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  • 96
    Integrated DNA Technologies antisense oligonucleotides
    Treatment with <t>Antisense</t> Oligonucleotide Targeting Ca v β 3 Improves [Ca 2+ ] i Dynamics and Insulin Secretion in ob/ob Islets (A) Left panel shows protein levels of Ca v β 3 in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. Right panel shows relative quantification of Ca v β 3 protein levels in left (n = 5; 40 islets in each case). (B and C) Effects of 11 mM glucose on [Ca 2+ ] i in ob/ob islets treated with scramble ASO (B) and Ca v β 3 ASO (C). Representative traces out of 30 are shown. (D) First peak ratios of glucose-induced [Ca 2+ ] i changes in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. (E) Oscillation periods of glucose-induced [Ca 2+ ] i changes in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. (F) Oscillation amplitudes of glucose-induced [Ca 2+ ] i changes in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. (G) Effects of 25 mM KCl on [Ca 2+ ] i in dissociated islet cells from scramble ASO (black) and Ca v β 3 -ASO-treated (red) ob/ob mice. Representative traces on dissociated islet cells are shown. (H) Peak ratios of [Ca 2+ ] i changes induced by 25 mM KCl in dissociated islet cells from scramble ASO (black) and Ca v β 3 -ASO-treated (red) ob/ob mice (n = 5; each experiment involved 50 single cells). (I) Peak ratios of [Ca 2+ ] i changes induced by 200 μM Cch in ob/ob-dissociated islet cells treated with scramble ASO and Ca v β 3 ASO (n = 5; each experiment involved 30 single cells). (J) Glucose-induced insulin release in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. The islets were treated with 3 mM or 11 mM glucose for 30 min (n = 5; 10 islets in each case). Data are presented as the means ± SEM; ∗ p
    Antisense Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 96/100, based on 281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher mir oligos against mouse atrx
    <t>Sertad1</t> downregulation by adenoviral shRNAs protects cortical neurons from DNA damage-evoked cell death. (A,B) <t>shRNA-mediated</t> knockdown of Sertad1 in cortical neurons. Cortical neurons were infected with two adenoviral shRNAs targeting mouse Sertad1 gene or a control virus at the time of plating, and 24 hrs after infection cells were treated with camptothecin for 16 hrs to induce Sertad1 expression. Cells were fixed and immunostaining was performed using Sertad1 chicken IgY antibody. (A) Both Sertad1 shRNA viruses repress the upregulation of Sertad1 in cortical neurons treated with camptothecin (Arrows indicate Sertad1 shRNA-expressing cells). (B) Quantification of fluorescence intensity of Sertad1-immunostained neurons infected with control and Sertad1 shRNA viruses. Values are mean ± SEM (n = 30–35). (C) Expression of two Sertad1 shRNAs diminishes death of primary cultured cortical neurons treated with camptothecin. Cortical neurons were infected with control and Sertad1 shRNA viruses (multiplicity of infection, 10) at the time of plating. 2 days after plating, cells were treated with 10 μM camptothecin for 16, 24, 36 and 48 hrs, fixed and stained with Hoechst 33258 (0.25 μg/ml). GFP-positive cells were counted based on nuclear integrity. Significance comparisons with camptothecin-treated controls, *p
    Mir Oligos Against Mouse Atrx, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Kaneka Corp mouse oligonucleotides primers
    <t>Sertad1</t> downregulation by adenoviral shRNAs protects cortical neurons from DNA damage-evoked cell death. (A,B) <t>shRNA-mediated</t> knockdown of Sertad1 in cortical neurons. Cortical neurons were infected with two adenoviral shRNAs targeting mouse Sertad1 gene or a control virus at the time of plating, and 24 hrs after infection cells were treated with camptothecin for 16 hrs to induce Sertad1 expression. Cells were fixed and immunostaining was performed using Sertad1 chicken IgY antibody. (A) Both Sertad1 shRNA viruses repress the upregulation of Sertad1 in cortical neurons treated with camptothecin (Arrows indicate Sertad1 shRNA-expressing cells). (B) Quantification of fluorescence intensity of Sertad1-immunostained neurons infected with control and Sertad1 shRNA viruses. Values are mean ± SEM (n = 30–35). (C) Expression of two Sertad1 shRNAs diminishes death of primary cultured cortical neurons treated with camptothecin. Cortical neurons were infected with control and Sertad1 shRNA viruses (multiplicity of infection, 10) at the time of plating. 2 days after plating, cells were treated with 10 μM camptothecin for 16, 24, 36 and 48 hrs, fixed and stained with Hoechst 33258 (0.25 μg/ml). GFP-positive cells were counted based on nuclear integrity. Significance comparisons with camptothecin-treated controls, *p
    Mouse Oligonucleotides Primers, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Illumina Inc mouse oligonucleotides
    <t>Sertad1</t> downregulation by adenoviral shRNAs protects cortical neurons from DNA damage-evoked cell death. (A,B) <t>shRNA-mediated</t> knockdown of Sertad1 in cortical neurons. Cortical neurons were infected with two adenoviral shRNAs targeting mouse Sertad1 gene or a control virus at the time of plating, and 24 hrs after infection cells were treated with camptothecin for 16 hrs to induce Sertad1 expression. Cells were fixed and immunostaining was performed using Sertad1 chicken IgY antibody. (A) Both Sertad1 shRNA viruses repress the upregulation of Sertad1 in cortical neurons treated with camptothecin (Arrows indicate Sertad1 shRNA-expressing cells). (B) Quantification of fluorescence intensity of Sertad1-immunostained neurons infected with control and Sertad1 shRNA viruses. Values are mean ± SEM (n = 30–35). (C) Expression of two Sertad1 shRNAs diminishes death of primary cultured cortical neurons treated with camptothecin. Cortical neurons were infected with control and Sertad1 shRNA viruses (multiplicity of infection, 10) at the time of plating. 2 days after plating, cells were treated with 10 μM camptothecin for 16, 24, 36 and 48 hrs, fixed and stained with Hoechst 33258 (0.25 μg/ml). GFP-positive cells were counted based on nuclear integrity. Significance comparisons with camptothecin-treated controls, *p
    Mouse Oligonucleotides, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sirna oligos against mouse snail
    <t>cGAS</t> is required for IFNβ expression in HeLa cells infected with C. muridarum or C. trachomatis (D and L2) HeLa cells were transfected with non-targeting <t>siRNA</t> (NT), 2 different siRNA for cGAS (cGAS1 and cGAS2), or a siRNA for STING as described in Methods. Significant knock down of STING (99%) and cGAS (90%) mRNA was achieved. Cells were infected with 1 MOI of C. muridarum (C.M) or 5 MOI of C.trachomatis -serovar D (C.T-D) or C. trachomatis L2 (C.T-L2). Cells were harvested at 24 h p.i and analyzed for expression of IFNβ (A), IFN λ (D), IL-8 (G), and chlamydial 16S rRNA (H). In parallel, cells were transfected with ISD (positive control) or poly IC-LyoVec (negative control) and harvested at 6 h post transfection and analyzed for expression of IFNβ (B, C), IFNλ (E, F). Culture supernatants from infected or transfected cells were collected at 24 h and CXCL10 protein levels assayed by ELISA (I). Mean ± SD of samples from 3 independent experiments are shown for ELISA. A representative western blot showing the levels of STING and cGAS following siRNA knock down in uninfected HeLa cells (J). A representative of five independent experiments is presented in A–H for qRT-PCR data and error bars represent range in technical replicates. Statistical significance for qPCR data between multiple experiments was determined by one way ANOVA with multiple comparison tests on the percent decrease in IFNβ levels for the siRNA used relative to NT siRNA in each experiment (K). UT=Un-treated.
    Sirna Oligos Against Mouse Snail, 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
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    86
    Horizon Discovery oligos against mouse ripk
    <t>cGAS</t> is required for IFNβ expression in HeLa cells infected with C. muridarum or C. trachomatis (D and L2) HeLa cells were transfected with non-targeting <t>siRNA</t> (NT), 2 different siRNA for cGAS (cGAS1 and cGAS2), or a siRNA for STING as described in Methods. Significant knock down of STING (99%) and cGAS (90%) mRNA was achieved. Cells were infected with 1 MOI of C. muridarum (C.M) or 5 MOI of C.trachomatis -serovar D (C.T-D) or C. trachomatis L2 (C.T-L2). Cells were harvested at 24 h p.i and analyzed for expression of IFNβ (A), IFN λ (D), IL-8 (G), and chlamydial 16S rRNA (H). In parallel, cells were transfected with ISD (positive control) or poly IC-LyoVec (negative control) and harvested at 6 h post transfection and analyzed for expression of IFNβ (B, C), IFNλ (E, F). Culture supernatants from infected or transfected cells were collected at 24 h and CXCL10 protein levels assayed by ELISA (I). Mean ± SD of samples from 3 independent experiments are shown for ELISA. A representative western blot showing the levels of STING and cGAS following siRNA knock down in uninfected HeLa cells (J). A representative of five independent experiments is presented in A–H for qRT-PCR data and error bars represent range in technical replicates. Statistical significance for qPCR data between multiple experiments was determined by one way ANOVA with multiple comparison tests on the percent decrease in IFNβ levels for the siRNA used relative to NT siRNA in each experiment (K). UT=Un-treated.
    Oligos Against Mouse Ripk, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher mouse trpv4 specific sirna oligonucleotides
    PDD-induced <t>TRPV4</t> activation triggered Ca 2+ influx. ( a ) Intracellular Ca 2+ measurement in control or Trpv4 knocked-down 4T07 cells treated with 10 μ m of 4α-PDD or DMSO. Luc -DMSO=cells transfected with Luc control <t>siRNA</t> and treated with DMSO; Luc -PDD, cells transfected with Luc control siRNA and treated with 4α-PDD; S1-DMSO, cells transfected with Trpv4 siRNA (sequence 1) and treated with DMSO; S1-PDD, cells transfected with Trpv4 siRNA (sequence 1) and treated with 4α-PDD; S3-DMSO, cells transfected with Trpv4 siRNA (sequence 3) and treated with DMSO; S3-PDD, cells transfected with TRPV4 siRNA (sequence 3) and treated with 4α-PDD. ( b ) Effects of chelating intracellular (by BAPTA-AM) and extracellular (by EGTA) Ca 2+ on 4α-PDD-induced, TRPV4-mediated signal transduction. 4T07 cells were untreated or pre-treated with 20 μ m of BAPTA-AM or 2 m m of EGTA for 1 h before being stimulated with 4α-PDD for 15 min (to assay for AKT activation) or 16 h (to assay for FAK activation and effects on E-cadherin and β-catenin). Cell lysates were then subject to immunoblotting using the indicated antibodies and bands densitometry performed using the ‘Image J’ software. The fold induction (ratio) was then obtained by dividing the values at PDD 15 min or PDD16 h over DMSO. Data points represent mean±s.e.m. of three independent experiments, * P
    Mouse Trpv4 Specific Sirna Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Horizon Discovery mouse tdp 43 sigenome smartpool oligos
    PDD-induced <t>TRPV4</t> activation triggered Ca 2+ influx. ( a ) Intracellular Ca 2+ measurement in control or Trpv4 knocked-down 4T07 cells treated with 10 μ m of 4α-PDD or DMSO. Luc -DMSO=cells transfected with Luc control <t>siRNA</t> and treated with DMSO; Luc -PDD, cells transfected with Luc control siRNA and treated with 4α-PDD; S1-DMSO, cells transfected with Trpv4 siRNA (sequence 1) and treated with DMSO; S1-PDD, cells transfected with Trpv4 siRNA (sequence 1) and treated with 4α-PDD; S3-DMSO, cells transfected with Trpv4 siRNA (sequence 3) and treated with DMSO; S3-PDD, cells transfected with TRPV4 siRNA (sequence 3) and treated with 4α-PDD. ( b ) Effects of chelating intracellular (by BAPTA-AM) and extracellular (by EGTA) Ca 2+ on 4α-PDD-induced, TRPV4-mediated signal transduction. 4T07 cells were untreated or pre-treated with 20 μ m of BAPTA-AM or 2 m m of EGTA for 1 h before being stimulated with 4α-PDD for 15 min (to assay for AKT activation) or 16 h (to assay for FAK activation and effects on E-cadherin and β-catenin). Cell lysates were then subject to immunoblotting using the indicated antibodies and bands densitometry performed using the ‘Image J’ software. The fold induction (ratio) was then obtained by dividing the values at PDD 15 min or PDD16 h over DMSO. Data points represent mean±s.e.m. of three independent experiments, * P
    Mouse Tdp 43 Sigenome Smartpool Oligos, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Integrated DNA Technologies ssdna ultramer oligonucleotides
    PDD-induced <t>TRPV4</t> activation triggered Ca 2+ influx. ( a ) Intracellular Ca 2+ measurement in control or Trpv4 knocked-down 4T07 cells treated with 10 μ m of 4α-PDD or DMSO. Luc -DMSO=cells transfected with Luc control <t>siRNA</t> and treated with DMSO; Luc -PDD, cells transfected with Luc control siRNA and treated with 4α-PDD; S1-DMSO, cells transfected with Trpv4 siRNA (sequence 1) and treated with DMSO; S1-PDD, cells transfected with Trpv4 siRNA (sequence 1) and treated with 4α-PDD; S3-DMSO, cells transfected with Trpv4 siRNA (sequence 3) and treated with DMSO; S3-PDD, cells transfected with TRPV4 siRNA (sequence 3) and treated with 4α-PDD. ( b ) Effects of chelating intracellular (by BAPTA-AM) and extracellular (by EGTA) Ca 2+ on 4α-PDD-induced, TRPV4-mediated signal transduction. 4T07 cells were untreated or pre-treated with 20 μ m of BAPTA-AM or 2 m m of EGTA for 1 h before being stimulated with 4α-PDD for 15 min (to assay for AKT activation) or 16 h (to assay for FAK activation and effects on E-cadherin and β-catenin). Cell lysates were then subject to immunoblotting using the indicated antibodies and bands densitometry performed using the ‘Image J’ software. The fold induction (ratio) was then obtained by dividing the values at PDD 15 min or PDD16 h over DMSO. Data points represent mean±s.e.m. of three independent experiments, * P
    Ssdna Ultramer Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 96/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Sigma-Genosys complementary oligos
    PDD-induced <t>TRPV4</t> activation triggered Ca 2+ influx. ( a ) Intracellular Ca 2+ measurement in control or Trpv4 knocked-down 4T07 cells treated with 10 μ m of 4α-PDD or DMSO. Luc -DMSO=cells transfected with Luc control <t>siRNA</t> and treated with DMSO; Luc -PDD, cells transfected with Luc control siRNA and treated with 4α-PDD; S1-DMSO, cells transfected with Trpv4 siRNA (sequence 1) and treated with DMSO; S1-PDD, cells transfected with Trpv4 siRNA (sequence 1) and treated with 4α-PDD; S3-DMSO, cells transfected with Trpv4 siRNA (sequence 3) and treated with DMSO; S3-PDD, cells transfected with TRPV4 siRNA (sequence 3) and treated with 4α-PDD. ( b ) Effects of chelating intracellular (by BAPTA-AM) and extracellular (by EGTA) Ca 2+ on 4α-PDD-induced, TRPV4-mediated signal transduction. 4T07 cells were untreated or pre-treated with 20 μ m of BAPTA-AM or 2 m m of EGTA for 1 h before being stimulated with 4α-PDD for 15 min (to assay for AKT activation) or 16 h (to assay for FAK activation and effects on E-cadherin and β-catenin). Cell lysates were then subject to immunoblotting using the indicated antibodies and bands densitometry performed using the ‘Image J’ software. The fold induction (ratio) was then obtained by dividing the values at PDD 15 min or PDD16 h over DMSO. Data points represent mean±s.e.m. of three independent experiments, * P
    Complementary Oligos, supplied by Sigma-Genosys, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Horizon Discovery mouse evi1 small interfering rna sirna oligonucleotides
    <t>EVI1</t> negatively regulates bacteria-induced NF-κB activation via inhibition of p65 acetylation at lysine 310 (A B) A549 cells co-transfected with EVI1 and p65 were treated with NTHi, and the cell lysates were immnoprecipitated with anti-p65 antibody and analyzed by western blot analysis with the indicated antibodies.(C) A549 cells were co-transfected with NF-κB-luciferase reporter plasmid together with EVI1 <t>siRNA</t> and p65 WT or p65 K310R constructs, and NF-κB-dependent promoter activity was measured by luciferase assay.* p
    Mouse Evi1 Small Interfering Rna Sirna Oligonucleotides, 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
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    88
    Ribobio nc oligos
    Overexpression of <t>miR-29</t> in mdx muscles downregulates fibrotic genes . ( a ) Differentially expressed genes in mdx muscles injected with NC and miR-29 <t>oligos</t> as determined by mRNA-seq. X- and Y-axis represent the log2 based FPKM values for expressed genes in NC and miR-29 samples, respectively. ( b ) Over-represented GO terms by GO analysis of downregulated list of genes. BP, biological process; CC, cellular component; KEGG, Kyoto Encyclopedia of Genes and Genomes; SP_PIR, a database of protein super-family names. ( c ) Coverage plot showing a 56-kb region encompassing the ACTA2 (α-SMA) gene on chromosome (Chr) 19; the gene structure is shown in blue below the graph. ( d ) Expressions of collagen 1A1 (Col 1A1), collagen 1A2 (Col 1A2), collagen 3A1 (Col 3A1), α-smooth muscle actin (α-SMA) or vimentin (VIM) in NC or miR-29 injected muscles. ( e ) Expressions of the above genes in TA muscles injected with negative control (Anti-NC) or miR-29 inhibitor oligos (Anti-miR-29). FPKM, fragments per kilobase of transcript per million fragments mapped; GO, gene ontology; mRNA, messenger RNA; NC, negative control; TA, tibialis anterior.
    Nc Oligos, supplied by Ribobio, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology sirna oligos against mouse yy1
    <t>YY1</t> negatively regulates miR-1 during CTX-induced muscle regeneration. (A) Tibialis anterior (TA) muscles from six-week old C57/BL6 background mice were injected with 10 µM cardiotoxin (CTX). RNAs and proteins were then extracted from injected muscles at the indicated days post-injection, and qRT-PCR was performed to measure the expression of miR-1 and miR-133, normalized to U6. Expression folds are shown with respect to day 0 where miR-1 and miR-133 levels were set to a value of 1. Quantitative values are represented as means ± S.D. (B) YY1 expression was measured by Western blotting. α-Tubulin was used as a loading control. Numbers below indicates the quantification by densitometry. (C) TA muscles from 6 week C57/BL6 background mice were injected CTX at day 0, followed by injection with siNC (left leg) and siYY1 <t>oligos</t> (right leg) 6 hours later. And re-injection of <t>siRNA</t> oligos was performed every other day for two more times. The injected muscles were harvested at the indicated days. N = 6 for each group. (D) Expressions of miR-1 and miR-133 were detected by qRT-PCR in CTX/siRNA injected muscles at day 2, 4 and 6, normalized to U6. Expression folds are shown with respect to siNC where miR-1 and miR-133 levels were set to a value of 1. (E) Western blotting was performed to analyze the expression of YY1, Pax7, MyoD and Myogenin. α-Tubulin was used as a loading control. Data is representative of 6 mice. (F) Expression of Pax7 and MyoD RNA levels were also detected by qRT-PCR normalized with GAPDH. Expression folds are shown with respect to siNC where Pax7 and MyoD levels were set to a value of 1. Quantitative values are represented as mean ± S.D. The p value was determined by Student's T-test: *p
    Sirna Oligos Against Mouse Yy1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Ionis Pharma asos antisense oligonucleotides
    Localization of <t>ASOs</t> in auditory hair cells at P30 after systemic treatment in Ush1c mice . Immunofluorescent labeling of ASO-29 (red) in HCs (green) at the a , b apex-mid turn at P30 after ASO treatment at P1 ( a ) or P5 ( b ). c Immunofluorescent image at the apex-middle turn of P5 vehicle-treated (saline), control littermate 1 week after P5 ASO treatment (P12). Scale bars indicate 10 μm. IHC, inner hair cell; OHC, outer hair cell; ASO, <t>antisense</t> oligonucleotide
    Asos Antisense Oligonucleotides, supplied by Ionis Pharma, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    OriGene sirna oligos against mouse ppargc1a
    Effects of SR-18292 on PGC-1 α acetylation and gluconeogenic genes in isolated primary hepatocytes . (A) SR-18292 is identical to C-80 except the methyl group on the 3′ position on the indole group. (B) Western blot analysis of PGC-1α acetylation following treatment with SR-18292 in the presence of the SIRT1 inhibitor, Ex527 (5μM) and the pan HDACs inhibitor TSA (1μM). Hepatocytes were infected with Ad-PGC-1α and Ad-GCN5 and treated with SR-18292 for 18 h. IP, immunoprecipitation. (C) – (D) qPCR analysis of <t>Ppargc1a</t> , Pck1 and G6pc mRNA expression levels following treatment with SR-18292 (20μM) in isolated primary hepatocytes. (E) qPCR analysis of Ppargc1a , Pck1 and G6pc mRNA expression levels following SR-18292 (20μM) treatment and <t>siRNA</t> to reduce to expression of Ppargc1a . Difference in % repression of Pck1 was calculated using two-way ANOVA with Sidak posttest. (F) – (G) Glucose production by primary hepatocytes following treatment with SR-18292 (20μM). Hepatocytes were treated with either glucagon (200nM) or infected with Ad-PGC-1α to induce glucose production. All data presented as mean +/− S.E.M. n=3, one-way ANOVA with Tuckey posttest. Representative of at least 2 independent experiments. *P
    Sirna Oligos Against Mouse Ppargc1a, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    SuperArray Bioscience Corporation mouse oligo gearrays
    Effects of SR-18292 on PGC-1 α acetylation and gluconeogenic genes in isolated primary hepatocytes . (A) SR-18292 is identical to C-80 except the methyl group on the 3′ position on the indole group. (B) Western blot analysis of PGC-1α acetylation following treatment with SR-18292 in the presence of the SIRT1 inhibitor, Ex527 (5μM) and the pan HDACs inhibitor TSA (1μM). Hepatocytes were infected with Ad-PGC-1α and Ad-GCN5 and treated with SR-18292 for 18 h. IP, immunoprecipitation. (C) – (D) qPCR analysis of <t>Ppargc1a</t> , Pck1 and G6pc mRNA expression levels following treatment with SR-18292 (20μM) in isolated primary hepatocytes. (E) qPCR analysis of Ppargc1a , Pck1 and G6pc mRNA expression levels following SR-18292 (20μM) treatment and <t>siRNA</t> to reduce to expression of Ppargc1a . Difference in % repression of Pck1 was calculated using two-way ANOVA with Sidak posttest. (F) – (G) Glucose production by primary hepatocytes following treatment with SR-18292 (20μM). Hepatocytes were treated with either glucagon (200nM) or infected with Ad-PGC-1α to induce glucose production. All data presented as mean +/− S.E.M. n=3, one-way ANOVA with Tuckey posttest. Representative of at least 2 independent experiments. *P
    Mouse Oligo Gearrays, supplied by SuperArray Bioscience Corporation, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Treatment with Antisense Oligonucleotide Targeting Ca v β 3 Improves [Ca 2+ ] i Dynamics and Insulin Secretion in ob/ob Islets (A) Left panel shows protein levels of Ca v β 3 in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. Right panel shows relative quantification of Ca v β 3 protein levels in left (n = 5; 40 islets in each case). (B and C) Effects of 11 mM glucose on [Ca 2+ ] i in ob/ob islets treated with scramble ASO (B) and Ca v β 3 ASO (C). Representative traces out of 30 are shown. (D) First peak ratios of glucose-induced [Ca 2+ ] i changes in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. (E) Oscillation periods of glucose-induced [Ca 2+ ] i changes in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. (F) Oscillation amplitudes of glucose-induced [Ca 2+ ] i changes in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. (G) Effects of 25 mM KCl on [Ca 2+ ] i in dissociated islet cells from scramble ASO (black) and Ca v β 3 -ASO-treated (red) ob/ob mice. Representative traces on dissociated islet cells are shown. (H) Peak ratios of [Ca 2+ ] i changes induced by 25 mM KCl in dissociated islet cells from scramble ASO (black) and Ca v β 3 -ASO-treated (red) ob/ob mice (n = 5; each experiment involved 50 single cells). (I) Peak ratios of [Ca 2+ ] i changes induced by 200 μM Cch in ob/ob-dissociated islet cells treated with scramble ASO and Ca v β 3 ASO (n = 5; each experiment involved 30 single cells). (J) Glucose-induced insulin release in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. The islets were treated with 3 mM or 11 mM glucose for 30 min (n = 5; 10 islets in each case). Data are presented as the means ± SEM; ∗ p

    Journal: Cell Reports

    Article Title: Blocking Ca2+ Channel β3 Subunit Reverses Diabetes

    doi: 10.1016/j.celrep.2018.06.086

    Figure Lengend Snippet: Treatment with Antisense Oligonucleotide Targeting Ca v β 3 Improves [Ca 2+ ] i Dynamics and Insulin Secretion in ob/ob Islets (A) Left panel shows protein levels of Ca v β 3 in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. Right panel shows relative quantification of Ca v β 3 protein levels in left (n = 5; 40 islets in each case). (B and C) Effects of 11 mM glucose on [Ca 2+ ] i in ob/ob islets treated with scramble ASO (B) and Ca v β 3 ASO (C). Representative traces out of 30 are shown. (D) First peak ratios of glucose-induced [Ca 2+ ] i changes in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. (E) Oscillation periods of glucose-induced [Ca 2+ ] i changes in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. (F) Oscillation amplitudes of glucose-induced [Ca 2+ ] i changes in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. (G) Effects of 25 mM KCl on [Ca 2+ ] i in dissociated islet cells from scramble ASO (black) and Ca v β 3 -ASO-treated (red) ob/ob mice. Representative traces on dissociated islet cells are shown. (H) Peak ratios of [Ca 2+ ] i changes induced by 25 mM KCl in dissociated islet cells from scramble ASO (black) and Ca v β 3 -ASO-treated (red) ob/ob mice (n = 5; each experiment involved 50 single cells). (I) Peak ratios of [Ca 2+ ] i changes induced by 200 μM Cch in ob/ob-dissociated islet cells treated with scramble ASO and Ca v β 3 ASO (n = 5; each experiment involved 30 single cells). (J) Glucose-induced insulin release in ob/ob islets treated with scramble ASO and Ca v β 3 ASO. The islets were treated with 3 mM or 11 mM glucose for 30 min (n = 5; 10 islets in each case). Data are presented as the means ± SEM; ∗ p

    Article Snippet: Antisense oligonucleotides, a series of chimeric 20-mer phosphorothioate oligonucleotides containing 2′-O-methoxyethyl groups at positions 1–5 and 16–20 targeted to mouse Cav β3 , were synthesized and purified (Integrated DNA Technologies, Coralville, IA, USA).

    Techniques: Allele-specific Oligonucleotide, Mouse Assay

    MLL4 regulates cell-cycle regulatory cyclins, p-protein and HOX genes. SW480 cells were transfected with MLL4 antisense for 48 h and the subjected to RNA extraction or ChIP assay. ( A ) RNA from MLL4-knocked down or control cells analysed by RT–PCR by using primers specific to selected cell-cycle regulatory genes ( cyclin A–E , p27 , HOXA5 and HOXB7 ) and GAPDH. GAPDH expression and rRNA levels were used as loading control. ( B ) ChIP assay: MLL4 and scramble antisense-treated and control cells were subjected to ChIP with MLL4, H3K4 tri-methyl, RNAPII and β -actin (control) antibodies. The immunoprecipitated DNA fragments were PCR amplified by using primers specific to the β -actin (ORF, as control) and promoters of cyclin B , cyclin D , p27 , HOXA5 and HOXB7 genes. The real-time quantification is on the bottom panel. Bars indicate standard errors ( n =3).

    Journal: British Journal of Cancer

    Article Title: Mixed lineage leukaemia-4 regulates cell-cycle progression and cell viability and its depletion suppresses growth of xenografted tumour in vivo

    doi: 10.1038/bjc.2012.263

    Figure Lengend Snippet: MLL4 regulates cell-cycle regulatory cyclins, p-protein and HOX genes. SW480 cells were transfected with MLL4 antisense for 48 h and the subjected to RNA extraction or ChIP assay. ( A ) RNA from MLL4-knocked down or control cells analysed by RT–PCR by using primers specific to selected cell-cycle regulatory genes ( cyclin A–E , p27 , HOXA5 and HOXB7 ) and GAPDH. GAPDH expression and rRNA levels were used as loading control. ( B ) ChIP assay: MLL4 and scramble antisense-treated and control cells were subjected to ChIP with MLL4, H3K4 tri-methyl, RNAPII and β -actin (control) antibodies. The immunoprecipitated DNA fragments were PCR amplified by using primers specific to the β -actin (ORF, as control) and promoters of cyclin B , cyclin D , p27 , HOXA5 and HOXB7 genes. The real-time quantification is on the bottom panel. Bars indicate standard errors ( n =3).

    Article Snippet: Two antisense oligonucleotide sequences for MLL4 (MLL4-A1: 5′-CCTTCTCTTCTCCCTCCTTGT-3′ MLL4-A2: 5′-CTTCCTTCTCTTCTCCCTCCT-3′) corresponding to +1123–1144, and +1126–1147 nt of MLL4 open reading frame (ORF) were designed and commercially synthesised (IDT-DNA).

    Techniques: Transfection, RNA Extraction, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunoprecipitation, Polymerase Chain Reaction, Amplification

    Regression of colon cancer xenograft by MLL4 antisense. SW480 cells were subcutaneously injected on the right hinge region of 6-week-old athymic nude (nu/nu) mice. Once the tumour reached about 32 mm 2 of cross-sectional area, mice were intraperitoneally administered with MLL4 antisense (300 μ g per 20 g body weight) on the left hinge region at 4 days interval for 4 weeks. Control mice were administered with either PBS (diluents) or a same doze of scramble antisense (with no homology with MLL4). Tumour sizes were measured using a slid caliper at every 2 days intervals. ( A ) Cross-sectional area of the tumours was plotted against time. Bars indicated standard errors. ( B ) Representative pictures of the control and antisense-treated mice at different stages. ( C ) Tumour xenografts excised at 28th day of treatment. The cross-section of tumours showing internal issue is in bottom panel. ( D ) The RNA extract of the excised tumour was analysed by RT–PCR with primer specific to MLL4 and MLL1 (specificity control). rRNA was used as quantitative control. The real-time quantification is on the right panel. Bars indicate standard errors ( n =3). ( E ) The western blot of the protein extract of the excised tumour was done with MLL4 and β -actin (control) antibody.

    Journal: British Journal of Cancer

    Article Title: Mixed lineage leukaemia-4 regulates cell-cycle progression and cell viability and its depletion suppresses growth of xenografted tumour in vivo

    doi: 10.1038/bjc.2012.263

    Figure Lengend Snippet: Regression of colon cancer xenograft by MLL4 antisense. SW480 cells were subcutaneously injected on the right hinge region of 6-week-old athymic nude (nu/nu) mice. Once the tumour reached about 32 mm 2 of cross-sectional area, mice were intraperitoneally administered with MLL4 antisense (300 μ g per 20 g body weight) on the left hinge region at 4 days interval for 4 weeks. Control mice were administered with either PBS (diluents) or a same doze of scramble antisense (with no homology with MLL4). Tumour sizes were measured using a slid caliper at every 2 days intervals. ( A ) Cross-sectional area of the tumours was plotted against time. Bars indicated standard errors. ( B ) Representative pictures of the control and antisense-treated mice at different stages. ( C ) Tumour xenografts excised at 28th day of treatment. The cross-section of tumours showing internal issue is in bottom panel. ( D ) The RNA extract of the excised tumour was analysed by RT–PCR with primer specific to MLL4 and MLL1 (specificity control). rRNA was used as quantitative control. The real-time quantification is on the right panel. Bars indicate standard errors ( n =3). ( E ) The western blot of the protein extract of the excised tumour was done with MLL4 and β -actin (control) antibody.

    Article Snippet: Two antisense oligonucleotide sequences for MLL4 (MLL4-A1: 5′-CCTTCTCTTCTCCCTCCTTGT-3′ MLL4-A2: 5′-CTTCCTTCTCTTCTCCCTCCT-3′) corresponding to +1123–1144, and +1126–1147 nt of MLL4 open reading frame (ORF) were designed and commercially synthesised (IDT-DNA).

    Techniques: Injection, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

    MLL4 knockdown affects nuclear integrity and expression of growth and angiogenic factors in xenografted tumour. The MLL4 antisense-treated or control mice with colon cancer xenograft were either euthanised to excise the tumour xenograft or perfused with 4% paraformaldehyde at 28th day of treatment. ( A ) Immunolocalisation of MLL4 and nuclear integrity: Formaldehyde-fixed tumours were sectioned, immunostained with MLL4 antibody and DAPI (for nuclear staining) and analysed under fluorescence microscope. Representative images showing the cellular morphology (DIC), nuclear integrity (DAPI) and MLL4 expression in the control and MLL4 antisense-treated tumours are shown. ( B ) MLL4 regulates expression of growth and angiogenic factors in vivo : RT–PCR analysis of the RNA extracted from MLL4 antisense-treated tumour tissues by using primers specific to MLL4, VEGF, bFGF, TGF β 2, CD31, HIF1 α and β -actin (control). The real-time quantification (relative to GAPDH) is shown in right panel. Bars indicate standard error. ( C ) The tumour tissue was analysed by ChIP assay using MLL4, H3K4-trimethyl and RNAP II antibodies. The ChIP DNA was analysed by PCR with primers specific to the promoter regions of CD31, TGF β 2 and NRG1. qPCR data are the right panel.

    Journal: British Journal of Cancer

    Article Title: Mixed lineage leukaemia-4 regulates cell-cycle progression and cell viability and its depletion suppresses growth of xenografted tumour in vivo

    doi: 10.1038/bjc.2012.263

    Figure Lengend Snippet: MLL4 knockdown affects nuclear integrity and expression of growth and angiogenic factors in xenografted tumour. The MLL4 antisense-treated or control mice with colon cancer xenograft were either euthanised to excise the tumour xenograft or perfused with 4% paraformaldehyde at 28th day of treatment. ( A ) Immunolocalisation of MLL4 and nuclear integrity: Formaldehyde-fixed tumours were sectioned, immunostained with MLL4 antibody and DAPI (for nuclear staining) and analysed under fluorescence microscope. Representative images showing the cellular morphology (DIC), nuclear integrity (DAPI) and MLL4 expression in the control and MLL4 antisense-treated tumours are shown. ( B ) MLL4 regulates expression of growth and angiogenic factors in vivo : RT–PCR analysis of the RNA extracted from MLL4 antisense-treated tumour tissues by using primers specific to MLL4, VEGF, bFGF, TGF β 2, CD31, HIF1 α and β -actin (control). The real-time quantification (relative to GAPDH) is shown in right panel. Bars indicate standard error. ( C ) The tumour tissue was analysed by ChIP assay using MLL4, H3K4-trimethyl and RNAP II antibodies. The ChIP DNA was analysed by PCR with primers specific to the promoter regions of CD31, TGF β 2 and NRG1. qPCR data are the right panel.

    Article Snippet: Two antisense oligonucleotide sequences for MLL4 (MLL4-A1: 5′-CCTTCTCTTCTCCCTCCTTGT-3′ MLL4-A2: 5′-CTTCCTTCTCTTCTCCCTCCT-3′) corresponding to +1123–1144, and +1126–1147 nt of MLL4 open reading frame (ORF) were designed and commercially synthesised (IDT-DNA).

    Techniques: Expressing, Mouse Assay, Staining, Fluorescence, Microscopy, In Vivo, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    MLL4 knockdown and its effect on cell viability. ( A ) Effect of MLL4 knockdown on the expression of MLL1–4: colon cancer (SW480) cells were transfected with varying concentrations of MLL4 antisense for 48 h. RNA was reverse transcribed and subjected to real-time PCR analysis using primers specific to MLL1, MLL2, MLL3, MLL4 and GAPDH. The expression of different MLLs relative to GAPDH was plotted. GAPDH expression and rRNA levels were used as loading control. Each experiment was performed in three replicates for at least two times. Bars indicate standard errors. ( B ) RNA from the control and MLL4 antisense-treated cells were subjected to RT–PCR using primer specific to MLL4, MLL1 (specificity control) and GAPDH (loading control) and then analysed by agarose gel electrophoresis. Lane 1: control cells; lane 2: cells transfected with scramble antisense; lanes 3–5: cell transfected with 3–7 μ g of MLL4 antisense. ( C ) Western blotting: proteins from the control and MLL4 antisense-treated cells were subjected to western blotting using anti-MLL4 and anti- β -actin antibodies (loading control). ( D ) Microscopic images showing different cancer and non-cancer cells transfected with MLL4 or scramble antisense for 48 h. Control cells were treated with PBS (buffer) alone. ( E ) MTT assay: cells were transfected with 7 μ g MLL4 or scramble antisense for 48 h and then subjected to MTT assay. The relative (%) cell viability (MLL4 antisense vs scramble antisense) was plotted for different cell lines. Bars indicate standard errors.

    Journal: British Journal of Cancer

    Article Title: Mixed lineage leukaemia-4 regulates cell-cycle progression and cell viability and its depletion suppresses growth of xenografted tumour in vivo

    doi: 10.1038/bjc.2012.263

    Figure Lengend Snippet: MLL4 knockdown and its effect on cell viability. ( A ) Effect of MLL4 knockdown on the expression of MLL1–4: colon cancer (SW480) cells were transfected with varying concentrations of MLL4 antisense for 48 h. RNA was reverse transcribed and subjected to real-time PCR analysis using primers specific to MLL1, MLL2, MLL3, MLL4 and GAPDH. The expression of different MLLs relative to GAPDH was plotted. GAPDH expression and rRNA levels were used as loading control. Each experiment was performed in three replicates for at least two times. Bars indicate standard errors. ( B ) RNA from the control and MLL4 antisense-treated cells were subjected to RT–PCR using primer specific to MLL4, MLL1 (specificity control) and GAPDH (loading control) and then analysed by agarose gel electrophoresis. Lane 1: control cells; lane 2: cells transfected with scramble antisense; lanes 3–5: cell transfected with 3–7 μ g of MLL4 antisense. ( C ) Western blotting: proteins from the control and MLL4 antisense-treated cells were subjected to western blotting using anti-MLL4 and anti- β -actin antibodies (loading control). ( D ) Microscopic images showing different cancer and non-cancer cells transfected with MLL4 or scramble antisense for 48 h. Control cells were treated with PBS (buffer) alone. ( E ) MTT assay: cells were transfected with 7 μ g MLL4 or scramble antisense for 48 h and then subjected to MTT assay. The relative (%) cell viability (MLL4 antisense vs scramble antisense) was plotted for different cell lines. Bars indicate standard errors.

    Article Snippet: Two antisense oligonucleotide sequences for MLL4 (MLL4-A1: 5′-CCTTCTCTTCTCCCTCCTTGT-3′ MLL4-A2: 5′-CTTCCTTCTCTTCTCCCTCCT-3′) corresponding to +1123–1144, and +1126–1147 nt of MLL4 open reading frame (ORF) were designed and commercially synthesised (IDT-DNA).

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, MTT Assay

    MLL4 knockdown induced apoptosis in colon cancer cell. SW480 cells were transfected with MLL4 antisense or scramble for 48 h then subjected to different analysis. ( A ) FACS analysis: MLL4 and scramble antisense-transfected cell was fixed, stained with propidium iodide (PI) and analysed by using Beckman Coulter Cytomics FC500 flow cytometry analyser. Percent cell populations at different stages of cell cycle are listed within the panels. ( B ) TUNEL assay. MLL4 antisense-treated and control cells were subjected to terminal nicked end-labelling using fluorescent dUTP, stained with DAPI (nuclear staining, blue fluorescence) and PI (PI that stains nucleus of dead cells, red colour) and analysed under fluorescence microscope. dUTP-stained green speckles represent apoptotic cells with fragmented nuclei. ( C ) Immunostaining of cytochrome-c: MLL4 antisense-treated and control cells were immunostained with cytochrome- c antibody followed by FITC-labelled secondary antibody, stained with DAPI and visualised under fluorescence microscope. ( D ) Caspase analysis: MLL4 antisense (or scramble antisense) cell lysates were analysed for caspase activity using Caspase-3/7 assay kit. Bars indicated standard error.

    Journal: British Journal of Cancer

    Article Title: Mixed lineage leukaemia-4 regulates cell-cycle progression and cell viability and its depletion suppresses growth of xenografted tumour in vivo

    doi: 10.1038/bjc.2012.263

    Figure Lengend Snippet: MLL4 knockdown induced apoptosis in colon cancer cell. SW480 cells were transfected with MLL4 antisense or scramble for 48 h then subjected to different analysis. ( A ) FACS analysis: MLL4 and scramble antisense-transfected cell was fixed, stained with propidium iodide (PI) and analysed by using Beckman Coulter Cytomics FC500 flow cytometry analyser. Percent cell populations at different stages of cell cycle are listed within the panels. ( B ) TUNEL assay. MLL4 antisense-treated and control cells were subjected to terminal nicked end-labelling using fluorescent dUTP, stained with DAPI (nuclear staining, blue fluorescence) and PI (PI that stains nucleus of dead cells, red colour) and analysed under fluorescence microscope. dUTP-stained green speckles represent apoptotic cells with fragmented nuclei. ( C ) Immunostaining of cytochrome-c: MLL4 antisense-treated and control cells were immunostained with cytochrome- c antibody followed by FITC-labelled secondary antibody, stained with DAPI and visualised under fluorescence microscope. ( D ) Caspase analysis: MLL4 antisense (or scramble antisense) cell lysates were analysed for caspase activity using Caspase-3/7 assay kit. Bars indicated standard error.

    Article Snippet: Two antisense oligonucleotide sequences for MLL4 (MLL4-A1: 5′-CCTTCTCTTCTCCCTCCTTGT-3′ MLL4-A2: 5′-CTTCCTTCTCTTCTCCCTCCT-3′) corresponding to +1123–1144, and +1126–1147 nt of MLL4 open reading frame (ORF) were designed and commercially synthesised (IDT-DNA).

    Techniques: Transfection, FACS, Staining, Flow Cytometry, Cytometry, TUNEL Assay, Fluorescence, Microscopy, Immunostaining, Activity Assay

    Sertad1 downregulation by adenoviral shRNAs protects cortical neurons from DNA damage-evoked cell death. (A,B) shRNA-mediated knockdown of Sertad1 in cortical neurons. Cortical neurons were infected with two adenoviral shRNAs targeting mouse Sertad1 gene or a control virus at the time of plating, and 24 hrs after infection cells were treated with camptothecin for 16 hrs to induce Sertad1 expression. Cells were fixed and immunostaining was performed using Sertad1 chicken IgY antibody. (A) Both Sertad1 shRNA viruses repress the upregulation of Sertad1 in cortical neurons treated with camptothecin (Arrows indicate Sertad1 shRNA-expressing cells). (B) Quantification of fluorescence intensity of Sertad1-immunostained neurons infected with control and Sertad1 shRNA viruses. Values are mean ± SEM (n = 30–35). (C) Expression of two Sertad1 shRNAs diminishes death of primary cultured cortical neurons treated with camptothecin. Cortical neurons were infected with control and Sertad1 shRNA viruses (multiplicity of infection, 10) at the time of plating. 2 days after plating, cells were treated with 10 μM camptothecin for 16, 24, 36 and 48 hrs, fixed and stained with Hoechst 33258 (0.25 μg/ml). GFP-positive cells were counted based on nuclear integrity. Significance comparisons with camptothecin-treated controls, *p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: SERTAD1 PLAYS AN ESSENTIAL ROLE IN DEVELOPMENTAL AND PATHOLOGICAL NEURON DEATH

    doi: 10.1523/JNEUROSCI.6421-09.2010

    Figure Lengend Snippet: Sertad1 downregulation by adenoviral shRNAs protects cortical neurons from DNA damage-evoked cell death. (A,B) shRNA-mediated knockdown of Sertad1 in cortical neurons. Cortical neurons were infected with two adenoviral shRNAs targeting mouse Sertad1 gene or a control virus at the time of plating, and 24 hrs after infection cells were treated with camptothecin for 16 hrs to induce Sertad1 expression. Cells were fixed and immunostaining was performed using Sertad1 chicken IgY antibody. (A) Both Sertad1 shRNA viruses repress the upregulation of Sertad1 in cortical neurons treated with camptothecin (Arrows indicate Sertad1 shRNA-expressing cells). (B) Quantification of fluorescence intensity of Sertad1-immunostained neurons infected with control and Sertad1 shRNA viruses. Values are mean ± SEM (n = 30–35). (C) Expression of two Sertad1 shRNAs diminishes death of primary cultured cortical neurons treated with camptothecin. Cortical neurons were infected with control and Sertad1 shRNA viruses (multiplicity of infection, 10) at the time of plating. 2 days after plating, cells were treated with 10 μM camptothecin for 16, 24, 36 and 48 hrs, fixed and stained with Hoechst 33258 (0.25 μg/ml). GFP-positive cells were counted based on nuclear integrity. Significance comparisons with camptothecin-treated controls, *p

    Article Snippet: Mouse Sertad1 specific shRNA oligos (Applied Biosystems Inc, Foster City, CA) were cloned into pSilencer3.0-H1 vector (Applied Biosystems Inc, Foster City, CA).

    Techniques: shRNA, Infection, Expressing, Immunostaining, Fluorescence, Cell Culture, Staining

    shRNAs targeted to Sertad1 block cortical neuron death in vivo during development. (A) Distribution of transfected neurons in cortex of the electroporated brain. GFP expressing DNA constructs of control shRNA or Sertad1 shRNA #4 were electroporated into the left lateral ventricles of E16 rat embryos which were subsequently harvested at p5. The brains were fixed and subjected to staining for eGFP expression. Morphologically identified eGFP expressing neurons are mostly present in cortical layers II, III, and IV of the electroporated side of the brain in each case. (B) Location of TUNEL positive eGFP expressing neurons in cortices of brains electroporated with control and Sertad1 shRNA. Note that TUNEL positive eGFP expressing neurons are encircled in each case. (C) Sertad1 shRNA inhibits developmental cell death of cortical neurons. The cortices of animals electroporated with control and Sertad1 shRNAs were evaluated for proportions of transfected cells (eGFP positive) that show TUNEL staining. Data represent means (SEM of 3 animals, each from an independent electroporation. Approximately 2000–3000 cells were evaluated per animal. Asterisks denote statistically significant differences between the two mean values * p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: SERTAD1 PLAYS AN ESSENTIAL ROLE IN DEVELOPMENTAL AND PATHOLOGICAL NEURON DEATH

    doi: 10.1523/JNEUROSCI.6421-09.2010

    Figure Lengend Snippet: shRNAs targeted to Sertad1 block cortical neuron death in vivo during development. (A) Distribution of transfected neurons in cortex of the electroporated brain. GFP expressing DNA constructs of control shRNA or Sertad1 shRNA #4 were electroporated into the left lateral ventricles of E16 rat embryos which were subsequently harvested at p5. The brains were fixed and subjected to staining for eGFP expression. Morphologically identified eGFP expressing neurons are mostly present in cortical layers II, III, and IV of the electroporated side of the brain in each case. (B) Location of TUNEL positive eGFP expressing neurons in cortices of brains electroporated with control and Sertad1 shRNA. Note that TUNEL positive eGFP expressing neurons are encircled in each case. (C) Sertad1 shRNA inhibits developmental cell death of cortical neurons. The cortices of animals electroporated with control and Sertad1 shRNAs were evaluated for proportions of transfected cells (eGFP positive) that show TUNEL staining. Data represent means (SEM of 3 animals, each from an independent electroporation. Approximately 2000–3000 cells were evaluated per animal. Asterisks denote statistically significant differences between the two mean values * p

    Article Snippet: Mouse Sertad1 specific shRNA oligos (Applied Biosystems Inc, Foster City, CA) were cloned into pSilencer3.0-H1 vector (Applied Biosystems Inc, Foster City, CA).

    Techniques: Blocking Assay, In Vivo, Transfection, Expressing, Construct, shRNA, Staining, TUNEL Assay, Electroporation

    shRNAs targeted to Sertad1 specifically block gene expression and neuron death in response to NGF deprivation. (A) Sertad1 shRNAs suppress Sertad1 expression. HEK 293 cells were co-transfected with pCDNA-Sertad1-V5 along with pSIREN-Luc (Luciferase)-Zsgreen shRNA, or pSIREN-Sertad1-Zsgreen shRNAs. After 48 hrs, cells were lysed and subjected to western immunoblotting using enhanced chemiluminescence for the detection of V5 and Zsgreen. (B) Sertad1 knockdown prevents neuronal degeneration and death after NGF deprivation. Neuronal PC12 cells were transfected with pSIREN-Random-Zsgreen shRNA (Control ShRNA) or pSIREN-Sertad1#5-ZsGreen shRNA (Sertad1 shRNA) maintained for 48h, then washed twice with RMPI 1640 medium and maintained with or without NGF. Representative images of transfected cells were taken in each case before and after 24 and 48 hrs of treatment. (C,D) Sertad1 shRNAs provide protection against death evoked by NGF deprivation of neuronal PC12 cells or SCG neurons. Neuronal cells were transfected with pSIREN-Luc-Zsgreen shRNA (shLuc), pSIREN-Sertad1-ZsGreen shRNA (shRnad) or pSIREN-Sertad1-ZsGreen shRNAs (shSertad1#2, shSertad1#4 and shSertad1#5), maintained for 48h and then deprived of NGF as indicated. Numbers of surviving transfected (green) cells were counted just before NGF deprivation and after 24 and 48 hrs of NGF deprivation. Data are from 1 of 3 independent experiments, each with comparable results and are shown as means ± SEM, performed in triplicates. Asterisks denote statistically significant differences from Control (shLuc or shRand): * p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: SERTAD1 PLAYS AN ESSENTIAL ROLE IN DEVELOPMENTAL AND PATHOLOGICAL NEURON DEATH

    doi: 10.1523/JNEUROSCI.6421-09.2010

    Figure Lengend Snippet: shRNAs targeted to Sertad1 specifically block gene expression and neuron death in response to NGF deprivation. (A) Sertad1 shRNAs suppress Sertad1 expression. HEK 293 cells were co-transfected with pCDNA-Sertad1-V5 along with pSIREN-Luc (Luciferase)-Zsgreen shRNA, or pSIREN-Sertad1-Zsgreen shRNAs. After 48 hrs, cells were lysed and subjected to western immunoblotting using enhanced chemiluminescence for the detection of V5 and Zsgreen. (B) Sertad1 knockdown prevents neuronal degeneration and death after NGF deprivation. Neuronal PC12 cells were transfected with pSIREN-Random-Zsgreen shRNA (Control ShRNA) or pSIREN-Sertad1#5-ZsGreen shRNA (Sertad1 shRNA) maintained for 48h, then washed twice with RMPI 1640 medium and maintained with or without NGF. Representative images of transfected cells were taken in each case before and after 24 and 48 hrs of treatment. (C,D) Sertad1 shRNAs provide protection against death evoked by NGF deprivation of neuronal PC12 cells or SCG neurons. Neuronal cells were transfected with pSIREN-Luc-Zsgreen shRNA (shLuc), pSIREN-Sertad1-ZsGreen shRNA (shRnad) or pSIREN-Sertad1-ZsGreen shRNAs (shSertad1#2, shSertad1#4 and shSertad1#5), maintained for 48h and then deprived of NGF as indicated. Numbers of surviving transfected (green) cells were counted just before NGF deprivation and after 24 and 48 hrs of NGF deprivation. Data are from 1 of 3 independent experiments, each with comparable results and are shown as means ± SEM, performed in triplicates. Asterisks denote statistically significant differences from Control (shLuc or shRand): * p

    Article Snippet: Mouse Sertad1 specific shRNA oligos (Applied Biosystems Inc, Foster City, CA) were cloned into pSilencer3.0-H1 vector (Applied Biosystems Inc, Foster City, CA).

    Techniques: Blocking Assay, Expressing, Transfection, Luciferase, shRNA, Western Blot

    Serum gonadotropin levels, ovarian morphology, and number of offspring in WT and CD36−/− mice. A . ELISA was used to quantify changes FSH and LH in serum collected from CD36 −/− and WT mice. CD36 −/− mice had significantly (p

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: The thrombospondin-1 receptor CD36 is an important mediator of ovarian angiogenesis and folliculogenesis

    doi: 10.1186/1477-7827-12-21

    Figure Lengend Snippet: Serum gonadotropin levels, ovarian morphology, and number of offspring in WT and CD36−/− mice. A . ELISA was used to quantify changes FSH and LH in serum collected from CD36 −/− and WT mice. CD36 −/− mice had significantly (p

    Article Snippet: In addition to the CD36 knockdown oligonucleotides, scrambled sequence oligos with similar G/C content (Life Technologies) were also used as recommended.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Expression of survival factors and the effects of the TSP-1 mimetic peptide ABT-898 in vitro. A . siRNA inhibition reduced expression of CD36 in spontaneously immortalized rat granulosa cells (SIGC) in vitro, as observed using brightfield/darkfield immunofluorescence overlay and Western blotting. B . Western blotting revealed that CD36 knockdown cells had increased expression of survival factors (VEGF, pVEGFR2, Bcl-2, and pAkt, compared to WT SIGC, or those transfected with a scrambled RNAi sequence. * - denotes statistically different (p

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: The thrombospondin-1 receptor CD36 is an important mediator of ovarian angiogenesis and folliculogenesis

    doi: 10.1186/1477-7827-12-21

    Figure Lengend Snippet: Expression of survival factors and the effects of the TSP-1 mimetic peptide ABT-898 in vitro. A . siRNA inhibition reduced expression of CD36 in spontaneously immortalized rat granulosa cells (SIGC) in vitro, as observed using brightfield/darkfield immunofluorescence overlay and Western blotting. B . Western blotting revealed that CD36 knockdown cells had increased expression of survival factors (VEGF, pVEGFR2, Bcl-2, and pAkt, compared to WT SIGC, or those transfected with a scrambled RNAi sequence. * - denotes statistically different (p

    Article Snippet: In addition to the CD36 knockdown oligonucleotides, scrambled sequence oligos with similar G/C content (Life Technologies) were also used as recommended.

    Techniques: Expressing, In Vitro, Inhibition, Immunofluorescence, Western Blot, Transfection, Sequencing

    Ovarian expression of VEGF, its receptor VEGFR-2, and TSP-1 in CD36−/− and WT mice. A . Ovaries from CD36−/− and WT mice were immunostained for VEGF, VEGFR-2 and TSP-1. B . Immunohistochemical analysis showed that ovaries from CD36−/− mice had higher (p

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: The thrombospondin-1 receptor CD36 is an important mediator of ovarian angiogenesis and folliculogenesis

    doi: 10.1186/1477-7827-12-21

    Figure Lengend Snippet: Ovarian expression of VEGF, its receptor VEGFR-2, and TSP-1 in CD36−/− and WT mice. A . Ovaries from CD36−/− and WT mice were immunostained for VEGF, VEGFR-2 and TSP-1. B . Immunohistochemical analysis showed that ovaries from CD36−/− mice had higher (p

    Article Snippet: In addition to the CD36 knockdown oligonucleotides, scrambled sequence oligos with similar G/C content (Life Technologies) were also used as recommended.

    Techniques: Expressing, Mouse Assay, Immunohistochemistry

    Cell proliferation and blood vessel density in ovaries from CD36−/− and WT mice. A . Immunohistochemistry for Ki67 showed that pre-antral and antral follicles from CD36−/− mice had a significant (p

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: The thrombospondin-1 receptor CD36 is an important mediator of ovarian angiogenesis and folliculogenesis

    doi: 10.1186/1477-7827-12-21

    Figure Lengend Snippet: Cell proliferation and blood vessel density in ovaries from CD36−/− and WT mice. A . Immunohistochemistry for Ki67 showed that pre-antral and antral follicles from CD36−/− mice had a significant (p

    Article Snippet: In addition to the CD36 knockdown oligonucleotides, scrambled sequence oligos with similar G/C content (Life Technologies) were also used as recommended.

    Techniques: Mouse Assay, Immunohistochemistry

    VASP regulates the interaction between β1 and talin. (A) Example FLIM images of β1-GFP–expressing fibroblasts cotransfected with mCherry-talin and either WT-VASP-myc or S153A VASP-myc. Cells were fixed and stained with an anti–myc-Cy5 antibody to detect VASP expression. Cells were then subjected to FRET analysis by FLIM. Images show the GFP multiphoton intensity image and (where appropriate) corresponding wide-field image of the acceptor and myc-tagged VASP. Lifetime images mapping spatial FRET across the cells are depicted using a pseudocolor scale (blue, normal lifetime; red, FRET). Bars, 10 µm. (B) Example FLIM images of β1-GFP–expressing fibroblasts cotransfected with mCherry-talin and either control siRNA (con) or siRNA directed against β3 integrin, RIAM, or VASP. Cells were fixed and subjected to FRET analysis by FLIM. Images show the GFP multiphoton intensity image and corresponding wide-field image of the talin acceptor. Lifetime images mapping spatial FRET across the cells are depicted using a pseudocolor scale (blue, normal lifetime; red, FRET). (C) Quantification of FRET efficiency from FLIM data in A and B. The graphs represent mean FRET efficiency of > 12 cells of each type over three independent experiments. Error bars indicate ± SEM. *, P

    Journal: The Journal of Cell Biology

    Article Title: ?v?3 integrin spatially regulates VASP and RIAM to control adhesion dynamics and migration

    doi: 10.1083/jcb.200912014

    Figure Lengend Snippet: VASP regulates the interaction between β1 and talin. (A) Example FLIM images of β1-GFP–expressing fibroblasts cotransfected with mCherry-talin and either WT-VASP-myc or S153A VASP-myc. Cells were fixed and stained with an anti–myc-Cy5 antibody to detect VASP expression. Cells were then subjected to FRET analysis by FLIM. Images show the GFP multiphoton intensity image and (where appropriate) corresponding wide-field image of the acceptor and myc-tagged VASP. Lifetime images mapping spatial FRET across the cells are depicted using a pseudocolor scale (blue, normal lifetime; red, FRET). Bars, 10 µm. (B) Example FLIM images of β1-GFP–expressing fibroblasts cotransfected with mCherry-talin and either control siRNA (con) or siRNA directed against β3 integrin, RIAM, or VASP. Cells were fixed and subjected to FRET analysis by FLIM. Images show the GFP multiphoton intensity image and corresponding wide-field image of the talin acceptor. Lifetime images mapping spatial FRET across the cells are depicted using a pseudocolor scale (blue, normal lifetime; red, FRET). (C) Quantification of FRET efficiency from FLIM data in A and B. The graphs represent mean FRET efficiency of > 12 cells of each type over three independent experiments. Error bars indicate ± SEM. *, P

    Article Snippet: Control and On-target siRNA oligos against mouse VASP and mouse or human β3 integrin were obtained from Thermo Fisher Scientific.

    Techniques: Expressing, Staining

    cGAS is required for IFNβ expression in HeLa cells infected with C. muridarum or C. trachomatis (D and L2) HeLa cells were transfected with non-targeting siRNA (NT), 2 different siRNA for cGAS (cGAS1 and cGAS2), or a siRNA for STING as described in Methods. Significant knock down of STING (99%) and cGAS (90%) mRNA was achieved. Cells were infected with 1 MOI of C. muridarum (C.M) or 5 MOI of C.trachomatis -serovar D (C.T-D) or C. trachomatis L2 (C.T-L2). Cells were harvested at 24 h p.i and analyzed for expression of IFNβ (A), IFN λ (D), IL-8 (G), and chlamydial 16S rRNA (H). In parallel, cells were transfected with ISD (positive control) or poly IC-LyoVec (negative control) and harvested at 6 h post transfection and analyzed for expression of IFNβ (B, C), IFNλ (E, F). Culture supernatants from infected or transfected cells were collected at 24 h and CXCL10 protein levels assayed by ELISA (I). Mean ± SD of samples from 3 independent experiments are shown for ELISA. A representative western blot showing the levels of STING and cGAS following siRNA knock down in uninfected HeLa cells (J). A representative of five independent experiments is presented in A–H for qRT-PCR data and error bars represent range in technical replicates. Statistical significance for qPCR data between multiple experiments was determined by one way ANOVA with multiple comparison tests on the percent decrease in IFNβ levels for the siRNA used relative to NT siRNA in each experiment (K). UT=Un-treated.

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

    Article Title: The DNA sensor, cyclic GMP-AMP synthase (cGAS) is essential for induction of IFN beta during Chlamydia trachomatis infection

    doi: 10.4049/jimmunol.1302718

    Figure Lengend Snippet: cGAS is required for IFNβ expression in HeLa cells infected with C. muridarum or C. trachomatis (D and L2) HeLa cells were transfected with non-targeting siRNA (NT), 2 different siRNA for cGAS (cGAS1 and cGAS2), or a siRNA for STING as described in Methods. Significant knock down of STING (99%) and cGAS (90%) mRNA was achieved. Cells were infected with 1 MOI of C. muridarum (C.M) or 5 MOI of C.trachomatis -serovar D (C.T-D) or C. trachomatis L2 (C.T-L2). Cells were harvested at 24 h p.i and analyzed for expression of IFNβ (A), IFN λ (D), IL-8 (G), and chlamydial 16S rRNA (H). In parallel, cells were transfected with ISD (positive control) or poly IC-LyoVec (negative control) and harvested at 6 h post transfection and analyzed for expression of IFNβ (B, C), IFNλ (E, F). Culture supernatants from infected or transfected cells were collected at 24 h and CXCL10 protein levels assayed by ELISA (I). Mean ± SD of samples from 3 independent experiments are shown for ELISA. A representative western blot showing the levels of STING and cGAS following siRNA knock down in uninfected HeLa cells (J). A representative of five independent experiments is presented in A–H for qRT-PCR data and error bars represent range in technical replicates. Statistical significance for qPCR data between multiple experiments was determined by one way ANOVA with multiple comparison tests on the percent decrease in IFNβ levels for the siRNA used relative to NT siRNA in each experiment (K). UT=Un-treated.

    Article Snippet: For J774 cells, two mouse cGAS (MB21D1) siRNA oligos (Cat# 2675, SIGMA), described earlier ( ) were used in parallel with the corresponding non-targeting siRNA from SIGMA (Cat# SIC001).

    Techniques: Expressing, Infection, Transfection, Positive Control, Negative Control, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    cGAS is required for IFNβ expression during Chlamydia spp. infection of mouse oviduct epithelial cells (BM1.11 cells) and human oviduct epithelial cells (OE-E6/E7) siRNA knock down in mouse BM1.11 cells were carried out using accell™ NT, mouse cGAS or STING siRNA pools. Seventy-two hours after transfection, cells were infected with C. muridarum or transfected with ISD (DNA) 6 h before harvest, and analyzed simultaneously at 24 h p.i for expression of mouse IFNβ (A), cGAS (B) and STING (C). Human oviduct epithelial cells (OE-E6/E7) were transfected with non-targeting (NT), human cGAS (cGAS siRNA 1 and 2) or human STING siRNA. Seventy-two hours after transfection, cells were infected with C. trachomatis (serovar D) at 5 MOI or transfected with ISD (DNA) 6 h before harvest and analyzed concurrently at 24 h p.i for expression of human IFNβ (E), cGAS (F) and STING (G). A representative of three experiments for BM1.11 cells and OE cells is presented for qRT-PCR data. Statistical significance for qPCR data between multiple experiments was determined by one way ANOVA with multiple comparison tests on the percent decrease in IFNβ levels for the siRNA used relative to NT siRNA in each experiment (D and H).

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

    Article Title: The DNA sensor, cyclic GMP-AMP synthase (cGAS) is essential for induction of IFN beta during Chlamydia trachomatis infection

    doi: 10.4049/jimmunol.1302718

    Figure Lengend Snippet: cGAS is required for IFNβ expression during Chlamydia spp. infection of mouse oviduct epithelial cells (BM1.11 cells) and human oviduct epithelial cells (OE-E6/E7) siRNA knock down in mouse BM1.11 cells were carried out using accell™ NT, mouse cGAS or STING siRNA pools. Seventy-two hours after transfection, cells were infected with C. muridarum or transfected with ISD (DNA) 6 h before harvest, and analyzed simultaneously at 24 h p.i for expression of mouse IFNβ (A), cGAS (B) and STING (C). Human oviduct epithelial cells (OE-E6/E7) were transfected with non-targeting (NT), human cGAS (cGAS siRNA 1 and 2) or human STING siRNA. Seventy-two hours after transfection, cells were infected with C. trachomatis (serovar D) at 5 MOI or transfected with ISD (DNA) 6 h before harvest and analyzed concurrently at 24 h p.i for expression of human IFNβ (E), cGAS (F) and STING (G). A representative of three experiments for BM1.11 cells and OE cells is presented for qRT-PCR data. Statistical significance for qPCR data between multiple experiments was determined by one way ANOVA with multiple comparison tests on the percent decrease in IFNβ levels for the siRNA used relative to NT siRNA in each experiment (D and H).

    Article Snippet: For J774 cells, two mouse cGAS (MB21D1) siRNA oligos (Cat# 2675, SIGMA), described earlier ( ) were used in parallel with the corresponding non-targeting siRNA from SIGMA (Cat# SIC001).

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

    Amiodarone alleviated liver injury after PHx. Wild-type mice were intraperitoneally injected with vehicle (Veh), amiodarone (AD), or chloroquine (CQ) at 0.5 h before PHx or sham operation and then once per day until 72 h. Plasma samples were collected at 0–72 h after PHx and analyzed for ALT levels ( A ). Wild-type mice were given control or Atg7-specific siRNA before PHx and plasma samples were analyzed for ALT levels at 24 h ( B ). Liver tissues were harvested at 0–48 h after surgery, and Caspase-8 cleavage and Caspase-3 cleavage was assessed by Western blot analysis ( C , D ). Samples were collected at 0–48 h after PHx and analyzed for IL-6 and IL-8 levels in plasma ( E,F ) and hepatocytes ( G , H ). The values are shown as the mean ± SD in the bar graph and compared by Student’s t test; # P

    Journal: Scientific Reports

    Article Title: Amiodarone as an autophagy promoter reduces liver injury and enhances liver regeneration and survival in mice after partial hepatectomy

    doi: 10.1038/srep15807

    Figure Lengend Snippet: Amiodarone alleviated liver injury after PHx. Wild-type mice were intraperitoneally injected with vehicle (Veh), amiodarone (AD), or chloroquine (CQ) at 0.5 h before PHx or sham operation and then once per day until 72 h. Plasma samples were collected at 0–72 h after PHx and analyzed for ALT levels ( A ). Wild-type mice were given control or Atg7-specific siRNA before PHx and plasma samples were analyzed for ALT levels at 24 h ( B ). Liver tissues were harvested at 0–48 h after surgery, and Caspase-8 cleavage and Caspase-3 cleavage was assessed by Western blot analysis ( C , D ). Samples were collected at 0–48 h after PHx and analyzed for IL-6 and IL-8 levels in plasma ( E,F ) and hepatocytes ( G , H ). The values are shown as the mean ± SD in the bar graph and compared by Student’s t test; # P

    Article Snippet: Mouse Atg7-specific siRNA (5′ CUGUGAACUUCUCUGACGU[dT][dT] 3′) (Sigma, Oligo #: 8015025863-000020) and negative siRNA (5′ GAUCAUACGUGCGAUCAGA[dT][dT] 3′) (Sigma, Oligo #: 8013122026-000020) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Mouse Assay, Injection, Western Blot

    Inhibition of autophagy reduced liver growth and hepatocyte proliferation in the early phase of liver regeneration after PHx. Wild-type mice were given control or Atg7-specific siRNA for 48 h before treatment with PHx. Liver tissues were harvested at 24 h after surgery and tissue extracts were analyzed for Atg7, LC3-II, and β-actin by Western blotting ( A ). The liver-to-body-weight ratios were calculated ( B ). Representative immunohistochemical staining of Ki67 is shown. Scale bar, 50 μm ( C ). The percentage of Ki67-positive nuclei in hepatocytes was counted in low-power field (200X) in 15 random sections from 3 different mice ( D ). Tissue extracts were analyzed for PCNA, cyclin D1, TGF-β1, and β-actin by Western blotting ( E ). Immunohistochemical staining of senescence-associated β-galactosidase (SA-β-gal) in hepatocytes. Scale bar, 100 μm ( F ). Fold-changes in IL-6 ( G ) and IL-8 ( H ) mRNA expression at 24 h after 70% PHx. Wild-type mice were intraperitoneally injected with vehicle (Veh) or chloroquine (CQ) at 0.5 h before PHx or the sham operation and then once per day until 48 h. Liver tissues were harvested at 0–48 h after surgery and tissue extracts were analyzed for PCNA, cyclin D1, TGF-β1, and β-actin by Western blotting ( I,J ). The values are shown as the mean ± SD in the bar graph and compared using Student’s t test. #P

    Journal: Scientific Reports

    Article Title: Amiodarone as an autophagy promoter reduces liver injury and enhances liver regeneration and survival in mice after partial hepatectomy

    doi: 10.1038/srep15807

    Figure Lengend Snippet: Inhibition of autophagy reduced liver growth and hepatocyte proliferation in the early phase of liver regeneration after PHx. Wild-type mice were given control or Atg7-specific siRNA for 48 h before treatment with PHx. Liver tissues were harvested at 24 h after surgery and tissue extracts were analyzed for Atg7, LC3-II, and β-actin by Western blotting ( A ). The liver-to-body-weight ratios were calculated ( B ). Representative immunohistochemical staining of Ki67 is shown. Scale bar, 50 μm ( C ). The percentage of Ki67-positive nuclei in hepatocytes was counted in low-power field (200X) in 15 random sections from 3 different mice ( D ). Tissue extracts were analyzed for PCNA, cyclin D1, TGF-β1, and β-actin by Western blotting ( E ). Immunohistochemical staining of senescence-associated β-galactosidase (SA-β-gal) in hepatocytes. Scale bar, 100 μm ( F ). Fold-changes in IL-6 ( G ) and IL-8 ( H ) mRNA expression at 24 h after 70% PHx. Wild-type mice were intraperitoneally injected with vehicle (Veh) or chloroquine (CQ) at 0.5 h before PHx or the sham operation and then once per day until 48 h. Liver tissues were harvested at 0–48 h after surgery and tissue extracts were analyzed for PCNA, cyclin D1, TGF-β1, and β-actin by Western blotting ( I,J ). The values are shown as the mean ± SD in the bar graph and compared using Student’s t test. #P

    Article Snippet: Mouse Atg7-specific siRNA (5′ CUGUGAACUUCUCUGACGU[dT][dT] 3′) (Sigma, Oligo #: 8015025863-000020) and negative siRNA (5′ GAUCAUACGUGCGAUCAGA[dT][dT] 3′) (Sigma, Oligo #: 8013122026-000020) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Inhibition, Mouse Assay, Western Blot, Immunohistochemistry, Staining, Expressing, Injection

    PDD-induced TRPV4 activation triggered Ca 2+ influx. ( a ) Intracellular Ca 2+ measurement in control or Trpv4 knocked-down 4T07 cells treated with 10 μ m of 4α-PDD or DMSO. Luc -DMSO=cells transfected with Luc control siRNA and treated with DMSO; Luc -PDD, cells transfected with Luc control siRNA and treated with 4α-PDD; S1-DMSO, cells transfected with Trpv4 siRNA (sequence 1) and treated with DMSO; S1-PDD, cells transfected with Trpv4 siRNA (sequence 1) and treated with 4α-PDD; S3-DMSO, cells transfected with Trpv4 siRNA (sequence 3) and treated with DMSO; S3-PDD, cells transfected with TRPV4 siRNA (sequence 3) and treated with 4α-PDD. ( b ) Effects of chelating intracellular (by BAPTA-AM) and extracellular (by EGTA) Ca 2+ on 4α-PDD-induced, TRPV4-mediated signal transduction. 4T07 cells were untreated or pre-treated with 20 μ m of BAPTA-AM or 2 m m of EGTA for 1 h before being stimulated with 4α-PDD for 15 min (to assay for AKT activation) or 16 h (to assay for FAK activation and effects on E-cadherin and β-catenin). Cell lysates were then subject to immunoblotting using the indicated antibodies and bands densitometry performed using the ‘Image J’ software. The fold induction (ratio) was then obtained by dividing the values at PDD 15 min or PDD16 h over DMSO. Data points represent mean±s.e.m. of three independent experiments, * P

    Journal: Oncogenesis

    Article Title: TRPV4 plays a role in breast cancer cell migration via Ca2+-dependent activation of AKT and downregulation of E-cadherin cell cortex protein

    doi: 10.1038/oncsis.2017.39

    Figure Lengend Snippet: PDD-induced TRPV4 activation triggered Ca 2+ influx. ( a ) Intracellular Ca 2+ measurement in control or Trpv4 knocked-down 4T07 cells treated with 10 μ m of 4α-PDD or DMSO. Luc -DMSO=cells transfected with Luc control siRNA and treated with DMSO; Luc -PDD, cells transfected with Luc control siRNA and treated with 4α-PDD; S1-DMSO, cells transfected with Trpv4 siRNA (sequence 1) and treated with DMSO; S1-PDD, cells transfected with Trpv4 siRNA (sequence 1) and treated with 4α-PDD; S3-DMSO, cells transfected with Trpv4 siRNA (sequence 3) and treated with DMSO; S3-PDD, cells transfected with TRPV4 siRNA (sequence 3) and treated with 4α-PDD. ( b ) Effects of chelating intracellular (by BAPTA-AM) and extracellular (by EGTA) Ca 2+ on 4α-PDD-induced, TRPV4-mediated signal transduction. 4T07 cells were untreated or pre-treated with 20 μ m of BAPTA-AM or 2 m m of EGTA for 1 h before being stimulated with 4α-PDD for 15 min (to assay for AKT activation) or 16 h (to assay for FAK activation and effects on E-cadherin and β-catenin). Cell lysates were then subject to immunoblotting using the indicated antibodies and bands densitometry performed using the ‘Image J’ software. The fold induction (ratio) was then obtained by dividing the values at PDD 15 min or PDD16 h over DMSO. Data points represent mean±s.e.m. of three independent experiments, * P

    Article Snippet: Mouse TRPV4-specific siRNA oligonucleotides were purchased from Invitrogen (Carlsbad, CA, USA) and the siRNA sequences are as following: Luciferase GL2: 5′-CGUACG CGGAAUACUUCGA-3′ Trpv4 siRNA1: 5′-AGAAGCAGCAGGUCGUACAUCUUGG-3′ Trpv4 siRNA3: 5′-AAACUUGGUGUUCUCUCGGGUGUUG-3′.

    Techniques: Activation Assay, Transfection, Sequencing, Transduction, Software

    Investigating AKT and FAK as potential mediators of TRPV4-mediated downregulation of E-cadherin and β-catenin expression. Cells were pre-treated or not with 5 μ m of AKT inhibitor IV ( a and b ) or 10 μ m of FAK inhibitor ( c and d ) for 1 h before stimulation with 10 μ m 4α-PDD for 15 min or 16 h as shown. Lysates were then probed with the indicated antibodies. Actin was used as a loading control. The levels of each protein shown in the bar charts were expressed using Actin as the denominator. ( e ) Effects of constitutively active AKT on Trpv4 siRNA-mediated downregulation of E-cadherin expression. ( f ) 4T07 cells transfected with Luc or Trpv4 siRNAs were overexpressed with or without Myr-AKT. 48 h post-transfection, cells were subject to transendothelial migration assay over a time course of 8 h duration. Data points represent mean±s.e.m. of three independent experiments.

    Journal: Oncogenesis

    Article Title: TRPV4 plays a role in breast cancer cell migration via Ca2+-dependent activation of AKT and downregulation of E-cadherin cell cortex protein

    doi: 10.1038/oncsis.2017.39

    Figure Lengend Snippet: Investigating AKT and FAK as potential mediators of TRPV4-mediated downregulation of E-cadherin and β-catenin expression. Cells were pre-treated or not with 5 μ m of AKT inhibitor IV ( a and b ) or 10 μ m of FAK inhibitor ( c and d ) for 1 h before stimulation with 10 μ m 4α-PDD for 15 min or 16 h as shown. Lysates were then probed with the indicated antibodies. Actin was used as a loading control. The levels of each protein shown in the bar charts were expressed using Actin as the denominator. ( e ) Effects of constitutively active AKT on Trpv4 siRNA-mediated downregulation of E-cadherin expression. ( f ) 4T07 cells transfected with Luc or Trpv4 siRNAs were overexpressed with or without Myr-AKT. 48 h post-transfection, cells were subject to transendothelial migration assay over a time course of 8 h duration. Data points represent mean±s.e.m. of three independent experiments.

    Article Snippet: Mouse TRPV4-specific siRNA oligonucleotides were purchased from Invitrogen (Carlsbad, CA, USA) and the siRNA sequences are as following: Luciferase GL2: 5′-CGUACG CGGAAUACUUCGA-3′ Trpv4 siRNA1: 5′-AGAAGCAGCAGGUCGUACAUCUUGG-3′ Trpv4 siRNA3: 5′-AAACUUGGUGUUCUCUCGGGUGUUG-3′.

    Techniques: Expressing, Transfection, Migration

    EVI1 negatively regulates bacteria-induced NF-κB activation via inhibition of p65 acetylation at lysine 310 (A B) A549 cells co-transfected with EVI1 and p65 were treated with NTHi, and the cell lysates were immnoprecipitated with anti-p65 antibody and analyzed by western blot analysis with the indicated antibodies.(C) A549 cells were co-transfected with NF-κB-luciferase reporter plasmid together with EVI1 siRNA and p65 WT or p65 K310R constructs, and NF-κB-dependent promoter activity was measured by luciferase assay.* p

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

    Article Title: EVI1 Acts As An Inducible Negative Feedback Regulator of NF-?B by inhibiting p65 Acetylation

    doi: 10.4049/jimmunol.1103527

    Figure Lengend Snippet: EVI1 negatively regulates bacteria-induced NF-κB activation via inhibition of p65 acetylation at lysine 310 (A B) A549 cells co-transfected with EVI1 and p65 were treated with NTHi, and the cell lysates were immnoprecipitated with anti-p65 antibody and analyzed by western blot analysis with the indicated antibodies.(C) A549 cells were co-transfected with NF-κB-luciferase reporter plasmid together with EVI1 siRNA and p65 WT or p65 K310R constructs, and NF-κB-dependent promoter activity was measured by luciferase assay.* p

    Article Snippet: Human and mouse EVI1 small interfering RNA (siRNA) oligonucleotides were purchased from Dharmacon and SantaCruz.

    Techniques: Activation Assay, Inhibition, Transfection, Western Blot, Luciferase, Plasmid Preparation, Construct, Activity Assay

    EVI1 negatively regulates bacteria-induced activation of NF-κB by inhibiting its DNA-binding activity, likely independently of p65 nuclear translocation (A) A549 cells were co-transfected with NF-κB-luciferase reporter plasmid together with a constitutively active form of IKKβ (IKKβ CA), and NF-κB-dependent promoter activity was measured by luciferase assay. (B C) A549 cells transfected either with EVI1 siRNA (B) or EVI1 WT construct (C) were treated with NTHi for the time indicated in the figure, and analyzed by western blot analysis with the indicated antibodies. (D) A549 cells co-transfected with p65 and EVI1 WT or control vector were treated with NTHi, and nuclear extracts were analyzed by western blot analysis with the indicated antibodies. (E) A549 cells co-transfected with EVI1 and p65 were treated with NTHi and immunostained with DAPI and anti-p65 antibodies. Cells were visualized by fluorescence microscopy. (F) A549 cells co-transfected with p65 and EVI1 WT or control vector were treated with NTHi, and DNA-binding activity of NF-κB was assessed by electrophoretic mobility shift assay (EMSA). (G) A549 cells transfected with EVI1 siRNA or control siRNA were treated with NTHi for the times indicated in the figure, and chromatin immunoprecipitation (ChIP) assay was conducted using antibody against p65 and analyzed by PCR using specific primers for the IL-8 promoter sequences spanning the κB binding sites. * p

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

    Article Title: EVI1 Acts As An Inducible Negative Feedback Regulator of NF-?B by inhibiting p65 Acetylation

    doi: 10.4049/jimmunol.1103527

    Figure Lengend Snippet: EVI1 negatively regulates bacteria-induced activation of NF-κB by inhibiting its DNA-binding activity, likely independently of p65 nuclear translocation (A) A549 cells were co-transfected with NF-κB-luciferase reporter plasmid together with a constitutively active form of IKKβ (IKKβ CA), and NF-κB-dependent promoter activity was measured by luciferase assay. (B C) A549 cells transfected either with EVI1 siRNA (B) or EVI1 WT construct (C) were treated with NTHi for the time indicated in the figure, and analyzed by western blot analysis with the indicated antibodies. (D) A549 cells co-transfected with p65 and EVI1 WT or control vector were treated with NTHi, and nuclear extracts were analyzed by western blot analysis with the indicated antibodies. (E) A549 cells co-transfected with EVI1 and p65 were treated with NTHi and immunostained with DAPI and anti-p65 antibodies. Cells were visualized by fluorescence microscopy. (F) A549 cells co-transfected with p65 and EVI1 WT or control vector were treated with NTHi, and DNA-binding activity of NF-κB was assessed by electrophoretic mobility shift assay (EMSA). (G) A549 cells transfected with EVI1 siRNA or control siRNA were treated with NTHi for the times indicated in the figure, and chromatin immunoprecipitation (ChIP) assay was conducted using antibody against p65 and analyzed by PCR using specific primers for the IL-8 promoter sequences spanning the κB binding sites. * p

    Article Snippet: Human and mouse EVI1 small interfering RNA (siRNA) oligonucleotides were purchased from Dharmacon and SantaCruz.

    Techniques: Activation Assay, Binding Assay, Activity Assay, Translocation Assay, Transfection, Luciferase, Plasmid Preparation, Construct, Western Blot, Fluorescence, Microscopy, Electrophoretic Mobility Shift Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    EVI1 negatively regulates bacteria-induced NF-κB-dependent inflammatory response in airway epithelial and middle ear epithelial cells (A-D) A549 or HMEEC-1 cells were co-transfected with NF-κB-luciferase reporter plasmid together with either EVI1 siRNA (A), EVI1 WT (B C), or EVI MT (D)construct for 40hours and then treated with or without NTHi for 5 hours. NF-κB-dependent promoter activity was measured by luciferase assay. (E-H) A549 or HMEEC-1 cells were transfected with either EVI1 siRNA (E), EVI1 WT (F G), or EVI MT (H) construct for 40 hours and then treated with or without NTHi for 5 hours. Total RNA was extracted, and mRNA expression of TNF-α, IL-1β and IL-8 was measured by real-time quantitative RT-PCR (Q-PCR) assay. Data represent the mean ± SD of at least three independent experiments, and each experiment was performed in triplicate. * p

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

    Article Title: EVI1 Acts As An Inducible Negative Feedback Regulator of NF-?B by inhibiting p65 Acetylation

    doi: 10.4049/jimmunol.1103527

    Figure Lengend Snippet: EVI1 negatively regulates bacteria-induced NF-κB-dependent inflammatory response in airway epithelial and middle ear epithelial cells (A-D) A549 or HMEEC-1 cells were co-transfected with NF-κB-luciferase reporter plasmid together with either EVI1 siRNA (A), EVI1 WT (B C), or EVI MT (D)construct for 40hours and then treated with or without NTHi for 5 hours. NF-κB-dependent promoter activity was measured by luciferase assay. (E-H) A549 or HMEEC-1 cells were transfected with either EVI1 siRNA (E), EVI1 WT (F G), or EVI MT (H) construct for 40 hours and then treated with or without NTHi for 5 hours. Total RNA was extracted, and mRNA expression of TNF-α, IL-1β and IL-8 was measured by real-time quantitative RT-PCR (Q-PCR) assay. Data represent the mean ± SD of at least three independent experiments, and each experiment was performed in triplicate. * p

    Article Snippet: Human and mouse EVI1 small interfering RNA (siRNA) oligonucleotides were purchased from Dharmacon and SantaCruz.

    Techniques: Transfection, Luciferase, Plasmid Preparation, Activity Assay, Construct, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction

    Overexpression of miR-29 in mdx muscles downregulates fibrotic genes . ( a ) Differentially expressed genes in mdx muscles injected with NC and miR-29 oligos as determined by mRNA-seq. X- and Y-axis represent the log2 based FPKM values for expressed genes in NC and miR-29 samples, respectively. ( b ) Over-represented GO terms by GO analysis of downregulated list of genes. BP, biological process; CC, cellular component; KEGG, Kyoto Encyclopedia of Genes and Genomes; SP_PIR, a database of protein super-family names. ( c ) Coverage plot showing a 56-kb region encompassing the ACTA2 (α-SMA) gene on chromosome (Chr) 19; the gene structure is shown in blue below the graph. ( d ) Expressions of collagen 1A1 (Col 1A1), collagen 1A2 (Col 1A2), collagen 3A1 (Col 3A1), α-smooth muscle actin (α-SMA) or vimentin (VIM) in NC or miR-29 injected muscles. ( e ) Expressions of the above genes in TA muscles injected with negative control (Anti-NC) or miR-29 inhibitor oligos (Anti-miR-29). FPKM, fragments per kilobase of transcript per million fragments mapped; GO, gene ontology; mRNA, messenger RNA; NC, negative control; TA, tibialis anterior.

    Journal: Molecular Therapy

    Article Title: Loss of miR-29 in Myoblasts Contributes to Dystrophic Muscle Pathogenesis

    doi: 10.1038/mt.2012.35

    Figure Lengend Snippet: Overexpression of miR-29 in mdx muscles downregulates fibrotic genes . ( a ) Differentially expressed genes in mdx muscles injected with NC and miR-29 oligos as determined by mRNA-seq. X- and Y-axis represent the log2 based FPKM values for expressed genes in NC and miR-29 samples, respectively. ( b ) Over-represented GO terms by GO analysis of downregulated list of genes. BP, biological process; CC, cellular component; KEGG, Kyoto Encyclopedia of Genes and Genomes; SP_PIR, a database of protein super-family names. ( c ) Coverage plot showing a 56-kb region encompassing the ACTA2 (α-SMA) gene on chromosome (Chr) 19; the gene structure is shown in blue below the graph. ( d ) Expressions of collagen 1A1 (Col 1A1), collagen 1A2 (Col 1A2), collagen 3A1 (Col 3A1), α-smooth muscle actin (α-SMA) or vimentin (VIM) in NC or miR-29 injected muscles. ( e ) Expressions of the above genes in TA muscles injected with negative control (Anti-NC) or miR-29 inhibitor oligos (Anti-miR-29). FPKM, fragments per kilobase of transcript per million fragments mapped; GO, gene ontology; mRNA, messenger RNA; NC, negative control; TA, tibialis anterior.

    Article Snippet: For systemic delivery of miR-29 mimics, 9-month-old mdx mice were injected with miR-29c mimics or NC oligos (RiboBio, Guangzhou, China).

    Techniques: Over Expression, Injection, Negative Control

    Loss of miR-29 promotes myoblasts transdifferentiation into myofibroblasts. ( a ) Expressions of Col 1A1, Col 1A2, and Col 3A1 in primary myoblasts freshly isolated from WT or mdx muscles. ( b ) Primary myoblasts from mdx muscles were transfected with NC or miR-29 oligos and examined for Col 1A1, Col 1A2, and Col 3A1 expressions. ( c ) WT or mutant Col 1A1, Col 1A2, or Col 3A1-3′UTR luciferase reporter constructs were transfected into mdx primary myoblasts with NC or miR-29 oligos. Luciferase activities were determined at 48 hours post-transfection. Relative luciferase unit (RLU) is shown with respect to NC cells where normalized luciferase values were set to 1. The data represents the average of three independent experiments ± SD. ( d ) Predicted binding between mmu-miR-29c and mouse 3′UTR of Mfap5. ( e ) WT or mutant Mfap5-3′UTR luciferase reporter constructs were transfected into C2C12 cells with NC or miR-29 oligos. Luciferase activities were determined as above. ( f ) Expressions of Mfap5 in primary myoblasts from WT or mdx muscles. ( g ) Primary myoblasts from mdx muscles were transfected with NC or miR-29 oligos and examined for Mfap5 expression. ( h ) Expressions of the Mfap5 mRNAs in mdx TA muscles injected with negative control (Anti-NC) or miR-29 inhibitor oligos (Anti-miR-29). ( i ) IF staining for MyoD (green) and α-SMA (red) were performed on cryosections of mdx TA muscles. DAPI (blue) staining was also performed to visualize the nuclei. A field with three types of cells is shown on the left: arrows, MyoD-/α-SMA+ cells; arrowhead, MyoD+/α-SMA- cells; asterisk, MyoD+/α-SMA+ cells. Higher magnification of one of the MyoD+/α-SMA+ cells is presented on the right. ( j ) TA muscles from mdx mice were injected with NC or miR-29 mimics oligos. Quantification of the MyoD+/SMA+ cells on the above muscles were performed on a minimal of 15 sections for each group. N = 5 mice per group. Data are plotted as mean ± SD.* P

    Journal: Molecular Therapy

    Article Title: Loss of miR-29 in Myoblasts Contributes to Dystrophic Muscle Pathogenesis

    doi: 10.1038/mt.2012.35

    Figure Lengend Snippet: Loss of miR-29 promotes myoblasts transdifferentiation into myofibroblasts. ( a ) Expressions of Col 1A1, Col 1A2, and Col 3A1 in primary myoblasts freshly isolated from WT or mdx muscles. ( b ) Primary myoblasts from mdx muscles were transfected with NC or miR-29 oligos and examined for Col 1A1, Col 1A2, and Col 3A1 expressions. ( c ) WT or mutant Col 1A1, Col 1A2, or Col 3A1-3′UTR luciferase reporter constructs were transfected into mdx primary myoblasts with NC or miR-29 oligos. Luciferase activities were determined at 48 hours post-transfection. Relative luciferase unit (RLU) is shown with respect to NC cells where normalized luciferase values were set to 1. The data represents the average of three independent experiments ± SD. ( d ) Predicted binding between mmu-miR-29c and mouse 3′UTR of Mfap5. ( e ) WT or mutant Mfap5-3′UTR luciferase reporter constructs were transfected into C2C12 cells with NC or miR-29 oligos. Luciferase activities were determined as above. ( f ) Expressions of Mfap5 in primary myoblasts from WT or mdx muscles. ( g ) Primary myoblasts from mdx muscles were transfected with NC or miR-29 oligos and examined for Mfap5 expression. ( h ) Expressions of the Mfap5 mRNAs in mdx TA muscles injected with negative control (Anti-NC) or miR-29 inhibitor oligos (Anti-miR-29). ( i ) IF staining for MyoD (green) and α-SMA (red) were performed on cryosections of mdx TA muscles. DAPI (blue) staining was also performed to visualize the nuclei. A field with three types of cells is shown on the left: arrows, MyoD-/α-SMA+ cells; arrowhead, MyoD+/α-SMA- cells; asterisk, MyoD+/α-SMA+ cells. Higher magnification of one of the MyoD+/α-SMA+ cells is presented on the right. ( j ) TA muscles from mdx mice were injected with NC or miR-29 mimics oligos. Quantification of the MyoD+/SMA+ cells on the above muscles were performed on a minimal of 15 sections for each group. N = 5 mice per group. Data are plotted as mean ± SD.* P

    Article Snippet: For systemic delivery of miR-29 mimics, 9-month-old mdx mice were injected with miR-29c mimics or NC oligos (RiboBio, Guangzhou, China).

    Techniques: Isolation, Transfection, Mutagenesis, Luciferase, Construct, Binding Assay, Expressing, Injection, Negative Control, Staining, Mouse Assay

    Systemic delivery of miR-29 oligos reduces fibrosis in mdx diaphragm . ( a ) Administration scheme for miR-29 injection. NC or miR-29 oligos formulated with liposome were injected into mdx mice through tail vein at day (d) 0, 4, and 7. Mice were killed and diaphragm muscles were harvested at day 21 for histology and immunostaining. n = 5 mice for each treatment group. ( b ) Expressions of miR-29 in various tissues collected at day 3 after the injection. ( c ) Trichrome staining of the fibrotic areas in NC and miR-29 injected diaphragm muscles. The positively stained areas were quantified with Image-Pro Plus from a minimum of 15 sections. ( d ) IHC staining of the above muscles with collagen 1. The positively stained areas were quantified as the above. ( e ) IF staining of the above muscle with collagen 1. ( f ) Expressions of Pax7, MyoD, myogenin, and YY1 mRNAs in the above treated diaphragm muscles. ( g ) H E staining of the above NC or miR-29 injected mdx muscles. ( h ) Damaged areas were identified as fibrotic and fatty areas and quantified as the above. ** P

    Journal: Molecular Therapy

    Article Title: Loss of miR-29 in Myoblasts Contributes to Dystrophic Muscle Pathogenesis

    doi: 10.1038/mt.2012.35

    Figure Lengend Snippet: Systemic delivery of miR-29 oligos reduces fibrosis in mdx diaphragm . ( a ) Administration scheme for miR-29 injection. NC or miR-29 oligos formulated with liposome were injected into mdx mice through tail vein at day (d) 0, 4, and 7. Mice were killed and diaphragm muscles were harvested at day 21 for histology and immunostaining. n = 5 mice for each treatment group. ( b ) Expressions of miR-29 in various tissues collected at day 3 after the injection. ( c ) Trichrome staining of the fibrotic areas in NC and miR-29 injected diaphragm muscles. The positively stained areas were quantified with Image-Pro Plus from a minimum of 15 sections. ( d ) IHC staining of the above muscles with collagen 1. The positively stained areas were quantified as the above. ( e ) IF staining of the above muscle with collagen 1. ( f ) Expressions of Pax7, MyoD, myogenin, and YY1 mRNAs in the above treated diaphragm muscles. ( g ) H E staining of the above NC or miR-29 injected mdx muscles. ( h ) Damaged areas were identified as fibrotic and fatty areas and quantified as the above. ** P

    Article Snippet: For systemic delivery of miR-29 mimics, 9-month-old mdx mice were injected with miR-29c mimics or NC oligos (RiboBio, Guangzhou, China).

    Techniques: Injection, Mouse Assay, Immunostaining, Staining, Immunohistochemistry

    miR-29 is downregulated in mdx myoblasts . ( a ) Expression of miR-29 in primary myoblasts from WT or mdx muscles. ( b ) Left: primary myoblasts from mdx muscles were kept growing (DM 0 hour) or differentiated (DM) for 7, 24, or 48 hours at which times cells were immunostained for MyHC. Right: positively stained cells were quantified. Numbers indicate the average number of MyHC positive cells counted from a minimum of 10 randomly chosen fields. Graphs are plotted as mean ± SD. Images were taken at 24 hours. ( c ) Expressions of MyHC or troponin RNAs in WT or mdx myoblasts differentiated for the indicated times. ( d ) Expression of miR-29 in mdx primary myoblasts transfected with NC or miR-29 oligos. ( e ) Left: the above transfected cells were differentiated for 48 hours at which time the cells were photographed under phase contrasts or immunostained for MyHC. Right: positively stained cells were quantified as in b . ( f ) Expressions of MyHC and troponin RNAs in NC or miR-29 transfected cells at different time points of differentiation. ( g ) mdx myoblasts were transfected with MyHC-Luc or TnI-Luc reporter plasmids and NC or miR-29 oligos. Cells were then differentiated for 48 hours at which time luciferase activities were determined. The data represent the average of three independent experiments ± SD. * P

    Journal: Molecular Therapy

    Article Title: Loss of miR-29 in Myoblasts Contributes to Dystrophic Muscle Pathogenesis

    doi: 10.1038/mt.2012.35

    Figure Lengend Snippet: miR-29 is downregulated in mdx myoblasts . ( a ) Expression of miR-29 in primary myoblasts from WT or mdx muscles. ( b ) Left: primary myoblasts from mdx muscles were kept growing (DM 0 hour) or differentiated (DM) for 7, 24, or 48 hours at which times cells were immunostained for MyHC. Right: positively stained cells were quantified. Numbers indicate the average number of MyHC positive cells counted from a minimum of 10 randomly chosen fields. Graphs are plotted as mean ± SD. Images were taken at 24 hours. ( c ) Expressions of MyHC or troponin RNAs in WT or mdx myoblasts differentiated for the indicated times. ( d ) Expression of miR-29 in mdx primary myoblasts transfected with NC or miR-29 oligos. ( e ) Left: the above transfected cells were differentiated for 48 hours at which time the cells were photographed under phase contrasts or immunostained for MyHC. Right: positively stained cells were quantified as in b . ( f ) Expressions of MyHC and troponin RNAs in NC or miR-29 transfected cells at different time points of differentiation. ( g ) mdx myoblasts were transfected with MyHC-Luc or TnI-Luc reporter plasmids and NC or miR-29 oligos. Cells were then differentiated for 48 hours at which time luciferase activities were determined. The data represent the average of three independent experiments ± SD. * P

    Article Snippet: For systemic delivery of miR-29 mimics, 9-month-old mdx mice were injected with miR-29c mimics or NC oligos (RiboBio, Guangzhou, China).

    Techniques: Expressing, Staining, Transfection, Luciferase

    YY1 negatively regulates miR-1 during CTX-induced muscle regeneration. (A) Tibialis anterior (TA) muscles from six-week old C57/BL6 background mice were injected with 10 µM cardiotoxin (CTX). RNAs and proteins were then extracted from injected muscles at the indicated days post-injection, and qRT-PCR was performed to measure the expression of miR-1 and miR-133, normalized to U6. Expression folds are shown with respect to day 0 where miR-1 and miR-133 levels were set to a value of 1. Quantitative values are represented as means ± S.D. (B) YY1 expression was measured by Western blotting. α-Tubulin was used as a loading control. Numbers below indicates the quantification by densitometry. (C) TA muscles from 6 week C57/BL6 background mice were injected CTX at day 0, followed by injection with siNC (left leg) and siYY1 oligos (right leg) 6 hours later. And re-injection of siRNA oligos was performed every other day for two more times. The injected muscles were harvested at the indicated days. N = 6 for each group. (D) Expressions of miR-1 and miR-133 were detected by qRT-PCR in CTX/siRNA injected muscles at day 2, 4 and 6, normalized to U6. Expression folds are shown with respect to siNC where miR-1 and miR-133 levels were set to a value of 1. (E) Western blotting was performed to analyze the expression of YY1, Pax7, MyoD and Myogenin. α-Tubulin was used as a loading control. Data is representative of 6 mice. (F) Expression of Pax7 and MyoD RNA levels were also detected by qRT-PCR normalized with GAPDH. Expression folds are shown with respect to siNC where Pax7 and MyoD levels were set to a value of 1. Quantitative values are represented as mean ± S.D. The p value was determined by Student's T-test: *p

    Journal: PLoS ONE

    Article Title: A Novel YY1-miR-1 Regulatory Circuit in Skeletal Myogenesis Revealed by Genome-Wide Prediction of YY1-miRNA Network

    doi: 10.1371/journal.pone.0027596

    Figure Lengend Snippet: YY1 negatively regulates miR-1 during CTX-induced muscle regeneration. (A) Tibialis anterior (TA) muscles from six-week old C57/BL6 background mice were injected with 10 µM cardiotoxin (CTX). RNAs and proteins were then extracted from injected muscles at the indicated days post-injection, and qRT-PCR was performed to measure the expression of miR-1 and miR-133, normalized to U6. Expression folds are shown with respect to day 0 where miR-1 and miR-133 levels were set to a value of 1. Quantitative values are represented as means ± S.D. (B) YY1 expression was measured by Western blotting. α-Tubulin was used as a loading control. Numbers below indicates the quantification by densitometry. (C) TA muscles from 6 week C57/BL6 background mice were injected CTX at day 0, followed by injection with siNC (left leg) and siYY1 oligos (right leg) 6 hours later. And re-injection of siRNA oligos was performed every other day for two more times. The injected muscles were harvested at the indicated days. N = 6 for each group. (D) Expressions of miR-1 and miR-133 were detected by qRT-PCR in CTX/siRNA injected muscles at day 2, 4 and 6, normalized to U6. Expression folds are shown with respect to siNC where miR-1 and miR-133 levels were set to a value of 1. (E) Western blotting was performed to analyze the expression of YY1, Pax7, MyoD and Myogenin. α-Tubulin was used as a loading control. Data is representative of 6 mice. (F) Expression of Pax7 and MyoD RNA levels were also detected by qRT-PCR normalized with GAPDH. Expression folds are shown with respect to siNC where Pax7 and MyoD levels were set to a value of 1. Quantitative values are represented as mean ± S.D. The p value was determined by Student's T-test: *p

    Article Snippet: Oligonucleotides Precursor miRNA oligos were obtained from Ambion. siRNA oligos against mouse YY1 were obtained from Santa Cruz technologies.

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

    YY1 repression of miR-1/133 is mediated through multiple enhancers. (A) Two conserved enhancers (E1 and E2) were identified in the promoter region and intragenic region of miR-1-2/miR-133a-1 cluster, respectively. Three putative YY1 binding sites, A, B and C, were identified. (B) One conserved enhancer (E3) was identified in between miR-1-1 and miR-133a-2 with a putative YY1 binding site, D, identified. (C) One conserved enhancer (E4) was identified upstream of miR-206 and miR-133b cluster with a putative YY1 binding site, E, identified. Binding sites for MyoD, MEF2 and SRF were also shown. (D) C2C12 cells were transfected with 250 ng of E1, E2, E3 or E4 reporter plasmid along with Renilla and control vector (YY1 0 ng) or 50 ng, 200 ng, 500 ng YY1 expressing plasmid. Cells were then cultured for 48 h at which time luciferase activities were determined and normalized to Renilla protein. The data represent the average of three independent experiments ± S.D. (E) C2C12 cells were transfected with 0.25 µg of E1, E2, E3 or E4 reporter plasmid along with Renilla luciferase vector and siYY1 or siNC oligos. Luciferase activity was determined as in (D). (F) Chromatins were harvested from C2C12 myoblasts growing in growth medium (GM) or myotubes maintained in differentiation medium (DM) and subjected to ChIP-PCR analysis. Primers were designed to amplify regions encompassing putative YY1 binding sites A, B, C, D, or E. MyHC and Tnni2 are known YY1 targets and used as positive controls. A genomic region that contains no YY1 binding sites was included as a negative control (NC). (G) Site A (Mut A) or both A and B (Mut A+B) were mutated in E1 luciferase reporter plasmid and luciferase reporter assay was performed to measure the response of mutants to YY1 over-expression as in (D). Relative luciferase unit (RLU) is shown with respect to Vector transfection where luciferase activities were set to a value of 1. (H) ChIP-PCR for Ezh2 or H3K27me3 was performed as in (F). The p value was determined by Student's T-test: *p

    Journal: PLoS ONE

    Article Title: A Novel YY1-miR-1 Regulatory Circuit in Skeletal Myogenesis Revealed by Genome-Wide Prediction of YY1-miRNA Network

    doi: 10.1371/journal.pone.0027596

    Figure Lengend Snippet: YY1 repression of miR-1/133 is mediated through multiple enhancers. (A) Two conserved enhancers (E1 and E2) were identified in the promoter region and intragenic region of miR-1-2/miR-133a-1 cluster, respectively. Three putative YY1 binding sites, A, B and C, were identified. (B) One conserved enhancer (E3) was identified in between miR-1-1 and miR-133a-2 with a putative YY1 binding site, D, identified. (C) One conserved enhancer (E4) was identified upstream of miR-206 and miR-133b cluster with a putative YY1 binding site, E, identified. Binding sites for MyoD, MEF2 and SRF were also shown. (D) C2C12 cells were transfected with 250 ng of E1, E2, E3 or E4 reporter plasmid along with Renilla and control vector (YY1 0 ng) or 50 ng, 200 ng, 500 ng YY1 expressing plasmid. Cells were then cultured for 48 h at which time luciferase activities were determined and normalized to Renilla protein. The data represent the average of three independent experiments ± S.D. (E) C2C12 cells were transfected with 0.25 µg of E1, E2, E3 or E4 reporter plasmid along with Renilla luciferase vector and siYY1 or siNC oligos. Luciferase activity was determined as in (D). (F) Chromatins were harvested from C2C12 myoblasts growing in growth medium (GM) or myotubes maintained in differentiation medium (DM) and subjected to ChIP-PCR analysis. Primers were designed to amplify regions encompassing putative YY1 binding sites A, B, C, D, or E. MyHC and Tnni2 are known YY1 targets and used as positive controls. A genomic region that contains no YY1 binding sites was included as a negative control (NC). (G) Site A (Mut A) or both A and B (Mut A+B) were mutated in E1 luciferase reporter plasmid and luciferase reporter assay was performed to measure the response of mutants to YY1 over-expression as in (D). Relative luciferase unit (RLU) is shown with respect to Vector transfection where luciferase activities were set to a value of 1. (H) ChIP-PCR for Ezh2 or H3K27me3 was performed as in (F). The p value was determined by Student's T-test: *p

    Article Snippet: Oligonucleotides Precursor miRNA oligos were obtained from Ambion. siRNA oligos against mouse YY1 were obtained from Santa Cruz technologies.

    Techniques: Binding Assay, Transfection, Plasmid Preparation, Expressing, Cell Culture, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control, Reporter Assay, Over Expression

    YY1 represses miR-1 functionally during C2C12 myogenesis. (A) C2C12 cells were transfected with the indicated combination of NC or miR-1 oligos and Vector or YY1 expression plasmids. Cells were then differentiated (DM) for 2 days, at which time cells were immunostained for MyHC. Cell morphology was visualized by phase-contrast microscopy. (B) MyHC positive cells were quantified by counting positively stained cells from 10 randomly chosen fields and are represented as mean ± S.D. (C) Total proteins were isolated from the above transfected cells and Western blotting was performed to probe for α-Actin. α-Tubulin was used as a loading control. (D) Total RNAs were extracted from the above transfected cells and used for qRT-PCR analysis of myogenic markers, MyHC, Troponin, α-Actin, and Myogenin normalized with GAPDH. YY1 and miR-1 levels were also measured to show the transfection efficiency. Quantitative values are represented as mean ± S.D. The p value was determined by Student's T-test: *p

    Journal: PLoS ONE

    Article Title: A Novel YY1-miR-1 Regulatory Circuit in Skeletal Myogenesis Revealed by Genome-Wide Prediction of YY1-miRNA Network

    doi: 10.1371/journal.pone.0027596

    Figure Lengend Snippet: YY1 represses miR-1 functionally during C2C12 myogenesis. (A) C2C12 cells were transfected with the indicated combination of NC or miR-1 oligos and Vector or YY1 expression plasmids. Cells were then differentiated (DM) for 2 days, at which time cells were immunostained for MyHC. Cell morphology was visualized by phase-contrast microscopy. (B) MyHC positive cells were quantified by counting positively stained cells from 10 randomly chosen fields and are represented as mean ± S.D. (C) Total proteins were isolated from the above transfected cells and Western blotting was performed to probe for α-Actin. α-Tubulin was used as a loading control. (D) Total RNAs were extracted from the above transfected cells and used for qRT-PCR analysis of myogenic markers, MyHC, Troponin, α-Actin, and Myogenin normalized with GAPDH. YY1 and miR-1 levels were also measured to show the transfection efficiency. Quantitative values are represented as mean ± S.D. The p value was determined by Student's T-test: *p

    Article Snippet: Oligonucleotides Precursor miRNA oligos were obtained from Ambion. siRNA oligos against mouse YY1 were obtained from Santa Cruz technologies.

    Techniques: Transfection, Plasmid Preparation, Expressing, Microscopy, Staining, Isolation, Western Blot, Quantitative RT-PCR

    miR-1 targets Pax7 during C2C12 differentiation. (A) Predicted target sites, A and B, of miR-1 in the 3′UTR of mouse Pax7. (B) A luciferase reporter plasmid was generated by cloning a ∼800 bp region of Pax7 3′UTR encompassing both site A and B downstream of the luciferase (Luc) reporter gene. The reporter construct was then transfected into C2C12 cells with negative control (NC) or miR-1 oligos along with Renilla luciferase plasmid. Luciferase activities were determined at 48 h post-transfection and normalized to Renilla readings. The data represent the average of three independent experiments ± S.D. (C) A mutation was introduced in either site A (Mut A) or site B (Mut B) or both (Mut A+B). Their responses to miR-1 over-expression were tested as above. (D) C2C12 myoblasts were transfected with either NC or miR-1 oligos. Both miR-1 and Pax7 mRNAs levels were then measured 48 hr post-transfection. (E) HDAC4 or Pax7 proteins were probed in extracts from cells 48 hr after transfection. Blots were stripped and reprobed for α-Tubulin as the loading control. (F) Proteins extracted from C2C12 differentiated (DM) for 0 d, 1 d, 3 d and 5 d were used for Western blotting assay of Pax7. α-Tubulin was used as a loading control. (G) Left: C2C12 myoblasts were transfected with Vector or YY1 expression plasmid. Pax7 mRNA expression was then measured in extracts from cells 48 hr after transfection using GAPDH as normalization. Right: C2C12 myoblasts were transfected with siNC or siYY1 oligos. Pax7 mRNA expression was then measured in extracts from cells 48 hr after transfection using GAPDH as normalization. Expression folds are shown with respect to Vector or siNC control where Pax7 levels were set to a value of 1. Values are represented as mean ± S.D. The p value was determined by Student's T-test: *p

    Journal: PLoS ONE

    Article Title: A Novel YY1-miR-1 Regulatory Circuit in Skeletal Myogenesis Revealed by Genome-Wide Prediction of YY1-miRNA Network

    doi: 10.1371/journal.pone.0027596

    Figure Lengend Snippet: miR-1 targets Pax7 during C2C12 differentiation. (A) Predicted target sites, A and B, of miR-1 in the 3′UTR of mouse Pax7. (B) A luciferase reporter plasmid was generated by cloning a ∼800 bp region of Pax7 3′UTR encompassing both site A and B downstream of the luciferase (Luc) reporter gene. The reporter construct was then transfected into C2C12 cells with negative control (NC) or miR-1 oligos along with Renilla luciferase plasmid. Luciferase activities were determined at 48 h post-transfection and normalized to Renilla readings. The data represent the average of three independent experiments ± S.D. (C) A mutation was introduced in either site A (Mut A) or site B (Mut B) or both (Mut A+B). Their responses to miR-1 over-expression were tested as above. (D) C2C12 myoblasts were transfected with either NC or miR-1 oligos. Both miR-1 and Pax7 mRNAs levels were then measured 48 hr post-transfection. (E) HDAC4 or Pax7 proteins were probed in extracts from cells 48 hr after transfection. Blots were stripped and reprobed for α-Tubulin as the loading control. (F) Proteins extracted from C2C12 differentiated (DM) for 0 d, 1 d, 3 d and 5 d were used for Western blotting assay of Pax7. α-Tubulin was used as a loading control. (G) Left: C2C12 myoblasts were transfected with Vector or YY1 expression plasmid. Pax7 mRNA expression was then measured in extracts from cells 48 hr after transfection using GAPDH as normalization. Right: C2C12 myoblasts were transfected with siNC or siYY1 oligos. Pax7 mRNA expression was then measured in extracts from cells 48 hr after transfection using GAPDH as normalization. Expression folds are shown with respect to Vector or siNC control where Pax7 levels were set to a value of 1. Values are represented as mean ± S.D. The p value was determined by Student's T-test: *p

    Article Snippet: Oligonucleotides Precursor miRNA oligos were obtained from Ambion. siRNA oligos against mouse YY1 were obtained from Santa Cruz technologies.

    Techniques: Luciferase, Plasmid Preparation, Generated, Clone Assay, Construct, Transfection, Negative Control, Mutagenesis, Over Expression, Western Blot, Expressing

    YY1 negatively regulates miR-1/133 expression both in vitro and in vivo . (A) C2C12 myoblasts were grown in growth medium (GM) or differentiation medium (DM) for 2, 3 or 5 days. Total RNAs or proteins were extracted and used for real-time RT-PCR assay (left) or Western blot analysis (right panel), respectively. (B) Primary myoblasts were isolated from limb muscles of 1 week old C57/BL6 mice and maintained in GM or induced to differentiate in DM. Cell morphology was visualized under light microscopy (left). Real-time PCR was performed to measure the expression levels of miR-1 and miR-133 normalized to U6 (middle). Semi-quantitative RT-PCR analysis was performed to measure the expression levels of Troponin, MyHC, α-Actin and YY1. Water (H 2 O) was used as negative control and GAPDH was used as a normalization. (C) Total proteins were isolated from lower limb muscles at post-natal day (P) 3 and 8 or tibialis anterior (TA) muscles from 2, 4, or 5 week old C57/BL6 background mice and Western blotting was used to probe for YY1 protein expression with GAPDH as a loading control (left). Total RNAs were isolated and qRT-PCR was subsequently performed to measure the expression of miR-1 and miR-133, normalized to U6 (middle and right). Expression folds are shown with respect to 3 day old mice where miR-1 and miR-133 levels were set to a value of 1. (D) TA muscles were isolated from 3 w, 4 w, 5 w, 8 w and 10 w old C57BL/6 wild type mice or mdx mice. RNAs were extracted and used for qRT-PCR assay of miR-1 (left), miR-133 (middle) or YY1 (right). Expression folds are shown with respect to wild type where miR-1, miR-133 or YY1 levels were set to a value of 1. (E) C2C12 myoblasts or (F) primary myoblasts were transfected with either negative control (siNC) or siRNA oligos against YY1 (siYY1). Cells were then cultured for 48 hours, at which time miR-1 and miR-133 expressions were measured by qRT-PCR and normalized to U6. Expression folds are shown with respect to siNC where miR-1 and miR-133 levels were set to a value of 1. (G) Expression of the primary transcripts of miR-1-2/miR-133a-1 was detected by qRT-PCR in C2C12 transfected with siYY1 or siNC oligos, and normalized to GAPDH. All quantitative data are represented as mean ± S.D. The p value was determined by Student's T-test: *p

    Journal: PLoS ONE

    Article Title: A Novel YY1-miR-1 Regulatory Circuit in Skeletal Myogenesis Revealed by Genome-Wide Prediction of YY1-miRNA Network

    doi: 10.1371/journal.pone.0027596

    Figure Lengend Snippet: YY1 negatively regulates miR-1/133 expression both in vitro and in vivo . (A) C2C12 myoblasts were grown in growth medium (GM) or differentiation medium (DM) for 2, 3 or 5 days. Total RNAs or proteins were extracted and used for real-time RT-PCR assay (left) or Western blot analysis (right panel), respectively. (B) Primary myoblasts were isolated from limb muscles of 1 week old C57/BL6 mice and maintained in GM or induced to differentiate in DM. Cell morphology was visualized under light microscopy (left). Real-time PCR was performed to measure the expression levels of miR-1 and miR-133 normalized to U6 (middle). Semi-quantitative RT-PCR analysis was performed to measure the expression levels of Troponin, MyHC, α-Actin and YY1. Water (H 2 O) was used as negative control and GAPDH was used as a normalization. (C) Total proteins were isolated from lower limb muscles at post-natal day (P) 3 and 8 or tibialis anterior (TA) muscles from 2, 4, or 5 week old C57/BL6 background mice and Western blotting was used to probe for YY1 protein expression with GAPDH as a loading control (left). Total RNAs were isolated and qRT-PCR was subsequently performed to measure the expression of miR-1 and miR-133, normalized to U6 (middle and right). Expression folds are shown with respect to 3 day old mice where miR-1 and miR-133 levels were set to a value of 1. (D) TA muscles were isolated from 3 w, 4 w, 5 w, 8 w and 10 w old C57BL/6 wild type mice or mdx mice. RNAs were extracted and used for qRT-PCR assay of miR-1 (left), miR-133 (middle) or YY1 (right). Expression folds are shown with respect to wild type where miR-1, miR-133 or YY1 levels were set to a value of 1. (E) C2C12 myoblasts or (F) primary myoblasts were transfected with either negative control (siNC) or siRNA oligos against YY1 (siYY1). Cells were then cultured for 48 hours, at which time miR-1 and miR-133 expressions were measured by qRT-PCR and normalized to U6. Expression folds are shown with respect to siNC where miR-1 and miR-133 levels were set to a value of 1. (G) Expression of the primary transcripts of miR-1-2/miR-133a-1 was detected by qRT-PCR in C2C12 transfected with siYY1 or siNC oligos, and normalized to GAPDH. All quantitative data are represented as mean ± S.D. The p value was determined by Student's T-test: *p

    Article Snippet: Oligonucleotides Precursor miRNA oligos were obtained from Ambion. siRNA oligos against mouse YY1 were obtained from Santa Cruz technologies.

    Techniques: Expressing, In Vitro, In Vivo, Quantitative RT-PCR, Western Blot, Isolation, Mouse Assay, Light Microscopy, Real-time Polymerase Chain Reaction, Negative Control, Transfection, Cell Culture

    miR-1 inhibits YY1 expression through targeting its 3′UTR. (A) Predicted target site of miR-1 in the 3′UTR of mouse YY1. (B) A wild type (WT) luciferase reporter plasmid was generated by fusing a ∼500 bp fragment of the YY1 3′UTR encompassing the miR-1 binding site downstream of the luciferase (Luc) reporter gene. The mutant plasmid was generated by mutating the miR-1 binding site from ACAUUCU to GGGCCUU. WT or Mutant reporter construct was transfected into C2C12 cells with indicated miRNA oligos and Renilla luciferase reporter plasmid. Luciferase activities were determined at 48 h post-transfection and normalized to Renilla readings. Relative Luciferase Unit (RLU) is shown with respect to wild type and NC transfection where luciferase activities were set to a value of 1. The data represent the average of three independent experiments ± S.D. (C) Upper: C2C12 myoblasts were transfected with either NC or miR-1 oligos. Total RNAs were used to detect YY1 expression level with GAPDH as normalization. Expression folds are shown with respect to negative control where YY1 levels were set to a value of 1. Quantitative values are represented as mean ± S.D. Lower: YY1 protein was then probed in extracts from cells 48 hr after transfection. Blots were stripped and reprobed for α-Tubulin as the loading control. The p value was determined by Student's T-test: *p

    Journal: PLoS ONE

    Article Title: A Novel YY1-miR-1 Regulatory Circuit in Skeletal Myogenesis Revealed by Genome-Wide Prediction of YY1-miRNA Network

    doi: 10.1371/journal.pone.0027596

    Figure Lengend Snippet: miR-1 inhibits YY1 expression through targeting its 3′UTR. (A) Predicted target site of miR-1 in the 3′UTR of mouse YY1. (B) A wild type (WT) luciferase reporter plasmid was generated by fusing a ∼500 bp fragment of the YY1 3′UTR encompassing the miR-1 binding site downstream of the luciferase (Luc) reporter gene. The mutant plasmid was generated by mutating the miR-1 binding site from ACAUUCU to GGGCCUU. WT or Mutant reporter construct was transfected into C2C12 cells with indicated miRNA oligos and Renilla luciferase reporter plasmid. Luciferase activities were determined at 48 h post-transfection and normalized to Renilla readings. Relative Luciferase Unit (RLU) is shown with respect to wild type and NC transfection where luciferase activities were set to a value of 1. The data represent the average of three independent experiments ± S.D. (C) Upper: C2C12 myoblasts were transfected with either NC or miR-1 oligos. Total RNAs were used to detect YY1 expression level with GAPDH as normalization. Expression folds are shown with respect to negative control where YY1 levels were set to a value of 1. Quantitative values are represented as mean ± S.D. Lower: YY1 protein was then probed in extracts from cells 48 hr after transfection. Blots were stripped and reprobed for α-Tubulin as the loading control. The p value was determined by Student's T-test: *p

    Article Snippet: Oligonucleotides Precursor miRNA oligos were obtained from Ambion. siRNA oligos against mouse YY1 were obtained from Santa Cruz technologies.

    Techniques: Expressing, Luciferase, Plasmid Preparation, Generated, Binding Assay, Mutagenesis, Construct, Transfection, Negative Control

    Localization of ASOs in auditory hair cells at P30 after systemic treatment in Ush1c mice . Immunofluorescent labeling of ASO-29 (red) in HCs (green) at the a , b apex-mid turn at P30 after ASO treatment at P1 ( a ) or P5 ( b ). c Immunofluorescent image at the apex-middle turn of P5 vehicle-treated (saline), control littermate 1 week after P5 ASO treatment (P12). Scale bars indicate 10 μm. IHC, inner hair cell; OHC, outer hair cell; ASO, antisense oligonucleotide

    Journal: JARO: Journal of the Association for Research in Otolaryngology

    Article Title: Rescue of Outer Hair Cells with Antisense Oligonucleotides in Usher Mice Is Dependent on Age of Treatment

    doi: 10.1007/s10162-017-0640-x

    Figure Lengend Snippet: Localization of ASOs in auditory hair cells at P30 after systemic treatment in Ush1c mice . Immunofluorescent labeling of ASO-29 (red) in HCs (green) at the a , b apex-mid turn at P30 after ASO treatment at P1 ( a ) or P5 ( b ). c Immunofluorescent image at the apex-middle turn of P5 vehicle-treated (saline), control littermate 1 week after P5 ASO treatment (P12). Scale bars indicate 10 μm. IHC, inner hair cell; OHC, outer hair cell; ASO, antisense oligonucleotide

    Article Snippet: ASOs Antisense oligonucleotides (2′-O -methoxyethyl-modified, Ionis Pharmaceuticals, Inc.) targeting the Ush1c c.216G > A mutation (ASO-29) (5′-AGCTGATCATATTCTACC-3′) and a non-specific control (ASO-C) (5′-TTAGTTTAATCACGCTCG-3′) were generated as previously described by (Lentz et al. ).

    Techniques: Mouse Assay, Labeling, Allele-specific Oligonucleotide, Immunohistochemistry

    Localization of ASOs in auditory hair cells at P12 after systemic treatment in Ush1c mice . Immunofluorescent labeling of ASO-29 (red) in HCs (green) at the a , b apex, c , d middle turn, and e , f base at P12 after ASO treatment at P1 or P5. Distance from the apex tip is indicated. Scale bars indicate 10 μm. IHC, inner hair cell; OHC, outer hair cell; ASO, antisense oligonucleotide

    Journal: JARO: Journal of the Association for Research in Otolaryngology

    Article Title: Rescue of Outer Hair Cells with Antisense Oligonucleotides in Usher Mice Is Dependent on Age of Treatment

    doi: 10.1007/s10162-017-0640-x

    Figure Lengend Snippet: Localization of ASOs in auditory hair cells at P12 after systemic treatment in Ush1c mice . Immunofluorescent labeling of ASO-29 (red) in HCs (green) at the a , b apex, c , d middle turn, and e , f base at P12 after ASO treatment at P1 or P5. Distance from the apex tip is indicated. Scale bars indicate 10 μm. IHC, inner hair cell; OHC, outer hair cell; ASO, antisense oligonucleotide

    Article Snippet: ASOs Antisense oligonucleotides (2′-O -methoxyethyl-modified, Ionis Pharmaceuticals, Inc.) targeting the Ush1c c.216G > A mutation (ASO-29) (5′-AGCTGATCATATTCTACC-3′) and a non-specific control (ASO-C) (5′-TTAGTTTAATCACGCTCG-3′) were generated as previously described by (Lentz et al. ).

    Techniques: Mouse Assay, Labeling, Allele-specific Oligonucleotide, Immunohistochemistry

    Effects of SR-18292 on PGC-1 α acetylation and gluconeogenic genes in isolated primary hepatocytes . (A) SR-18292 is identical to C-80 except the methyl group on the 3′ position on the indole group. (B) Western blot analysis of PGC-1α acetylation following treatment with SR-18292 in the presence of the SIRT1 inhibitor, Ex527 (5μM) and the pan HDACs inhibitor TSA (1μM). Hepatocytes were infected with Ad-PGC-1α and Ad-GCN5 and treated with SR-18292 for 18 h. IP, immunoprecipitation. (C) – (D) qPCR analysis of Ppargc1a , Pck1 and G6pc mRNA expression levels following treatment with SR-18292 (20μM) in isolated primary hepatocytes. (E) qPCR analysis of Ppargc1a , Pck1 and G6pc mRNA expression levels following SR-18292 (20μM) treatment and siRNA to reduce to expression of Ppargc1a . Difference in % repression of Pck1 was calculated using two-way ANOVA with Sidak posttest. (F) – (G) Glucose production by primary hepatocytes following treatment with SR-18292 (20μM). Hepatocytes were treated with either glucagon (200nM) or infected with Ad-PGC-1α to induce glucose production. All data presented as mean +/− S.E.M. n=3, one-way ANOVA with Tuckey posttest. Representative of at least 2 independent experiments. *P

    Journal: Cell

    Article Title: Selective Chemical Inhibition of PGC-1α Gluconeogenic Activity Ameliorates Type 2 Diabetes

    doi: 10.1016/j.cell.2017.03.001

    Figure Lengend Snippet: Effects of SR-18292 on PGC-1 α acetylation and gluconeogenic genes in isolated primary hepatocytes . (A) SR-18292 is identical to C-80 except the methyl group on the 3′ position on the indole group. (B) Western blot analysis of PGC-1α acetylation following treatment with SR-18292 in the presence of the SIRT1 inhibitor, Ex527 (5μM) and the pan HDACs inhibitor TSA (1μM). Hepatocytes were infected with Ad-PGC-1α and Ad-GCN5 and treated with SR-18292 for 18 h. IP, immunoprecipitation. (C) – (D) qPCR analysis of Ppargc1a , Pck1 and G6pc mRNA expression levels following treatment with SR-18292 (20μM) in isolated primary hepatocytes. (E) qPCR analysis of Ppargc1a , Pck1 and G6pc mRNA expression levels following SR-18292 (20μM) treatment and siRNA to reduce to expression of Ppargc1a . Difference in % repression of Pck1 was calculated using two-way ANOVA with Sidak posttest. (F) – (G) Glucose production by primary hepatocytes following treatment with SR-18292 (20μM). Hepatocytes were treated with either glucagon (200nM) or infected with Ad-PGC-1α to induce glucose production. All data presented as mean +/− S.E.M. n=3, one-way ANOVA with Tuckey posttest. Representative of at least 2 independent experiments. *P

    Article Snippet: siRNA oligos against mouse Ppargc1a were purchased from OriGene (SR420231).

    Techniques: Pyrolysis Gas Chromatography, Isolation, Western Blot, Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing