools of 4 individual siRNAs in Huh7 cells. RNAi of SMAD7, SMAD4 and STAT3 served as a control. (B) Six regulators of hepcidin mRNA expression were validated by at least two independent siRNAs. Shown are mRNA levels of hepcidin and target genes after RNAi. Results are presented as a fold change (± 95% CI) compared to samples transfected with scrambled siRNA. The mean of at least three independent experiments is shown. Significant changes are indicated by asterisks (* P

    Journal: Haematologica

    Article Title: Imatinib and spironolactone suppress hepcidin expression

    doi: 10.3324/haematol.2016.162917

    Figure Lengend Snippet: Two-step validation RNAi assays identify six novel hepcidin regulators. (A) Thirteen screening hits were validated by the analysis of endogenous hepcidin mRNA levels upon knockdown with pools of 4 individual siRNAs in Huh7 cells. RNAi of SMAD7, SMAD4 and STAT3 served as a control. (B) Six regulators of hepcidin mRNA expression were validated by at least two independent siRNAs. Shown are mRNA levels of hepcidin and target genes after RNAi. Results are presented as a fold change (± 95% CI) compared to samples transfected with scrambled siRNA. The mean of at least three independent experiments is shown. Significant changes are indicated by asterisks (* P

    Article Snippet: Transfection of siRNAs and luciferase reporter constructs As reported previously, we reverse transfected Huh7 cells with 10 pmol siRNA (pooled or individual duplexes; Dharmacon, Online Supplementary Table S2 ) in a 96-well format.

    Techniques: Expressing, Transfection

    The midbody positions the apical surface during cyst development. (A) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue), tubulin (green), and aPKC (red). (top) A control cyst at the two-cell stage (note that abscission appears to have occurred symmetrically). (middle) A larger control cyst, with the midbody in the center of the developing structure (apical region of dividing cell) reflecting asymmetric abscission. (bottom) Cdc42 siRNA structure with one midbody positioned normally at the center and another midbody (located in a different z section) abnormally positioned. (B) Quantitation of midbodies at the center of the cyst from three independent experiments. The total number of midbodies is indicated (N). A midbody is regarded as being in the center if it is located at a distance from the centroid that is less than one third the radius of the structure.

    Journal: The Journal of Cell Biology

    Article Title: Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis

    doi: 10.1083/jcb.200807121

    Figure Lengend Snippet: The midbody positions the apical surface during cyst development. (A) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue), tubulin (green), and aPKC (red). (top) A control cyst at the two-cell stage (note that abscission appears to have occurred symmetrically). (middle) A larger control cyst, with the midbody in the center of the developing structure (apical region of dividing cell) reflecting asymmetric abscission. (bottom) Cdc42 siRNA structure with one midbody positioned normally at the center and another midbody (located in a different z section) abnormally positioned. (B) Quantitation of midbodies at the center of the cyst from three independent experiments. The total number of midbodies is indicated (N). A midbody is regarded as being in the center if it is located at a distance from the centroid that is less than one third the radius of the structure.

    Article Snippet: RNAi To deplete Cdc42, a SMARTpool (a mixture of four siRNA duplexes) and individual siRNA duplexes were purchased from Thermo Fisher Scientific.

    Techniques: Transfection, Staining, Quantitation Assay

    Cdc42 depletion disrupts mitotic spindle orientation. (A) Diagram depicting spindle angle measurement. The centroid of the cyst (dark blue circle) and the center of the spindle axis (pink circles) of a metaphase cell were drawn using ImageJ. The angle (red) between the spindle axis (black lines) and the line connecting the centroid of the cyst to the center of the spindle (dashed lines) was determined. To analyze spindle poles in different z sections, three z sections were taken so as to include both spindle poles and were merged as shown. Three schematic spindles are shown. The right and middle spindle examples represent correctly oriented spindles whose poles are positioned in one section (z2; middle spindle) or in different sections (z1 and z3; right spindle). The left spindle represents a misoriented spindle whose poles are in different z sections. Spindle microtubules, green; centrosomes, yellow; DNA, light blue. (B) Scatter diagram of metaphase spindle angles in cysts that were transfected with control or two Cdc42 siRNA duplexes from three independent experiments. Pink circles indicate mean values, green circles indicate individual data points, and error bars represent the SEM of the total number of spindles analyzed (N). (C) Caco-2 was transfected with control or Cdc42 siRNA and was fixed and stained for DNA (blue), tubulin (green), and filamentous actin (F-actin; red). Single confocal sections through the center of the cysts are shown. Three z sections are shown for the Cdc42 siRNA cyst to reveal both poles of the misoriented spindle. (D and E) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue) and aPKC (green). (D) Cdc42 siRNA structures contain cells in the middle, with apical domains present between inner and outer cells. (E) Cdc42 siRNA structures possess apical domains that are not in the center and single cells with more than one apical domain. Arrows indicate cells with multiple distinct apical patches on their surface. Bars, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis

    doi: 10.1083/jcb.200807121

    Figure Lengend Snippet: Cdc42 depletion disrupts mitotic spindle orientation. (A) Diagram depicting spindle angle measurement. The centroid of the cyst (dark blue circle) and the center of the spindle axis (pink circles) of a metaphase cell were drawn using ImageJ. The angle (red) between the spindle axis (black lines) and the line connecting the centroid of the cyst to the center of the spindle (dashed lines) was determined. To analyze spindle poles in different z sections, three z sections were taken so as to include both spindle poles and were merged as shown. Three schematic spindles are shown. The right and middle spindle examples represent correctly oriented spindles whose poles are positioned in one section (z2; middle spindle) or in different sections (z1 and z3; right spindle). The left spindle represents a misoriented spindle whose poles are in different z sections. Spindle microtubules, green; centrosomes, yellow; DNA, light blue. (B) Scatter diagram of metaphase spindle angles in cysts that were transfected with control or two Cdc42 siRNA duplexes from three independent experiments. Pink circles indicate mean values, green circles indicate individual data points, and error bars represent the SEM of the total number of spindles analyzed (N). (C) Caco-2 was transfected with control or Cdc42 siRNA and was fixed and stained for DNA (blue), tubulin (green), and filamentous actin (F-actin; red). Single confocal sections through the center of the cysts are shown. Three z sections are shown for the Cdc42 siRNA cyst to reveal both poles of the misoriented spindle. (D and E) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue) and aPKC (green). (D) Cdc42 siRNA structures contain cells in the middle, with apical domains present between inner and outer cells. (E) Cdc42 siRNA structures possess apical domains that are not in the center and single cells with more than one apical domain. Arrows indicate cells with multiple distinct apical patches on their surface. Bars, 10 μm.

    Article Snippet: RNAi To deplete Cdc42, a SMARTpool (a mixture of four siRNA duplexes) and individual siRNA duplexes were purchased from Thermo Fisher Scientific.

    Techniques: Transfection, Staining

    Cdc42 depletion induces multiple lumens. (A) Caco-2 was transfected with control or Cdc42 siRNA, plated in three dimensions, and treated with CTX at day 6 to induce luminal swelling. Phase images from cysts before (0 h) and after (12 h) treatment are shown. Note that about half of the Cdc42-depleted cysts lack a single central lumen. Bars, 50 μm. (B) Western blot of Caco-2 transfected with control siRNA, Cdc42 siRNA SMARTpool, or two different individual duplexes. Cdc42 levels are significantly reduced by 3 d and remain reduced for 7 d. (C) Caco-2 cultured as in A was fixed and stained with rhodamine phalloidin. Cysts were examined for single lumen (blue) or multiple lumens (red). The mean ± standard deviation for three independent experiments (at least 50 cysts each) is shown. (D and E) Caco-2 cultured as in A was fixed and stained for DNA (blue) and aPKC (green; D) or DNA (blue), E-cadherin (Ecad; green), and ZO-1 (red; E). Single confocal sections through the middle of the cysts are shown. DIC, differential interference contrast. Bars, 25 μm.

    Journal: The Journal of Cell Biology

    Article Title: Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis

    doi: 10.1083/jcb.200807121

    Figure Lengend Snippet: Cdc42 depletion induces multiple lumens. (A) Caco-2 was transfected with control or Cdc42 siRNA, plated in three dimensions, and treated with CTX at day 6 to induce luminal swelling. Phase images from cysts before (0 h) and after (12 h) treatment are shown. Note that about half of the Cdc42-depleted cysts lack a single central lumen. Bars, 50 μm. (B) Western blot of Caco-2 transfected with control siRNA, Cdc42 siRNA SMARTpool, or two different individual duplexes. Cdc42 levels are significantly reduced by 3 d and remain reduced for 7 d. (C) Caco-2 cultured as in A was fixed and stained with rhodamine phalloidin. Cysts were examined for single lumen (blue) or multiple lumens (red). The mean ± standard deviation for three independent experiments (at least 50 cysts each) is shown. (D and E) Caco-2 cultured as in A was fixed and stained for DNA (blue) and aPKC (green; D) or DNA (blue), E-cadherin (Ecad; green), and ZO-1 (red; E). Single confocal sections through the middle of the cysts are shown. DIC, differential interference contrast. Bars, 25 μm.

    Article Snippet: RNAi To deplete Cdc42, a SMARTpool (a mixture of four siRNA duplexes) and individual siRNA duplexes were purchased from Thermo Fisher Scientific.

    Techniques: Transfection, Western Blot, Cell Culture, Staining, Standard Deviation

    Increased NF-κB luciferase activity in neuronal cells with knockdown of RNF11. (A) Stable SH-SY5Y cell lines were generated using lentiviruses containing a control scramble small hairpin RNA (shRNA) sequence (shRNA-Scramble cells) or shRNA targeted against RNF11 (shRNA-RNF11 cells). Quantitative RT-PCR was used to measure relative RNF11 mRNA levels with glucuronidase β as an internal control. Values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. (B) Stable SH-SY5Y cell lines, as well as untransduced cells, were transiently cotransfected with luciferase and Renilla plasmids and stimulated with 10 ng/ml TNF-α or PBS for 6 hours. Values represent the fold change in NF-κB-dependent activity as measured by the ratio of luciferase to Renilla luminescence in stimulated over unstimulated samples ± SEM in triplicate experiments. * P

    Journal: Journal of Neuroinflammation

    Article Title: Neuronal RING finger protein 11 (RNF11) regulates canonical NF-?B signaling

    doi: 10.1186/1742-2094-9-67

    Figure Lengend Snippet: Increased NF-κB luciferase activity in neuronal cells with knockdown of RNF11. (A) Stable SH-SY5Y cell lines were generated using lentiviruses containing a control scramble small hairpin RNA (shRNA) sequence (shRNA-Scramble cells) or shRNA targeted against RNF11 (shRNA-RNF11 cells). Quantitative RT-PCR was used to measure relative RNF11 mRNA levels with glucuronidase β as an internal control. Values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. (B) Stable SH-SY5Y cell lines, as well as untransduced cells, were transiently cotransfected with luciferase and Renilla plasmids and stimulated with 10 ng/ml TNF-α or PBS for 6 hours. Values represent the fold change in NF-κB-dependent activity as measured by the ratio of luciferase to Renilla luminescence in stimulated over unstimulated samples ± SEM in triplicate experiments. * P

    Article Snippet: RNA interference Individual siRNA duplexes were purchased from Dharmacon Inc (Chicago, IL, USA) and tested for knockdown of RNF11 using quantitative RT-PCR (qRT-PCR) in SH-SY5Y cells (not shown).

    Techniques: Luciferase, Activity Assay, Generated, shRNA, Sequencing, Quantitative RT-PCR

    Prolonged nuclear p65 translocation after TNF-α stimulation observed in neuronal cells with knockdown of RNF11. (A) SH-SY5Y cell lines (untransduced, small hairpin RNA (shRNA)-RNF11, shRNA-Scramble) were stimulated with TNF-α for 0, 30, 60 or 120 minutes. Cells were immunostained for p65 (red) and nuclear DNA (Hoechst 333258, blue). Representative images obtained at 0, 30 and 120 minutes after stimulation are shown. (B) Cells were analyzed for overlap of p65-positive and Hoechst 333258-positive pixels. The fold change in percentage overlap of p65- and Hoechst 333258-positive pixels was calculated relative to unstimulated conditions. (C) Murine primary cortical neurons were treated with cytosine arabinoside and transduced with lentiviruses containing shRNA targeted against RNF11 (shRNA-RNF11 neurons) or scramble shRNA sequence (shRNA-Scramble neurons). Quantitative RT-PCR (qRT-PCR) was used to measure relative RNF11 mRNA levels. (D) shRNA-RNF11 or shRNA-Scramble neurons were stimulated for 0, 30 or 120 min before being immunostained for p65 and Hoechst 333258. Transduced cells were analyzed for overlap of p65- and Hoechst 333258-positive cells. The fold change in percentage overlap of p65- and Hoechst 333258-positive pixels was calculated relative to unstimulated conditions. (E) shRNA-Scramble and shRNA-RNF11 cells were stimulated for 0, 30 or 120 minutes. Cytoplasmic and nuclear fractions were resolved by SDS-PAGE. (F) ImageJ software was used to quantify the ratio of the densitometry of the p65 bands in the nuclear fractions to the density of histone 1 immunoreactivity. The ratio for each time point was compared relative to the steady-state ratio, which was set at 100%. (G) ImageJ software was used to quantify the ratio of the densitometry of the p65 bands in the cytoplasmic fractions in a manner similar to that described in (F). All p65 colocalization values are means ± SEM of triplicate experiments. qRT-PCR values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. Western blots of cytoplasmic and nuclear fractions were run from triplicate experiments. ** P

    Journal: Journal of Neuroinflammation

    Article Title: Neuronal RING finger protein 11 (RNF11) regulates canonical NF-?B signaling

    doi: 10.1186/1742-2094-9-67

    Figure Lengend Snippet: Prolonged nuclear p65 translocation after TNF-α stimulation observed in neuronal cells with knockdown of RNF11. (A) SH-SY5Y cell lines (untransduced, small hairpin RNA (shRNA)-RNF11, shRNA-Scramble) were stimulated with TNF-α for 0, 30, 60 or 120 minutes. Cells were immunostained for p65 (red) and nuclear DNA (Hoechst 333258, blue). Representative images obtained at 0, 30 and 120 minutes after stimulation are shown. (B) Cells were analyzed for overlap of p65-positive and Hoechst 333258-positive pixels. The fold change in percentage overlap of p65- and Hoechst 333258-positive pixels was calculated relative to unstimulated conditions. (C) Murine primary cortical neurons were treated with cytosine arabinoside and transduced with lentiviruses containing shRNA targeted against RNF11 (shRNA-RNF11 neurons) or scramble shRNA sequence (shRNA-Scramble neurons). Quantitative RT-PCR (qRT-PCR) was used to measure relative RNF11 mRNA levels. (D) shRNA-RNF11 or shRNA-Scramble neurons were stimulated for 0, 30 or 120 min before being immunostained for p65 and Hoechst 333258. Transduced cells were analyzed for overlap of p65- and Hoechst 333258-positive cells. The fold change in percentage overlap of p65- and Hoechst 333258-positive pixels was calculated relative to unstimulated conditions. (E) shRNA-Scramble and shRNA-RNF11 cells were stimulated for 0, 30 or 120 minutes. Cytoplasmic and nuclear fractions were resolved by SDS-PAGE. (F) ImageJ software was used to quantify the ratio of the densitometry of the p65 bands in the nuclear fractions to the density of histone 1 immunoreactivity. The ratio for each time point was compared relative to the steady-state ratio, which was set at 100%. (G) ImageJ software was used to quantify the ratio of the densitometry of the p65 bands in the cytoplasmic fractions in a manner similar to that described in (F). All p65 colocalization values are means ± SEM of triplicate experiments. qRT-PCR values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. Western blots of cytoplasmic and nuclear fractions were run from triplicate experiments. ** P

    Article Snippet: RNA interference Individual siRNA duplexes were purchased from Dharmacon Inc (Chicago, IL, USA) and tested for knockdown of RNF11 using quantitative RT-PCR (qRT-PCR) in SH-SY5Y cells (not shown).

    Techniques: Translocation Assay, shRNA, Transduction, Sequencing, Quantitative RT-PCR, SDS Page, Software, Western Blot

    Knockdown of RNF11 exaggerates inflammatory responses following TNF-α treatment. (A) and (B) Stably transduced SH-SY5Y short hairpin RNA (shRNA)-RNF11 and shRNA-Scramble cells were exposed to 0 or 10 ng/ml TNF-α for 24 hours. The relative amount of mRNA expression in stimulated and unstimulated cells was calculated using quantitative RT-PCR (qRT-PCR) and glucuronidase β (GUSB) as an internal control. (C) Stably transduced SH-SY5Y shRNA-RNF11 and shRNA-Scramble cells were exposed to 0 or 10 ng/ml TNF-α for 4 hours before cells were rinsed and fresh media were replaced in the plate. Media were collected after 20 hours and analyzed for TNF-α protein levels using a human TNF-α ELISA. ND, not detectable. (D) Murine primary cultures were harvested, and qRT-PCR was used to measure relative RNF11 mRNA levels. (E) Murine primary cortical neurons were treated with cytosine arabinoside and transduced with shRNA-Scramble or shRNA-RNF11 lentivirus. Cells were exposed to 0 or 10 ng/ml TNF-α for 4 or 24 hours, and mRNA levels were examined by qRT-PCR. (F) Murine primary cortical neurons were transduced with shRNA-Scramble or shRNA-RNF11 lentivirus. Cells were exposed to 0 or 10 ng/ml TNF-α for 4 or 24 hours, and protein levels of monocyte chemoattractant protein 1 (MCP-1) were measured using a mouse MCP-1 ELISA. qRT-PCR values are expressed as mean comparative cycle threshold ± SEM with GUSB (for SH-SY5Y cells) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (for primary cultures) levels as an internal control for three independent experiments. ELISA values are expressed as averages of protein levels extrapolated from standard curve analysis from three independent experiments. *** P

    Journal: Journal of Neuroinflammation

    Article Title: Neuronal RING finger protein 11 (RNF11) regulates canonical NF-?B signaling

    doi: 10.1186/1742-2094-9-67

    Figure Lengend Snippet: Knockdown of RNF11 exaggerates inflammatory responses following TNF-α treatment. (A) and (B) Stably transduced SH-SY5Y short hairpin RNA (shRNA)-RNF11 and shRNA-Scramble cells were exposed to 0 or 10 ng/ml TNF-α for 24 hours. The relative amount of mRNA expression in stimulated and unstimulated cells was calculated using quantitative RT-PCR (qRT-PCR) and glucuronidase β (GUSB) as an internal control. (C) Stably transduced SH-SY5Y shRNA-RNF11 and shRNA-Scramble cells were exposed to 0 or 10 ng/ml TNF-α for 4 hours before cells were rinsed and fresh media were replaced in the plate. Media were collected after 20 hours and analyzed for TNF-α protein levels using a human TNF-α ELISA. ND, not detectable. (D) Murine primary cultures were harvested, and qRT-PCR was used to measure relative RNF11 mRNA levels. (E) Murine primary cortical neurons were treated with cytosine arabinoside and transduced with shRNA-Scramble or shRNA-RNF11 lentivirus. Cells were exposed to 0 or 10 ng/ml TNF-α for 4 or 24 hours, and mRNA levels were examined by qRT-PCR. (F) Murine primary cortical neurons were transduced with shRNA-Scramble or shRNA-RNF11 lentivirus. Cells were exposed to 0 or 10 ng/ml TNF-α for 4 or 24 hours, and protein levels of monocyte chemoattractant protein 1 (MCP-1) were measured using a mouse MCP-1 ELISA. qRT-PCR values are expressed as mean comparative cycle threshold ± SEM with GUSB (for SH-SY5Y cells) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (for primary cultures) levels as an internal control for three independent experiments. ELISA values are expressed as averages of protein levels extrapolated from standard curve analysis from three independent experiments. *** P

    Article Snippet: RNA interference Individual siRNA duplexes were purchased from Dharmacon Inc (Chicago, IL, USA) and tested for knockdown of RNF11 using quantitative RT-PCR (qRT-PCR) in SH-SY5Y cells (not shown).

    Techniques: Stable Transfection, shRNA, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transduction

    RNF11 is essential for the termination of NF-κB signalling. ( A ) THP-1 cells were transfected on consecutive days with control scrambled or RNF11 siRNAs. At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK and RNF11. ( B ) THP-1 cells were transfected on consecutive days with control scrambled, RNF11 or A20 siRNAs. At 2 days after the second transfection, cells were treated with LPS (1 μg/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK, A20, RNF11 and β-actin. ( C ) THP-1 cells were transfected with control scrambled or RNF11 siRNA as described in (A). At 2 days after the second transfection, cells were stimulated with TNF (10 ng/ml) for various times and RNA was subjected to real-time PCR for IκBα and A20 expression. This experiment was repeated twice with similar results. ( D ) THP-1 cells were transfected with siRNAs as described in (A). Supernatants were subjected to an IL-6 ELISA. Error bars indicate s.e.m. of triplicate samples. Statistical analysis was performed by one-way ANOVA, followed by the Tukey–Kramer test for multiple comparisons. * P

    Journal: The EMBO Journal

    Article Title: The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-κB signalling

    doi: 10.1038/emboj.2008.285

    Figure Lengend Snippet: RNF11 is essential for the termination of NF-κB signalling. ( A ) THP-1 cells were transfected on consecutive days with control scrambled or RNF11 siRNAs. At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK and RNF11. ( B ) THP-1 cells were transfected on consecutive days with control scrambled, RNF11 or A20 siRNAs. At 2 days after the second transfection, cells were treated with LPS (1 μg/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK, A20, RNF11 and β-actin. ( C ) THP-1 cells were transfected with control scrambled or RNF11 siRNA as described in (A). At 2 days after the second transfection, cells were stimulated with TNF (10 ng/ml) for various times and RNA was subjected to real-time PCR for IκBα and A20 expression. This experiment was repeated twice with similar results. ( D ) THP-1 cells were transfected with siRNAs as described in (A). Supernatants were subjected to an IL-6 ELISA. Error bars indicate s.e.m. of triplicate samples. Statistical analysis was performed by one-way ANOVA, followed by the Tukey–Kramer test for multiple comparisons. * P

    Article Snippet: For RNF11 knockdown-reconstitution experiments in THP-1 cells, an individual RNF11 siRNA duplex was purchased from Dharmacon.

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

    RNF11 specifically inhibits NF-κB activation. ( A ) THP-1 cells were transfected on day 1 with no siRNA (mock), control scrambled siRNA or RNF11 siRNA. On day 2, the same cells were transfected with pRL-tk internal control Renilla luciferase plasmid, κB-TATA Luc and Myc–RNF11 as indicated. After an additional 2 days, cells were treated with TNF (10 ng/ml) for 8 h and lysates were subjected to dual luciferase assays. The lysates were also subjected to immunoblotting to examine the expression of RNF11 and β-actin. Error bars indicate s.e.m. of triplicate samples. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by the Tukey–Kramer test for multiple comparisons ( * P

    Journal: The EMBO Journal

    Article Title: The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-κB signalling

    doi: 10.1038/emboj.2008.285

    Figure Lengend Snippet: RNF11 specifically inhibits NF-κB activation. ( A ) THP-1 cells were transfected on day 1 with no siRNA (mock), control scrambled siRNA or RNF11 siRNA. On day 2, the same cells were transfected with pRL-tk internal control Renilla luciferase plasmid, κB-TATA Luc and Myc–RNF11 as indicated. After an additional 2 days, cells were treated with TNF (10 ng/ml) for 8 h and lysates were subjected to dual luciferase assays. The lysates were also subjected to immunoblotting to examine the expression of RNF11 and β-actin. Error bars indicate s.e.m. of triplicate samples. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by the Tukey–Kramer test for multiple comparisons ( * P

    Article Snippet: For RNF11 knockdown-reconstitution experiments in THP-1 cells, an individual RNF11 siRNA duplex was purchased from Dharmacon.

    Techniques: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing

    RNF11 requires the RING domain and PPXY motif to interact with the A20 ubiquitin-editing complex and to terminate NF-κB signalling. ( A ) Schematic diagram of RNF11 domain organization and mutants used in this figure. The PPXY motif is located between amino acids 37 and 40. The RING finger domain is located between amino acids 99 and 140. The RNF11 C99A mutant has a non-functional RING domain. The RNF11 Y40A mutant has a disrupted PPXY motif. ( B ) THP-1 cells were transfected with a control scrambled or a single duplex RNF11 siRNA. The following day, the cells were transfected with siRNA-resistant forms of Flag–RNF11 (WT R ), Flag–RNF11 C99A (C99A R ) and Flag–RNF11 Y40A (Y40A R ). After two more days, cells were treated with TNF (10 ng/ml) for the indicated times and lysates were immunoblotted with anti-pIκBα, IκBα, pJNK, JNK, Flag, RNF11 and β-actin. ( C ) THP-1 cells were transfected with siRNA and siRNA-resistant forms of RNF11 as described in (B). At 2 days after DNA transfections, cells were treated with TNF for 30 min where indicated. Lysates were subjected to immunoprecipitations with anti-RNF11 followed by immunoblotting with A20, TAX1BP1 and RIP1. The lysates were also used for immunoblotting using anti-IκBα, Flag, RNF11 and β-actin.

    Journal: The EMBO Journal

    Article Title: The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-κB signalling

    doi: 10.1038/emboj.2008.285

    Figure Lengend Snippet: RNF11 requires the RING domain and PPXY motif to interact with the A20 ubiquitin-editing complex and to terminate NF-κB signalling. ( A ) Schematic diagram of RNF11 domain organization and mutants used in this figure. The PPXY motif is located between amino acids 37 and 40. The RING finger domain is located between amino acids 99 and 140. The RNF11 C99A mutant has a non-functional RING domain. The RNF11 Y40A mutant has a disrupted PPXY motif. ( B ) THP-1 cells were transfected with a control scrambled or a single duplex RNF11 siRNA. The following day, the cells were transfected with siRNA-resistant forms of Flag–RNF11 (WT R ), Flag–RNF11 C99A (C99A R ) and Flag–RNF11 Y40A (Y40A R ). After two more days, cells were treated with TNF (10 ng/ml) for the indicated times and lysates were immunoblotted with anti-pIκBα, IκBα, pJNK, JNK, Flag, RNF11 and β-actin. ( C ) THP-1 cells were transfected with siRNA and siRNA-resistant forms of RNF11 as described in (B). At 2 days after DNA transfections, cells were treated with TNF for 30 min where indicated. Lysates were subjected to immunoprecipitations with anti-RNF11 followed by immunoblotting with A20, TAX1BP1 and RIP1. The lysates were also used for immunoblotting using anti-IκBα, Flag, RNF11 and β-actin.

    Article Snippet: For RNF11 knockdown-reconstitution experiments in THP-1 cells, an individual RNF11 siRNA duplex was purchased from Dharmacon.

    Techniques: Mutagenesis, Functional Assay, Transfection

    Drug specificity and final phenotypes. A: Drug specificity tests. HeLa H2B-GFP cells were transfected with siRNA pools as indicated, or “Mock” transfected in the absence of siRNA. 24 hours after transfection, medium containing either 150 nM of taxol or 300 nM of nocadazole was added to all of the samples. Cells were then incubated for an additional 24 hours and imaged for phenotypic changes. The pools were scored visually for cells in mitotic arrest, and this value was quantified for the pool by counting > 100 cells. (B) Phenotypic categorization of hits on the basis of data in Figure 7 and 8A . Category I) failure to enter mitosis, II) brief mitotic arrest without cytokinesis, and III) brief mitotic arrest with cytokinesis (true suppressors). Shown in bold are 5 expected genes, 4 known SAC genes and KifC1, which is a known true suppressor of loss of Kinesin-5 function [28] .

    Journal: PLoS ONE

    Article Title: An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor

    doi: 10.1371/journal.pone.0007339

    Figure Lengend Snippet: Drug specificity and final phenotypes. A: Drug specificity tests. HeLa H2B-GFP cells were transfected with siRNA pools as indicated, or “Mock” transfected in the absence of siRNA. 24 hours after transfection, medium containing either 150 nM of taxol or 300 nM of nocadazole was added to all of the samples. Cells were then incubated for an additional 24 hours and imaged for phenotypic changes. The pools were scored visually for cells in mitotic arrest, and this value was quantified for the pool by counting > 100 cells. (B) Phenotypic categorization of hits on the basis of data in Figure 7 and 8A . Category I) failure to enter mitosis, II) brief mitotic arrest without cytokinesis, and III) brief mitotic arrest with cytokinesis (true suppressors). Shown in bold are 5 expected genes, 4 known SAC genes and KifC1, which is a known true suppressor of loss of Kinesin-5 function [28] .

    Article Snippet: Taxol and Nocodazole Assays HeLa H2B-GFP cells were reverse transfected by hand with individual siRNA duplexes and pools (both at 10 nM final concentration) in 384 well plates (Corning Costar #3712).

    Techniques: Transfection, Incubation

    Sample strong hit phenotypes from the K5I Suppressor screen. Nuclei from each time point are color-coded to illustrate their CellProfiler classifications: yellow = interphase, red = monopolar, blue = unclassified objects. As illustrated in Fig. 2 , transfection occurred at T = 0. (A) untreated HeLa H2B-GFP cells, (B) untransfected cells treated with 1 uM K5I, and (C) mock transfected cells treated with HiPerFect transfection reagent and no K5I. In panels D–F, 1 uM K5I was added to cells 24 hours after transfection with (D) non-targeting siRNA, or siRNAs directed against (E) TTK or (F) Bub3.

    Journal: PLoS ONE

    Article Title: An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor

    doi: 10.1371/journal.pone.0007339

    Figure Lengend Snippet: Sample strong hit phenotypes from the K5I Suppressor screen. Nuclei from each time point are color-coded to illustrate their CellProfiler classifications: yellow = interphase, red = monopolar, blue = unclassified objects. As illustrated in Fig. 2 , transfection occurred at T = 0. (A) untreated HeLa H2B-GFP cells, (B) untransfected cells treated with 1 uM K5I, and (C) mock transfected cells treated with HiPerFect transfection reagent and no K5I. In panels D–F, 1 uM K5I was added to cells 24 hours after transfection with (D) non-targeting siRNA, or siRNAs directed against (E) TTK or (F) Bub3.

    Article Snippet: Taxol and Nocodazole Assays HeLa H2B-GFP cells were reverse transfected by hand with individual siRNA duplexes and pools (both at 10 nM final concentration) in 384 well plates (Corning Costar #3712).

    Techniques: Transfection

    Results of time-lapse experiments to examine duration of mitotic arrest and percent of cells entering cytokinesis after treatment with K5I suppressor siRNAs. HeLa H2B-GFP cells were transfected with siRNA pools and individual siRNA duplexes directed against the indicated genes or with a non-targeting control siRNA. “Mock” cells were treated with HiPerFect transfection agent in the absence of siRNA. 24 hours after transfection, K5I was added to all of the samples, so that the final inhibitor concentration was 1 uM. Cells were imaged at 10 minute intervals for 48 hours on an automated inverted fluorescence microscope and automated focus. 50–100 cells from each condition were scored by visual inspection for (A) the average duration of mitosis in the population, and (B) the percent of cells going through cytokinesis upon exit from mitosis. For each of the genes shown, both the siRNA pool and the individual siRNA duplexes were observed to cause the same phenotypes, but only data for the siRNA pools is graphed here.

    Journal: PLoS ONE

    Article Title: An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor

    doi: 10.1371/journal.pone.0007339

    Figure Lengend Snippet: Results of time-lapse experiments to examine duration of mitotic arrest and percent of cells entering cytokinesis after treatment with K5I suppressor siRNAs. HeLa H2B-GFP cells were transfected with siRNA pools and individual siRNA duplexes directed against the indicated genes or with a non-targeting control siRNA. “Mock” cells were treated with HiPerFect transfection agent in the absence of siRNA. 24 hours after transfection, K5I was added to all of the samples, so that the final inhibitor concentration was 1 uM. Cells were imaged at 10 minute intervals for 48 hours on an automated inverted fluorescence microscope and automated focus. 50–100 cells from each condition were scored by visual inspection for (A) the average duration of mitosis in the population, and (B) the percent of cells going through cytokinesis upon exit from mitosis. For each of the genes shown, both the siRNA pool and the individual siRNA duplexes were observed to cause the same phenotypes, but only data for the siRNA pools is graphed here.

    Article Snippet: Taxol and Nocodazole Assays HeLa H2B-GFP cells were reverse transfected by hand with individual siRNA duplexes and pools (both at 10 nM final concentration) in 384 well plates (Corning Costar #3712).

    Techniques: Transfection, Concentration Assay, Fluorescence, Microscopy

    Sample strong hit phenotypes from the K5I Enhancer screen. The nuclei from each time point are color-coded to illustrate their CellProfiler classifications: yellow = interphase, red = monopolar, blue = unclassified objects. As illustrated in Fig. 2 , transfection occurred at T = 0. (A) untreated HeLa H2B-GFP cells, (B) untransfected cells treated with 60 nM K5I, (C) mock transfected cells treated with HiPerFect transfection reagent and no K5I. In panels D–F, 60 nM K5I was added to cells 24 hours after transfection with (D) non-targeting siRNA, or siRNAs directed against (E) PLK1 or (F) AURKA.

    Journal: PLoS ONE

    Article Title: An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor

    doi: 10.1371/journal.pone.0007339

    Figure Lengend Snippet: Sample strong hit phenotypes from the K5I Enhancer screen. The nuclei from each time point are color-coded to illustrate their CellProfiler classifications: yellow = interphase, red = monopolar, blue = unclassified objects. As illustrated in Fig. 2 , transfection occurred at T = 0. (A) untreated HeLa H2B-GFP cells, (B) untransfected cells treated with 60 nM K5I, (C) mock transfected cells treated with HiPerFect transfection reagent and no K5I. In panels D–F, 60 nM K5I was added to cells 24 hours after transfection with (D) non-targeting siRNA, or siRNAs directed against (E) PLK1 or (F) AURKA.

    Article Snippet: Taxol and Nocodazole Assays HeLa H2B-GFP cells were reverse transfected by hand with individual siRNA duplexes and pools (both at 10 nM final concentration) in 384 well plates (Corning Costar #3712).

    Techniques: Transfection

    Examples of chromosomal phenotypes in the screening assay. All cells were grown in 384-well plates and treated according to the primary screen assay protocol outlined in Figure 2 and described in Materials and Methods . These samples were all transfected with a non-targeting siRNA duplexes. (A) Interphase cells and (B) Mitotic cells selected from control wells undergoing normal cell cycle progression; no Kinesin 5 inhibitor (K5I) added. (C) Monopolar spindles formed in the presence of 1 uM K5I. (D) Analysis of cells treated with 1 uM K5I after 48 hours.

    Journal: PLoS ONE

    Article Title: An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor

    doi: 10.1371/journal.pone.0007339

    Figure Lengend Snippet: Examples of chromosomal phenotypes in the screening assay. All cells were grown in 384-well plates and treated according to the primary screen assay protocol outlined in Figure 2 and described in Materials and Methods . These samples were all transfected with a non-targeting siRNA duplexes. (A) Interphase cells and (B) Mitotic cells selected from control wells undergoing normal cell cycle progression; no Kinesin 5 inhibitor (K5I) added. (C) Monopolar spindles formed in the presence of 1 uM K5I. (D) Analysis of cells treated with 1 uM K5I after 48 hours.

    Article Snippet: Taxol and Nocodazole Assays HeLa H2B-GFP cells were reverse transfected by hand with individual siRNA duplexes and pools (both at 10 nM final concentration) in 384 well plates (Corning Costar #3712).

    Techniques: Screening Assay, Transfection

    Effects of siRNA Treatment on Cellular UGT1A6 Activities and Expression. In , p-nitrophenol (50 µM) glucuronidation rates were measured in Caco-2 cells treated with Lipofectamine alone (control), negative control (nonsense) siRNA pool (Non-control)

    Journal: Molecular pharmaceutics

    Article Title: Disposition of Flavonoids via Enteric Recycling: Determination of the UDP-Glucuronosyltransferase (UGT) Isoforms Responsible for the Metabolism of Flavonoids in Intact Caco-2 TC7 Cells Using siRNA

    doi: 10.1021/mp0601190

    Figure Lengend Snippet: Effects of siRNA Treatment on Cellular UGT1A6 Activities and Expression. In , p-nitrophenol (50 µM) glucuronidation rates were measured in Caco-2 cells treated with Lipofectamine alone (control), negative control (nonsense) siRNA pool (Non-control)

    Article Snippet: A siRNA mixture or SMART pool against UGT1A6 (Cat# M-020196-00-0050), 4 individual duplex siRNA, and the negative control-pool (Cat.# D-001206-13-20) were products of DHARMACON Inc. (Dallas, TX).

    Techniques: Expressing, Negative Control

    Effect of Different siRNA Sequences on Cellular Glucuronidation Rates of p-Nitrophenol (50 µM) in Caco-2 Cell Lysates. All siRNAs were targeted to human UGT1A6 but had different sequences and each was used at equal molar amount for 72 hrs. Each

    Journal: Molecular pharmaceutics

    Article Title: Disposition of Flavonoids via Enteric Recycling: Determination of the UDP-Glucuronosyltransferase (UGT) Isoforms Responsible for the Metabolism of Flavonoids in Intact Caco-2 TC7 Cells Using siRNA

    doi: 10.1021/mp0601190

    Figure Lengend Snippet: Effect of Different siRNA Sequences on Cellular Glucuronidation Rates of p-Nitrophenol (50 µM) in Caco-2 Cell Lysates. All siRNAs were targeted to human UGT1A6 but had different sequences and each was used at equal molar amount for 72 hrs. Each

    Article Snippet: A siRNA mixture or SMART pool against UGT1A6 (Cat# M-020196-00-0050), 4 individual duplex siRNA, and the negative control-pool (Cat.# D-001206-13-20) were products of DHARMACON Inc. (Dallas, TX).

    Techniques:

    Kinome siRNA screen to determine genes involved in AT 1 R–EGFR transactivation. (A) Using the HMEC-LST-AT 1 R cell line, we performed a primary siRNA screen using the Dharmacon siGENOME SMARTpool siRNA library targeting kinase genes (720 in total,

    Journal: Journal of Cell Science

    Article Title: A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation

    doi: 10.1242/jcs.128280

    Figure Lengend Snippet: Kinome siRNA screen to determine genes involved in AT 1 R–EGFR transactivation. (A) Using the HMEC-LST-AT 1 R cell line, we performed a primary siRNA screen using the Dharmacon siGENOME SMARTpool siRNA library targeting kinase genes (720 in total,

    Article Snippet: Briefly, 40 nM Dharmacon SMARTpool siRNA or 25 nM Dharmacon SMARTpool deconvoluted siRNA (individual duplex) were complexed for 20 minutes at ambient temperature in a 200 µl volume of HuMEC basal medium with a final concentration of 1 µl per well of DharmaFECT 1 transfection lipid (ThermoFisher Scientific).

    Techniques:

    RGS12 coordinates a Ras/Raf/MEK/ERK complex and enhances signaling to ERK. ( A ) CHO-K1 cells with endogenous PDGFβR were transfected with ERK2–GFP and either empty vector or increasing amounts of HA-RGS12 vector, serum-starved overnight,

    Journal: The EMBO Journal

    Article Title: Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation

    doi: 10.1038/sj.emboj.7601659

    Figure Lengend Snippet: RGS12 coordinates a Ras/Raf/MEK/ERK complex and enhances signaling to ERK. ( A ) CHO-K1 cells with endogenous PDGFβR were transfected with ERK2–GFP and either empty vector or increasing amounts of HA-RGS12 vector, serum-starved overnight,

    Article Snippet: siRNAs against rat A-Raf, B-Raf, MEK2, and RGS12 (the latter including SMARTpool and constituent individual duplexes) were from Dharmacon (Lafayette, CO).

    Techniques: Transfection, Plasmid Preparation

    RGS12 and its isolated tandem RBDs preferentially interact with activated H-Ras. ( A, B ) HEK 293T cells were cotransfected with myc-RGS12 and indicated HA-Ras plasmids. Anti-myc was used to immunoprecipitate RGS12 and anti-HA used to detect associated

    Journal: The EMBO Journal

    Article Title: Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation

    doi: 10.1038/sj.emboj.7601659

    Figure Lengend Snippet: RGS12 and its isolated tandem RBDs preferentially interact with activated H-Ras. ( A, B ) HEK 293T cells were cotransfected with myc-RGS12 and indicated HA-Ras plasmids. Anti-myc was used to immunoprecipitate RGS12 and anti-HA used to detect associated

    Article Snippet: siRNAs against rat A-Raf, B-Raf, MEK2, and RGS12 (the latter including SMARTpool and constituent individual duplexes) were from Dharmacon (Lafayette, CO).

    Techniques: Isolation

    Prolonged ERK activation by NGF is reduced upon RGS12 depletion, expressed RGS12 is punctate and co-incident with endosomal markers, and translocation of endogenous RGS12 from membranes is regulated by NGF. ( A ) PC12 cells were transfected with non-specific

    Journal: The EMBO Journal

    Article Title: Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation

    doi: 10.1038/sj.emboj.7601659

    Figure Lengend Snippet: Prolonged ERK activation by NGF is reduced upon RGS12 depletion, expressed RGS12 is punctate and co-incident with endosomal markers, and translocation of endogenous RGS12 from membranes is regulated by NGF. ( A ) PC12 cells were transfected with non-specific

    Article Snippet: siRNAs against rat A-Raf, B-Raf, MEK2, and RGS12 (the latter including SMARTpool and constituent individual duplexes) were from Dharmacon (Lafayette, CO).

    Techniques: Activation Assay, Translocation Assay, Transfection

    RGS12 is critical for NGF-mediated and MAPK-dependent neurite outgrowth in PC12 cells. ( A ) PC12 lysates were immunoprecipitated with beads alone (no Ab), or anti-H-Ras, anti-Rap-1, anti-B-Raf, or anti-RGS12 antibodies. Anti-RGS12 was used to detect associated

    Journal: The EMBO Journal

    Article Title: Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation

    doi: 10.1038/sj.emboj.7601659

    Figure Lengend Snippet: RGS12 is critical for NGF-mediated and MAPK-dependent neurite outgrowth in PC12 cells. ( A ) PC12 lysates were immunoprecipitated with beads alone (no Ab), or anti-H-Ras, anti-Rap-1, anti-B-Raf, or anti-RGS12 antibodies. Anti-RGS12 was used to detect associated

    Article Snippet: siRNAs against rat A-Raf, B-Raf, MEK2, and RGS12 (the latter including SMARTpool and constituent individual duplexes) were from Dharmacon (Lafayette, CO).

    Techniques: Immunoprecipitation

    RGS12 forms a multiprotein complex containing TrkA and shows selectivity for TrkA signaling versus FGFR signaling. ( A ) HEK 293T cells were cotransfected with myc-RGS12, and either empty vector, FLAG-TrkA, or HA-FGFR1 plasmids. Anti-FLAG or anti-HA antibody

    Journal: The EMBO Journal

    Article Title: Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation

    doi: 10.1038/sj.emboj.7601659

    Figure Lengend Snippet: RGS12 forms a multiprotein complex containing TrkA and shows selectivity for TrkA signaling versus FGFR signaling. ( A ) HEK 293T cells were cotransfected with myc-RGS12, and either empty vector, FLAG-TrkA, or HA-FGFR1 plasmids. Anti-FLAG or anti-HA antibody

    Article Snippet: siRNAs against rat A-Raf, B-Raf, MEK2, and RGS12 (the latter including SMARTpool and constituent individual duplexes) were from Dharmacon (Lafayette, CO).

    Techniques: Plasmid Preparation

    Components of the mitogen-activated protein kinase (MAPK) cascade identified as RGS12 interactors. ( A ) RGS12 architecture. Numbering denotes amino-acid boundaries of domains within rat RGS12. Parentheses denote baits used in yeast two-hybrid screens against

    Journal: The EMBO Journal

    Article Title: Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation

    doi: 10.1038/sj.emboj.7601659

    Figure Lengend Snippet: Components of the mitogen-activated protein kinase (MAPK) cascade identified as RGS12 interactors. ( A ) RGS12 architecture. Numbering denotes amino-acid boundaries of domains within rat RGS12. Parentheses denote baits used in yeast two-hybrid screens against

    Article Snippet: siRNAs against rat A-Raf, B-Raf, MEK2, and RGS12 (the latter including SMARTpool and constituent individual duplexes) were from Dharmacon (Lafayette, CO).

    Techniques:

    RGS12 is critical for NGF-mediated axonal growth in primary mouse DRG neurons. ( A ) N1E-115 mouse neuroblastoma cells were transfected with non-specific or mouse RGS12-directed siRNA and lysed 72 h post-transection for immunoblotting of RGS12 and actin.

    Journal: The EMBO Journal

    Article Title: Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation

    doi: 10.1038/sj.emboj.7601659

    Figure Lengend Snippet: RGS12 is critical for NGF-mediated axonal growth in primary mouse DRG neurons. ( A ) N1E-115 mouse neuroblastoma cells were transfected with non-specific or mouse RGS12-directed siRNA and lysed 72 h post-transection for immunoblotting of RGS12 and actin.

    Article Snippet: siRNAs against rat A-Raf, B-Raf, MEK2, and RGS12 (the latter including SMARTpool and constituent individual duplexes) were from Dharmacon (Lafayette, CO).

    Techniques: Transfection

    Androgen/AR-dependent SGK3 transcription involves ER. A, LNCaP cells were transfected with the siRNA negative control (siNC) or ERβ siRNA (siERβ) for 72 hours. Cell extracts were subjected to Western blotting. B, After being hormone-stripped for 2 days, LNCaP cells were transfected with the siRNA negative control or ERβ siRNA for 48 hours and then were transfected with the luciferase reporters as indicated and cultured in the presence or absence of 1 nM DHT for 24 hours. Luciferase activity of cell extracts was measured and normalized to protein concentration. Data are expressed as means ± SD from 3 independent experiments. **, P

    Journal: Molecular Endocrinology

    Article Title: SGK3 Is an Androgen-Inducible Kinase Promoting Prostate Cancer Cell Proliferation Through Activation of p70 S6 Kinase and Up-Regulation of Cyclin D1

    doi: 10.1210/me.2013-1339

    Figure Lengend Snippet: Androgen/AR-dependent SGK3 transcription involves ER. A, LNCaP cells were transfected with the siRNA negative control (siNC) or ERβ siRNA (siERβ) for 72 hours. Cell extracts were subjected to Western blotting. B, After being hormone-stripped for 2 days, LNCaP cells were transfected with the siRNA negative control or ERβ siRNA for 48 hours and then were transfected with the luciferase reporters as indicated and cultured in the presence or absence of 1 nM DHT for 24 hours. Luciferase activity of cell extracts was measured and normalized to protein concentration. Data are expressed as means ± SD from 3 independent experiments. **, P

    Article Snippet: To this end, LNCaP cells were transfected with either pooled (sc-44852; Santa Cruz Biotechnology) or 3 individual SGK3 siRNA duplexes (sc-44852A, sc-44852B, and sc-44852C; Santa Cruz Biotechnology).

    Techniques: Transfection, Negative Control, Western Blot, Luciferase, Cell Culture, Activity Assay, Protein Concentration

    SGK3 promotes cell proliferation of AR-positive prostate cancer cells. A and B, LNCaP cells (A) or 22Rv1 cells (B) were transfected with the siRNA negative control (siNC) or 3 individual SGK3 siRNA duplexes (siSGK), respectively. Western blotting (insert) was performed to examine SGK3 level after the cells were transfected with siRNA for 72 hours. An MTT assay was performed to measure cell proliferation at the indicated time after siRNA transfection. Error bars represent SDs of 3 independent experiments. C, LNCaP/TetON/SGK3 cells cultured in phenol red–free RPMI 1640 medium with 2% CD-treated FBS were added with or without 100 ng/mL DOX. An MTT assay was performed to measure cell proliferation at the indicated time. D, LNCaP cells were transfected with the siRNA negative control or SGK3 siRNA and cultured in the phenol red–free RPMI 1640 medium with 5% CD-treated FBS in the presence or absence of DHT (1 nM). SGK3 protein levels were examined by Western blotting at 72 hours after siRNA transfection. The cell growth rate was measured by an MTT assay at the indicated time. **, P

    Journal: Molecular Endocrinology

    Article Title: SGK3 Is an Androgen-Inducible Kinase Promoting Prostate Cancer Cell Proliferation Through Activation of p70 S6 Kinase and Up-Regulation of Cyclin D1

    doi: 10.1210/me.2013-1339

    Figure Lengend Snippet: SGK3 promotes cell proliferation of AR-positive prostate cancer cells. A and B, LNCaP cells (A) or 22Rv1 cells (B) were transfected with the siRNA negative control (siNC) or 3 individual SGK3 siRNA duplexes (siSGK), respectively. Western blotting (insert) was performed to examine SGK3 level after the cells were transfected with siRNA for 72 hours. An MTT assay was performed to measure cell proliferation at the indicated time after siRNA transfection. Error bars represent SDs of 3 independent experiments. C, LNCaP/TetON/SGK3 cells cultured in phenol red–free RPMI 1640 medium with 2% CD-treated FBS were added with or without 100 ng/mL DOX. An MTT assay was performed to measure cell proliferation at the indicated time. D, LNCaP cells were transfected with the siRNA negative control or SGK3 siRNA and cultured in the phenol red–free RPMI 1640 medium with 5% CD-treated FBS in the presence or absence of DHT (1 nM). SGK3 protein levels were examined by Western blotting at 72 hours after siRNA transfection. The cell growth rate was measured by an MTT assay at the indicated time. **, P

    Article Snippet: To this end, LNCaP cells were transfected with either pooled (sc-44852; Santa Cruz Biotechnology) or 3 individual SGK3 siRNA duplexes (sc-44852A, sc-44852B, and sc-44852C; Santa Cruz Biotechnology).

    Techniques: Transfection, Negative Control, Western Blot, MTT Assay, Cell Culture

    Knockdown of SGK3 expression results in a decrease in p70S6K phosphorylation. A and B, LNCaP cells were transfected with the siRNA negative control (siNC), pooled (A) or individual (B) SGK3 siRNA duplexes (siSGK3), respectively. At 72 hours after transfection, the cells were harvested for Western blotting analysis with the relevant antibodies. C and D, LNCaP cells were treated with DMSO or the indicated concentrations of rapamycin (RAPA). An MTT assay (D) was performed to measure cell proliferation at the indicated time posttreatment. C, Western blotting was performed at 24 hours after treatment. **, P

    Journal: Molecular Endocrinology

    Article Title: SGK3 Is an Androgen-Inducible Kinase Promoting Prostate Cancer Cell Proliferation Through Activation of p70 S6 Kinase and Up-Regulation of Cyclin D1

    doi: 10.1210/me.2013-1339

    Figure Lengend Snippet: Knockdown of SGK3 expression results in a decrease in p70S6K phosphorylation. A and B, LNCaP cells were transfected with the siRNA negative control (siNC), pooled (A) or individual (B) SGK3 siRNA duplexes (siSGK3), respectively. At 72 hours after transfection, the cells were harvested for Western blotting analysis with the relevant antibodies. C and D, LNCaP cells were treated with DMSO or the indicated concentrations of rapamycin (RAPA). An MTT assay (D) was performed to measure cell proliferation at the indicated time posttreatment. C, Western blotting was performed at 24 hours after treatment. **, P

    Article Snippet: To this end, LNCaP cells were transfected with either pooled (sc-44852; Santa Cruz Biotechnology) or 3 individual SGK3 siRNA duplexes (sc-44852A, sc-44852B, and sc-44852C; Santa Cruz Biotechnology).

    Techniques: Expressing, Transfection, Negative Control, Western Blot, MTT Assay

    SGK3 promotes the G 1 to S phase cell cycle progression of LNCaP cells through up-regulation of cyclin D1. A and B, LNCaP cells were transfected with the siRNA negative control (siNC) or pooled SGK3 siRNA duplexes (siSGK3) for 4 days. The transfected cells were imaged under a microscope (A) or harvested for Western blotting (B) to determine PARP cleavage and cleaved caspase 3 levels. Scale bar corresponds to 50 μm; ×100 magnification. For Western blotting analysis of apoptosis markers, we also included a positive control, which are LNCaP cells treated with 20 μM proteasome inhibitor MG132 for 24 hours. C, LNCaP cells were transfected with siRNA negative control (LNCaP/siNC) or pooled SGK3 siRNA duplexes (LNCaP/siSGK3). Seventy-two hours after transfection, cells were harvested, fixed in 70% ethanol, and stained with propidium iodide and analyzed by flow cytometry. The percentage of DNA content in each phase was analyzed with ModFit LT 2.0 software. The representative result of 3 experiments was shown. D, Quantitative result of the percentage of DNA content in each cell cycle phase from 3 experiments. The data are expressed as means ± SD. *, P

    Journal: Molecular Endocrinology

    Article Title: SGK3 Is an Androgen-Inducible Kinase Promoting Prostate Cancer Cell Proliferation Through Activation of p70 S6 Kinase and Up-Regulation of Cyclin D1

    doi: 10.1210/me.2013-1339

    Figure Lengend Snippet: SGK3 promotes the G 1 to S phase cell cycle progression of LNCaP cells through up-regulation of cyclin D1. A and B, LNCaP cells were transfected with the siRNA negative control (siNC) or pooled SGK3 siRNA duplexes (siSGK3) for 4 days. The transfected cells were imaged under a microscope (A) or harvested for Western blotting (B) to determine PARP cleavage and cleaved caspase 3 levels. Scale bar corresponds to 50 μm; ×100 magnification. For Western blotting analysis of apoptosis markers, we also included a positive control, which are LNCaP cells treated with 20 μM proteasome inhibitor MG132 for 24 hours. C, LNCaP cells were transfected with siRNA negative control (LNCaP/siNC) or pooled SGK3 siRNA duplexes (LNCaP/siSGK3). Seventy-two hours after transfection, cells were harvested, fixed in 70% ethanol, and stained with propidium iodide and analyzed by flow cytometry. The percentage of DNA content in each phase was analyzed with ModFit LT 2.0 software. The representative result of 3 experiments was shown. D, Quantitative result of the percentage of DNA content in each cell cycle phase from 3 experiments. The data are expressed as means ± SD. *, P

    Article Snippet: To this end, LNCaP cells were transfected with either pooled (sc-44852; Santa Cruz Biotechnology) or 3 individual SGK3 siRNA duplexes (sc-44852A, sc-44852B, and sc-44852C; Santa Cruz Biotechnology).

    Techniques: Transfection, Negative Control, Microscopy, Western Blot, Positive Control, Staining, Flow Cytometry, Cytometry, Software

    Validation of the detection of GnRH-R in immunoblots. A. Representative immunoblots of GnRH-R detection (upper panel) after 48 h transfection with the Human cDNA clone pCMV6-XL5/GNRH-R in 16HBE14o − (1) and CFBE41o − (2) cells. pCMV6-XL5 empty plasmid was used as a control. B. The densitometric analysis after normalization by G3PDH expression and comparison with the controls, indicate that the GnRH-R expression in significantly increased, (n = 5). C. Representative immunoblots of GnRH-R detection after 72 h transfection with a siGENOME individual duplex targeting GnRH-R in 16HBE14o − (1) and CFBE41o − (2) cells. siGENOME Non-Targeting was used as control. A decreased expression of GnRH-R is observed in both cell types. D. The densitometric analysis after normalization by G3PDH expression and comparison with the controls, indicate that the GnRH-R expression is significantly decreased in 16HBE14o − (1) and CFBE41o − (2) cells (n = 7) in the presence of siRNA.

    Journal: PLoS ONE

    Article Title: Improvement of Chloride Transport Defect by Gonadotropin-Releasing Hormone (GnRH) in Cystic Fibrosis Epithelial Cells

    doi: 10.1371/journal.pone.0088964

    Figure Lengend Snippet: Validation of the detection of GnRH-R in immunoblots. A. Representative immunoblots of GnRH-R detection (upper panel) after 48 h transfection with the Human cDNA clone pCMV6-XL5/GNRH-R in 16HBE14o − (1) and CFBE41o − (2) cells. pCMV6-XL5 empty plasmid was used as a control. B. The densitometric analysis after normalization by G3PDH expression and comparison with the controls, indicate that the GnRH-R expression in significantly increased, (n = 5). C. Representative immunoblots of GnRH-R detection after 72 h transfection with a siGENOME individual duplex targeting GnRH-R in 16HBE14o − (1) and CFBE41o − (2) cells. siGENOME Non-Targeting was used as control. A decreased expression of GnRH-R is observed in both cell types. D. The densitometric analysis after normalization by G3PDH expression and comparison with the controls, indicate that the GnRH-R expression is significantly decreased in 16HBE14o − (1) and CFBE41o − (2) cells (n = 7) in the presence of siRNA.

    Article Snippet: Transfection For GnRH-R overexpression and inhibition, cells were transfected using Lipofectamine 2000 (Life Technologies Corporation, Carlsbad, CA, USA), according to the manufacturer’s instructions, with either the human cDNA clone pCMV6-XL5/GNRH-R (OriGene Technologies Inc., Rockville, MD, USA) or the human GnRH-R siRNA (5′-GGAAUUUGGUAUUGGUUUG-3′, siGENOME individual duplex (Thermo Fisher Scientific Inc., Waltham, MA, USA).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing