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  • 90
    Qiagen sirna oligos
    Ubiquitination of rΔF508 CFTR by CHIP occurs in the post-endocytic compartments. (A) CFBE41o-ΔF cells were transfected with control or CHIP <t>siRNA</t> <t>oligonucleotides</t> at 37°C and then incubated for 48 h at 27°C to rescue the ΔF508 CFTR to the cell surface. During the last 16 h of low-temperature incubation, 150 μg/ml of cycloheximide was added to the culture to inhibit protein synthesis and eliminate the internal biosynthetic pool of ΔF508 CFTR. The cells were then switched to 37°C to promote endocytosis of the rΔF508 CFTR for the time periods indicated. The CFTR was then pulled-down by immunoprecipitation with specific antibody (24–1) and blotted with an ubiquitin antibody. The ubiquitin level is increased after 15 to 60 min warm-up at 37°C incubator in control cells (upper panel). The increase of ubiquitinated rΔF508 CFTR was attenuated under CHIP depletion (middle panel). The lower panel shows that the ubiquitination of rescued ΔF508 CFTR was reduced in the presence of 5 μM Dyngo 4a, which inhibits endocytosis. The relative ubiquitin level of rΔF508 CFTR after 37°C warm-up was analyzed in control, CHIP depleted and Dyngo 4a-treated cells (n = 3). (B) The ubiquitinated pool of rΔF508 CFTR was analyzed after Dab2 or CHIP depletion as described above. Dab2 depletion increases the CHIP-mediated ubiquitination of rΔF508 CFTR by blocking the post-endocytic trafficking.
    Sirna Oligos, supplied by Qiagen, used in various techniques. Bioz Stars score: 90/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Millipore biotin labeled ssrna oligos
    ( A ) Schematic drawing of the HPV16 region around late 3′-splice site SA5639 and the 35-nucleotide biotinylated ssDNA <t>oligos</t> (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA5639 is indicated. ( B ) Upper and lower panels show pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the region of HPV16 late 3′-splice site SA5639 followed by Western blot analysis using antibodies indicated to the right. (–) mock pull downs using streptavidin beads in the absence of oligo. ( C ) Quantitation of some of the Western blots of the pull downs in (B). ( D and E ) Western blots of indicated proteins on pull downs using biotinylated <t>ssRNA</t> oligos of a subset of the ssDNA oligos shown in (B).
    Biotin Labeled Ssrna Oligos, supplied by Millipore, used in various techniques. Bioz Stars score: 83/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher sirna oligos
    Ubiquitination of rΔF508 CFTR by CHIP occurs in the post-endocytic compartments. (A) CFBE41o-ΔF cells were transfected with control or CHIP <t>siRNA</t> <t>oligonucleotides</t> at 37°C and then incubated for 48 h at 27°C to rescue the ΔF508 CFTR to the cell surface. During the last 16 h of low-temperature incubation, 150 μg/ml of cycloheximide was added to the culture to inhibit protein synthesis and eliminate the internal biosynthetic pool of ΔF508 CFTR. The cells were then switched to 37°C to promote endocytosis of the rΔF508 CFTR for the time periods indicated. The CFTR was then pulled-down by immunoprecipitation with specific antibody (24–1) and blotted with an ubiquitin antibody. The ubiquitin level is increased after 15 to 60 min warm-up at 37°C incubator in control cells (upper panel). The increase of ubiquitinated rΔF508 CFTR was attenuated under CHIP depletion (middle panel). The lower panel shows that the ubiquitination of rescued ΔF508 CFTR was reduced in the presence of 5 μM Dyngo 4a, which inhibits endocytosis. The relative ubiquitin level of rΔF508 CFTR after 37°C warm-up was analyzed in control, CHIP depleted and Dyngo 4a-treated cells (n = 3). (B) The ubiquitinated pool of rΔF508 CFTR was analyzed after Dab2 or CHIP depletion as described above. Dab2 depletion increases the CHIP-mediated ubiquitination of rΔF508 CFTR by blocking the post-endocytic trafficking.
    Sirna Oligos, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 571 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Gene Tools Inc stx1b morpholino oligos
    Larval seizure was aggravated by downregulation of <t>stx1b</t> transcription in the PTZ-induced seizure model. Levels of stx1b gene transcription ( n = 20) (A) and STX1B protein ( n = 3) (B) were reduced and c-fos gene transcription was increased in the larval (7 dpf) brain ( n = 20) (C) by stx1b <t>morpholino</t> <t>oligos</t> injection in the PTZ model, as compared with in the PTZ-only and the morpholino oligos-only injection models. The larval swimming experiment ( n = 24) (D) showed that average speed and total distance were not changed, but that the abnormal pathway and overspeed were increased following 20 min in the dark condition and that photosensitive seizure was aggravated under the condition of light–dark transition with 5 min in the dark and 10 s in the light for three cycles in the PTZ plus stx1b morpholino oligos larvae, as compared with the two groups of the PTZ-only and the stx1b morpholino oligos-only injection models. The data show average speeds during the 20 min in the dark and the 10 s in the dark–light transformation; the boxes indicate the difference of locomotion distances and speeds between the light–dark transitions. Swimming tracks were recorded at 2 min in the dark condition and the red trajectory indicates overactive movement and the green trajectory indicates active movement. stx1b -MO1 and stx1b -MO2 were two morpholino oligos that bound to the stx1b messenger RNA initiate sequence with a different sequence; by using two target oligos, their inhibition effect was confirmed with each other. ∗∗∗ P
    Stx1b Morpholino Oligos, supplied by Gene Tools Inc, used in various techniques. Bioz Stars score: 88/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Thermo Fisher synthetic ie3 dna oligos
    Summary of ICP4 domains and sites of <t>DNA</t> interaction and homo-dimerization. ( A ) The domain composition of HSV1 ICP4 with predicted secondary structure (SSpred) and ordered regions (ORDERpred) from PSIPRED ( 37 ). Predicted α-helices and β-sheets are shown as red and yellow respectively, with ordered regions colored green. ( B ) Sequence alignment of ICP4 DBDs from HSV1, HSV2 and VZV (Uniprot codes: P08392, P90493, Q8AZM1 respectively) from Clustal omega ( 36 ). Secondary structure elements determined here are labeled on the HSV-1 sequence. Residues previously probed by mutagenesis are highlighted blue with black arrows pointing to those with lower affinity for DNA ( 13 , 14 , 16 ). Below the sequences, blocks colored gray or purple indicate residues which form homo-dimer or DNA contacts respectively as observed in the crystallography data. Purple circles indicate protein–DNA contacts derived from NMR chemical shift perturbations in intrinsically disordered regions (IDRs). ( C ) Cartoon representation of the crystal structure with helices, sheets and loops shown as cylinders, arrows and coil respectively: (i) ICP4N · <t>IE3_19mer</t> with DNA colored purple and pink for sense and anti-sense strands respectively, one protein chain is colored gray and other blue through red from N-to-C termini. (ii) As with panel i with view rotated 90°.
    Synthetic Ie3 Dna Oligos, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Eurofins o me rna oligos
    ( A ) Schematic representation of HPV16 plasmid pHPV16ANE2fs that is integrated in the genome of the transfected and immortalized human keratinocyte cell line 3310 ( 42 ). Open reading frames are represented as rectangles, the early and late promoters p97 and p670 are indicated as arrows, and splice sites as triangles. The long control region (LCR) and the early and late polyA signals pAE and pAL are indicated. Two loxP sites and two SphI restriction sites flanking the HPV16 genome are indicated, as is the RSV-neoR-SV40pA cassette. The E2 frame-shift mutation is indicated. ( B ) Western blot analysis of differentiation marker involucrin, phosphorylated Akt kinase (p-Akt S473) and the Akt phosphorylation substrate p-GSK3b and b-actin in the absence (DMSO) or presence of Akt inhibitor GDC0068, and in the absence (–) or presence (+) of Ca-induced differentiation. ( C ) RT-PCR of HPV16 L1, L1i, E4 and GAPDH mRNAs in the absence (DMSO) or presence of Akt inhibitor GDC0068, and in the absence (–) or presence (+) of Ca-induced differentiation. ( D ) Upper left panel: RT-PCR of HPV16 E4 mRNAs spliced from SD880 to SA3358 in 3310 cells grown in the absence (DMSO) or presence of Akt inhibitor GDC0068, and in the absence (–) or presence (+) of Ca-induced differentiation. Upper right panel: extracts from UV irradiated 3310 cells grown in the absence (DMSO) or presence of Akt inhibitor GDC0068, and in the absence (–) or presence (+) of Ca-induced differentiation were subjected to immunoprecipitation with IgG or anti-hnRNP L antibody followed by <t>RNA</t> extraction and RT-PCR with <t>primers</t> that detect E4 mRNAs spliced from SD880 to SA3358. ( E ) RT-qPCR on the HPV16 E4 mRNAs spliced from SD880 to SA3358 shown in (D).
    O Me Rna Oligos, supplied by Eurofins, used in various techniques. Bioz Stars score: 83/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Eurofins biotin labeled ssdna oligos
    ( A ) Schematic drawing of the HPV16 region around late 3′-splice site SA5639 and the 35-nucleotide biotinylated <t>ssDNA</t> <t>oligos</t> (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA5639 is indicated. ( B ) Upper and lower panels show pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the region of HPV16 late 3′-splice site SA5639 followed by Western blot analysis using antibodies indicated to the right. (–) mock pull downs using streptavidin beads in the absence of oligo. ( C ) Quantitation of some of the Western blots of the pull downs in (B). ( D and E ) Western blots of indicated proteins on pull downs using biotinylated ssRNA oligos of a subset of the ssDNA oligos shown in (B).
    Biotin Labeled Ssdna Oligos, supplied by Eurofins, used in various techniques. Bioz Stars score: 83/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Millipore tpx2 sirna oligos pool
    Increase of sub-G1 and reduction of G1 to S phase cells via <t>TPX2</t> silencing. Notes: ( A ) PPC1 cells were synchronized by double thymidine and nocodazol (Thy–Thy–Noc) block or double thymidine (Thy–Thy) block and then released for the indicated time period. The phase of cell fracture during cell cycle progression was monitored through protein expression evolution. Cyclin B1 indicated G2/M phase progression, and cyclin A indicated S to G2/M phase progression. TPX2 protein levels were measured using a Western blot assay. ( B ) Scrambled <t>siRNA</t> (20 nM, si-CTL) or the siRNA <t>oligos</t> pool against TPX2 (20 nM, si-TPX2) was transfected into PPC1 cells for 72 h, followed by cell harvesting. The knockdown cells were fixed and stained with DAPI to examine the proportions of the cell cycle by using an image-flow cytometry assay. ( C ) Representative data for cell cycle proportions were analyzed and quantified using Nucleoview NC-3000 software. The results are expressed as mean ± SD from three individual experiments (Student’s t -test, P
    Tpx2 Sirna Oligos Pool, supplied by Millipore, used in various techniques. Bioz Stars score: 83/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Cell Signaling Technology Inc sirna oligos targeting ampk α2
    AMP-activated protein kinase <t>(AMPK)-phosphoprotein</t> enriched in astrocytes 15 kDa (PEA15) axis is critical for the anchorage-independent growth of breast cancer cells. (A) Michigan Cancer Foundation 7 breast cancer cell line (MCF7), BT-474, and primary breast cancer-derived cells cultured in adherent (ADH) condition or as cancer spheres (CS) for a week were harvested and subjected to immunoblotting; n = 3. (B) MCF7 cells cultured in methylcellulose for a week in the presence of 10 μM AMPK inhibitor Compound C or dimethyl sulphoxide (DMSO) (vehicle control) were subjected to immunoblotting. Graph represents number of spheres formed/20 fields. Error bars represent standard error of the mean (SEM); n = 3. (C-F) Adherent MCF7 breast cancer cells were transfected with specified small interfering RNA <t>(siRNA)</t> <t>oligos/plasmids</t> (see below). Two days posttransfection, 1 × 10 5 cells/35 mm dish were seeded in methylcellulose. After 48 hrs, cells were retrieved from some dishes and subjected to immunoblot analyses for the specified proteins. Parallel dishes were allowed to form spheres; graph represents sphere formation at the end of a week, error bar represents SEM: (C) control siRNA or siRNA targeting AMPK <t>α2,</t> n = 4; (D) control siRNA or siRNA targeting PEA15, (n = 4); (E) control siRNA or siRNA targeting PEA15 and seeded in methylcellulose in the presence of 100 μM AMPK activator, A769662; DMSO served as vehicle control, n = 4; (F) transfected with CSCG constructs expressing flag-tagged wild-type (WT) or S116A mutant of PEA15, n = 4. (G) MDAMB231 cells were treated with DMSO or 100 μM AMPK activator, A769662, and immunoprecipitated with anti-Fas-associated death domain protein (FADD) antibody. The immunoprecipates were resolved by SDS-PAGE followed by immunoblotting for specified proteins, n = 3. (H) BT 474 cells stably expressing flag-tagged WT-PEA15 or PEA15-S116A mutant were seeded in methyl cellulose for two days, retrieved and immunoprecipitated with anti-flag antibody. The immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting for specified proteins, n = 3.
    Sirna Oligos Targeting Ampk α2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 82/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher sirna oligos targeting rab10
    The <t>Rab10-EHBP1-EHD2</t> complex mediates the engulfment of LDs by autophagic organelles. ( A ) Live-cell confocal fluorescence microscopy of two distinct LDs (stained with MDH, blue) from Hep3B cells expressing either mCherry-Rab10 (top series) or GFP-Rab10 (lower series). Imaging reveals the association of LD-bound Rab10 at early time points with phagophore/autophagosome-associated Rab10 (0 s) extending to nearly surround the perimeter of LDs at later time points. Dashed outlines provide fiducial points of reference as the envelopment of the LD by the phagophore progresses. These events are representative of data from more than 30 individual cells examined by live-cell imaging. ( B and C ) Quantification of the percentage of LDs associated with LC3- or Atg16L1-positive structures in Hep3B hepatoma cells after 48-hour <t>siRNA</t> treatment with the indicated siRNAs (Tri-siRNA, triple knockdown). Cells were preloaded with 150 μM oleic acid overnight. ( D and E ) Quantification of the percentage of LDs associated with LC3- or Atg16L1-positive structures after culture in low-serum conditions in WT or Rab10 KO MEFs. Cells were preloaded with 400 μM oleic acid overnight. ( F ) Quantification of the percentage of LDs associated with LAMP1-positive structures after 48-hour siRNA treatment followed by 48-hour starvation from n = 3 independent experiments, measuring 20 cells per condition. Cells were preloaded with 150 μM oleic acid overnight. ( G and H ) LDs visualized from WT or Rab10 KO MEFs were divided into three groups on the basis of their association with LAMP-1: “none,” “attached,” or “engulfed” (G). Manual counting of LDs (H) in each group from WT and Rab10 KO MEFs. The graphs represent observations from n = 3 independent experiments, measuring 20 cells per condition. Cells were preloaded with 400 μM oleic acid overnight. Data are represented as means ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Scale bars, 1 μm.
    Sirna Oligos Targeting Rab10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Ribobio sirna oligos against mouse malat1
    <t>Malat1</t> interacts and recruits Suv39h1 to MyoD-binding loci. ( a ) The expression levels of Suv39h1, HDAC1 and Hp1β proteins in Vector or sh Malat1 C2C12 cells were determined by western blotting in proliferating myoblasts. α-Tubulin was used as a normalization control. ( b ) Nuclear extracts from the above cells were immunoprecipitated by IgG or an antibody against Suv39h1. The retrieved RNAs were detected by qRT-PCR. ( c ) In vitro -transcribed biotinylated Malat1 FL transcripts were used to pull down its binding proteins. The indicated proteins were detected by western blotting. Beads only or antisense Malat1 transcripts were used as negative controls. ( d ) Upper: schematic illustration of the Malat1 FL or fragments (F1, F2, F3 or F4) that were used in the RNA pull-down assays for mapping the functional domain. Beads only or GFP transcripts were used as negative controls. Middle: the retrieved Suv39h1 proteins were detected by western blotting. The numbers below indicate the quantification of the intensity of each band. Lower: the in vitro generated transcripts were visualized on an agarose gel. ( e ) EMSA assays were performed to detect the association between Malat1 FL or various fragments with purified GST-Suv39h1 protein (for 200 fmol Malat1 FL RNA, 100, 400, 800 or 1 600 n M GST-Suv39h1 were used. For 400 fmol F1–F4 fragments, 800, 1 600, 2 400 or 3 200 n M GST-Suv39h1 protein were used). Antisense Malat1 transcript was used as an unrelated negative control for RNA. GST only protein (3 200 n M ) was used as a negative control for GST-Suv39h1 protein. A shifted band (lanes 5 and 6) ran higher than the control bands (lanes 1 and 2) and also displayed lower intensity. ( f ) Malat1 ChIRP with both even and odd antisense <t>oligos</t> retrieved a significant amount of genomic DNAs corresponding to the promoter regions of Myogenin , MyHC , Tnni2 and Ccnd3 genes but not GAPDH locus. LacZ ChIRP retrieved no signal. ( g – k ) ChIP-PCR analysis of Suv39h1, HP1β, HDAC1, H3K9me3 and MyoD enrichment on the promoter or enhancer of Myogenin , MCK , MyHC , Troponin , Ccnd3 and p21 loci in Vector or sh Malat1 expressing C2C12 cells. The enrichment fold was calculated as a fraction of DNA present in the input samples. ( l ) 10T1/2 cells were transfected with the indicated combination of plasmids and si Malat1 oligos. The expression of the myogenic marker genes was analyzed by qRT-PCR. ( m ) 10T1/2 cells were co-transfected with a Myogenin or 4RE luciferase reporter, the indicated expression plasmids and si Malat1 oligos. Luciferase activities were measured after differentiating the cells for 48 h. All PCR data were normalized to GAPDH mRNA and represent the average of three independent experiments±s.d. All luciferase data were normalized to Renilla luciferase activities and represent the average of three independent experiments±s.d. * P
    Sirna Oligos Against Mouse Malat1, supplied by Ribobio, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    New England Biolabs spo11 oligo complexes
    <t>Spo11-oligo</t> complexes in msh5 and zip1 Representative time courses are shown.
    Spo11 Oligo Complexes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher sirna oligos targeting dyn2
    Model for a role of <t>Dyn2</t> in autophagic breakdown of lipid droplets in hepatocytes. After initiation of starvation, lipid droplets are enclosed by the double-membrane isolation membrane (phagophore), which engulfs the droplet, forming an autophagosome. Subsequent fusion with lysosomes results in the release of hydrolytic enzymes and lysosomal lipases into the autolysosomal compartment, which degrade the lipid droplets within. During the process of autophagic lysosomal reformation (top), nascent lysosomes are generated from membrane tubule extensions of this autolysosomal compartment. Dyn2 mediates the scission of these tubular structures. Depletion of Dyn2 by <t>siRNA</t> knockdown or inhibition of Dyn2 activity by pharmacological reagents (bottom) prevents tubular scission, eventually resulting in depleted lysosomal pools within the cell. Continued fusion of remaining lysosomes causes enlargement of the autolysosomal structures. A complete decrease in the recycled lysosomal population will prevent autophagic-mediated breakdown of hepatic lipid droplets.
    Sirna Oligos Targeting Dyn2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher custom designed sirna duplex oligos targeting gnrh e1 rna
    The effect of <t>GnRH-E1</t> RNA knockdown on Gnrh1 gene expression. (A-D) GT1-7 neurons were transfected with either negative <t>siRNA</t> control (Neg; black bars) or siRNA targeting both strands of the mouse GnRH-E1 RNA (siRNA; white bars). Total RNA was harvested at 36 hours, 48 hours, and 72 hours after siRNA transfection. RT-qPCR analysis was performed to quantify endogenous mouse GnRH-E1 RNA (A), transgene-derived rat (rTg) GnRH-E1 RNA (B), Gnrh1 pre-mRNA (C), and Gnrh1 mRNA (D) expression. Relative RNA expression is normalized to control histone 2A.Z ( H2afz ) mRNA expression at each time point. Data are displayed as the mean ± SD, where statistical significance was determined by Student’s t-test compared between negative control and siRNA treatment at each time point. Asterisk indicates statistical significance, where p
    Custom Designed Sirna Duplex Oligos Targeting Gnrh E1 Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher t oligo
    Increased reduction of MM-AN melanoma xenograft tumors by TOP complex in nu/nu mice. First, 2.5×10 6 MM-AN cells were injected into the flank of nu/nu mice (n=10). After 24 hours, the mice were treated daily with diluent, <t>T-oligo</t> (52 nmol), TOP complex (52 nmol T-oligo and 0.025 mg/mL <t>PVBLG)</t> intravenously for 3 weeks. Mice treated with T-oligo demonstrated reduced tumors, while most TOP complex treated mice showed no visible tumors. The graph shows a 3-fold and 9-fold reduction in tumor volume after treatment with T-oligo or the TOP complex, respectively, as compared with diluent ( P ,0.001). Abbreviations: TOP complex, T-oligo and PVBLG nanocomplex.
    T Oligo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Agilent technologies oligo array cgh platforms
    Detection of a homozygous deletion by the <t>oligo</t> array <t>CGH</t> platform. DNA from the cell lines MDA-MB-468 ( A ) and SUM159 ( C ) was hybridized with normal male reference DNA on a human oligo array. Log2ratios were calculated without moving average and are displayed in black as a function of their position on chromosome 13. The smoothed values of the log2ratios are displayed in red and the position of the RB1 oligo is indicated by the green circle in A and the arrow in C. Note the lack of the deletion in SUM159. Validation of the HD by FISH analysis in cell lines: MDA-MB-468 ( B ) and SUM159 ( D ). The green signal from the RB1 probe clearly shows the presence of RB1 in SUM159 (D), but is absent from MDA-MB-468 (B). Chromosome 13 paint (red) shows the presence of three normal copies of chromosome 13 and two marker chromosomes with chromosome 13 material.
    Oligo Array Cgh Platforms, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher cin directed sirna oligos duplexes
    Cofilin is required for the translocation of <t>CIN</t> at the cell leading edge. ( A , Left ) Depletion of cofilin in MTLn3 cells. MTLn3 cells were transfected with <t>shRNA</t> constructs, stimulated with EGF, and stained for endogenous cofilin (green in merge) and
    Cin Directed Sirna Oligos Duplexes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    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
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    Image Search Results


    Ubiquitination of rΔF508 CFTR by CHIP occurs in the post-endocytic compartments. (A) CFBE41o-ΔF cells were transfected with control or CHIP siRNA oligonucleotides at 37°C and then incubated for 48 h at 27°C to rescue the ΔF508 CFTR to the cell surface. During the last 16 h of low-temperature incubation, 150 μg/ml of cycloheximide was added to the culture to inhibit protein synthesis and eliminate the internal biosynthetic pool of ΔF508 CFTR. The cells were then switched to 37°C to promote endocytosis of the rΔF508 CFTR for the time periods indicated. The CFTR was then pulled-down by immunoprecipitation with specific antibody (24–1) and blotted with an ubiquitin antibody. The ubiquitin level is increased after 15 to 60 min warm-up at 37°C incubator in control cells (upper panel). The increase of ubiquitinated rΔF508 CFTR was attenuated under CHIP depletion (middle panel). The lower panel shows that the ubiquitination of rescued ΔF508 CFTR was reduced in the presence of 5 μM Dyngo 4a, which inhibits endocytosis. The relative ubiquitin level of rΔF508 CFTR after 37°C warm-up was analyzed in control, CHIP depleted and Dyngo 4a-treated cells (n = 3). (B) The ubiquitinated pool of rΔF508 CFTR was analyzed after Dab2 or CHIP depletion as described above. Dab2 depletion increases the CHIP-mediated ubiquitination of rΔF508 CFTR by blocking the post-endocytic trafficking.

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: Ubiquitination of rΔF508 CFTR by CHIP occurs in the post-endocytic compartments. (A) CFBE41o-ΔF cells were transfected with control or CHIP siRNA oligonucleotides at 37°C and then incubated for 48 h at 27°C to rescue the ΔF508 CFTR to the cell surface. During the last 16 h of low-temperature incubation, 150 μg/ml of cycloheximide was added to the culture to inhibit protein synthesis and eliminate the internal biosynthetic pool of ΔF508 CFTR. The cells were then switched to 37°C to promote endocytosis of the rΔF508 CFTR for the time periods indicated. The CFTR was then pulled-down by immunoprecipitation with specific antibody (24–1) and blotted with an ubiquitin antibody. The ubiquitin level is increased after 15 to 60 min warm-up at 37°C incubator in control cells (upper panel). The increase of ubiquitinated rΔF508 CFTR was attenuated under CHIP depletion (middle panel). The lower panel shows that the ubiquitination of rescued ΔF508 CFTR was reduced in the presence of 5 μM Dyngo 4a, which inhibits endocytosis. The relative ubiquitin level of rΔF508 CFTR after 37°C warm-up was analyzed in control, CHIP depleted and Dyngo 4a-treated cells (n = 3). (B) The ubiquitinated pool of rΔF508 CFTR was analyzed after Dab2 or CHIP depletion as described above. Dab2 depletion increases the CHIP-mediated ubiquitination of rΔF508 CFTR by blocking the post-endocytic trafficking.

    Article Snippet: siRNA duplexes corresponding to non-conserved regions of human Dab2, μ2 and c-Cbl were purchased from Qiagen Inc. siRNA oligos for Nedd4-2 knock-down were purchased from Ambion® (Life technologies).

    Techniques: Chromatin Immunoprecipitation, Transfection, Incubation, Immunoprecipitation, Blocking Assay

    c-Cbl, CHIP or Nedd4-2 depletion do not affect rΔF508 CFTR endocytosis. CFBE41o-ΔF cells were transfected with control or c-Cbl, CHIP, or Nedd4-2 siRNA oligonucleotides. At 24 h after transfection, cells were transferred to Transwell filters and cultured for an additional 4–5 days. The last 24 h, the cells were cultured at 27°C to promote ΔF508 CFTR delivery to the cell surface. (A) Cell surface expression of rΔF508 CFTR after siRNA transfection of control (C), c-Cbl, CHIP or Nedd4-2 as indicated. The knockdown efficiency of c-Cbl, CHIP, and Nedd4-2 was > 95%. β-actin was blotted as a loading control. (B) Representative blots showing the remaining surface rΔF508 CFTR after 2.5 min internalization at 37°C. (C) Quantitative analysis of rΔF508 CFTR internalization after 2.5 min warm-up following c-Cbl, CHIP, or Nedd4-2 siRNA depletion and low-temperature rescue. The rate of rΔF508 CFTR internalization was measured after 2.5 min warm-up as described in the Material and Methods section. Depletion of c-Cbl, CHIP, or Nedd4-2 had no significant (N.S.) effect on rΔF508 CFTR internalization (n = 3).

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: c-Cbl, CHIP or Nedd4-2 depletion do not affect rΔF508 CFTR endocytosis. CFBE41o-ΔF cells were transfected with control or c-Cbl, CHIP, or Nedd4-2 siRNA oligonucleotides. At 24 h after transfection, cells were transferred to Transwell filters and cultured for an additional 4–5 days. The last 24 h, the cells were cultured at 27°C to promote ΔF508 CFTR delivery to the cell surface. (A) Cell surface expression of rΔF508 CFTR after siRNA transfection of control (C), c-Cbl, CHIP or Nedd4-2 as indicated. The knockdown efficiency of c-Cbl, CHIP, and Nedd4-2 was > 95%. β-actin was blotted as a loading control. (B) Representative blots showing the remaining surface rΔF508 CFTR after 2.5 min internalization at 37°C. (C) Quantitative analysis of rΔF508 CFTR internalization after 2.5 min warm-up following c-Cbl, CHIP, or Nedd4-2 siRNA depletion and low-temperature rescue. The rate of rΔF508 CFTR internalization was measured after 2.5 min warm-up as described in the Material and Methods section. Depletion of c-Cbl, CHIP, or Nedd4-2 had no significant (N.S.) effect on rΔF508 CFTR internalization (n = 3).

    Article Snippet: siRNA duplexes corresponding to non-conserved regions of human Dab2, μ2 and c-Cbl were purchased from Qiagen Inc. siRNA oligos for Nedd4-2 knock-down were purchased from Ambion® (Life technologies).

    Techniques: Chromatin Immunoprecipitation, Transfection, Cell Culture, Expressing

    CHIP Depletion increases the function of rΔF508 CFTR. CFBE41o-ΔF cells were transfected with control or CHIP siRNA oligonucleotides, seeded on to Transwell filters and the I SC across the monolayers was measured in Ussing chambers as described in Fig 4 legend. (A) Representative tracings from control and CHIP-depleted monolayers. After a stable baseline was attained, 20 μM forskolin (F), 50 μM genistein (G) and 10 μM CFTR inh -172 were added at the indicated arrows. (B) Forskolin and genistein activated I SC . ΔI SC was calculated as an increase in I SC after forskolin and genistein addition over the base-line currents. C. CFTR inh -172 inhibited I SC . ΔI SC was calculated as a decrease in I SC after CFTR inh -172 addition. (n = 6)

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: CHIP Depletion increases the function of rΔF508 CFTR. CFBE41o-ΔF cells were transfected with control or CHIP siRNA oligonucleotides, seeded on to Transwell filters and the I SC across the monolayers was measured in Ussing chambers as described in Fig 4 legend. (A) Representative tracings from control and CHIP-depleted monolayers. After a stable baseline was attained, 20 μM forskolin (F), 50 μM genistein (G) and 10 μM CFTR inh -172 were added at the indicated arrows. (B) Forskolin and genistein activated I SC . ΔI SC was calculated as an increase in I SC after forskolin and genistein addition over the base-line currents. C. CFTR inh -172 inhibited I SC . ΔI SC was calculated as a decrease in I SC after CFTR inh -172 addition. (n = 6)

    Article Snippet: siRNA duplexes corresponding to non-conserved regions of human Dab2, μ2 and c-Cbl were purchased from Qiagen Inc. siRNA oligos for Nedd4-2 knock-down were purchased from Ambion® (Life technologies).

    Techniques: Chromatin Immunoprecipitation, Transfection

    Increased transepithelial chloride transport following Dab2 depletion in CFBE41o-ΔF monolayers. CFBE41o-ΔF cells were transfected with control or Dab2 siRNA oligonucleotides. At 24 h after transfection, cells were lifted, seeded on to Transwell filters and cultured for an additional 4–5 days. The last 24 h, the cells were cultured at 27°C to promote ΔF508 CFTR delivery to the cell surface. The I SC across the monolayers was measured in Ussing chambers as described in the Experimental Section. (A) Representative tracings from control and Dab2-depleted monolayers. After a stable baseline was attained, 20 μM forskolin (F), 50 μM genistein (G) and 10 μM CFTR inh -172 were added at the indicated arrows. (B) Forskolin and genistein activated I SC . ΔI SC was calculated as an increase in I SC after forskolin and genistein addition over the base-line currents (n = 4). C. CFTR inh -172 inhibited I SC . ΔI SC was calculated as a decrease in I SC after CFTR inh -172 addition.

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: Increased transepithelial chloride transport following Dab2 depletion in CFBE41o-ΔF monolayers. CFBE41o-ΔF cells were transfected with control or Dab2 siRNA oligonucleotides. At 24 h after transfection, cells were lifted, seeded on to Transwell filters and cultured for an additional 4–5 days. The last 24 h, the cells were cultured at 27°C to promote ΔF508 CFTR delivery to the cell surface. The I SC across the monolayers was measured in Ussing chambers as described in the Experimental Section. (A) Representative tracings from control and Dab2-depleted monolayers. After a stable baseline was attained, 20 μM forskolin (F), 50 μM genistein (G) and 10 μM CFTR inh -172 were added at the indicated arrows. (B) Forskolin and genistein activated I SC . ΔI SC was calculated as an increase in I SC after forskolin and genistein addition over the base-line currents (n = 4). C. CFTR inh -172 inhibited I SC . ΔI SC was calculated as a decrease in I SC after CFTR inh -172 addition.

    Article Snippet: siRNA duplexes corresponding to non-conserved regions of human Dab2, μ2 and c-Cbl were purchased from Qiagen Inc. siRNA oligos for Nedd4-2 knock-down were purchased from Ambion® (Life technologies).

    Techniques: Transfection, Cell Culture

    rΔF508 CFTR delivery to the late endosomes is inhibited in Dab2-depleted cells. CFBE41o-ΔF cells were treated with control (A and B) or Dab2-specific (C) siRNA oligonucleotides. At 24 h after transfection, the cells were cultured at 27°C for an additional 48 h to facilitate cell surface delivery of ΔF508 CFTR. After the low-temperature rescue, one set of the control cells was transferred to 37°C for 1 h (A), another set of the control (B) and the Dab2-depleted cells (C) were treated with 5 mM ammonium chloride for 1 h at 37°C followed by immunofluorescent staining of CFTR and M6PR. (A) r ΔF508 CFTR and mannose-6-phosphate receptor (M6PR) do not co-localize in control untreated cells. (B) Ammonium chloride treatment (inhibition of lysosomal degradation) resulted in rΔF508 CFTR and M6PR co-localization (right-hand panel, arrowheads) . (C) Dab2 depletion and ammonium chloride treatment together enhanced rΔF508 CFTR staining; however, no co-localization of rΔF508 CFTR and M6PR is apparent.

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: rΔF508 CFTR delivery to the late endosomes is inhibited in Dab2-depleted cells. CFBE41o-ΔF cells were treated with control (A and B) or Dab2-specific (C) siRNA oligonucleotides. At 24 h after transfection, the cells were cultured at 27°C for an additional 48 h to facilitate cell surface delivery of ΔF508 CFTR. After the low-temperature rescue, one set of the control cells was transferred to 37°C for 1 h (A), another set of the control (B) and the Dab2-depleted cells (C) were treated with 5 mM ammonium chloride for 1 h at 37°C followed by immunofluorescent staining of CFTR and M6PR. (A) r ΔF508 CFTR and mannose-6-phosphate receptor (M6PR) do not co-localize in control untreated cells. (B) Ammonium chloride treatment (inhibition of lysosomal degradation) resulted in rΔF508 CFTR and M6PR co-localization (right-hand panel, arrowheads) . (C) Dab2 depletion and ammonium chloride treatment together enhanced rΔF508 CFTR staining; however, no co-localization of rΔF508 CFTR and M6PR is apparent.

    Article Snippet: siRNA duplexes corresponding to non-conserved regions of human Dab2, μ2 and c-Cbl were purchased from Qiagen Inc. siRNA oligos for Nedd4-2 knock-down were purchased from Ambion® (Life technologies).

    Techniques: Transfection, Cell Culture, Staining, Inhibition

    CHIP depletion increases the surface half-life. CFBE41o-ΔF cells were transfected with control or c-Cbl (A) , CHIP (B), or Nedd4-2 (C) siRNA oligonucleotides and cultured for 48 h at 37°C. The cells were then transferred to 27°C and incubated for an additional 48 h. Protein synthesis was stopped by preincubation with cycloheximide during the last 2 h at 27°C. Fresh media containing cycloheximide (CHX) was added and the cells were incubated for 0 to 6 h at 37°C and the surface pool of rΔF508 CFTR was detected using biotinylation as described in the Material and Methods section. Quantitative analysis of the blots is shown at the bottom (n = 3). The CHIP depletion significantly increased the cell surface half-life of rΔF508 CFTR, whereas c-Cbl depletion had little and Nedd4-2 depletion had no effect. (D) Cell surface half-life of rΔF508 CFTR was analyzed after CHIP depletion in combination with c-Cbl and/or Nedd4-2 as indicated. Depletion of c-Cbl and Nedd4-2 in combination with CHIP depletion did not have additional effect on the surface half-life of rΔF508 CFTR.

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: CHIP depletion increases the surface half-life. CFBE41o-ΔF cells were transfected with control or c-Cbl (A) , CHIP (B), or Nedd4-2 (C) siRNA oligonucleotides and cultured for 48 h at 37°C. The cells were then transferred to 27°C and incubated for an additional 48 h. Protein synthesis was stopped by preincubation with cycloheximide during the last 2 h at 27°C. Fresh media containing cycloheximide (CHX) was added and the cells were incubated for 0 to 6 h at 37°C and the surface pool of rΔF508 CFTR was detected using biotinylation as described in the Material and Methods section. Quantitative analysis of the blots is shown at the bottom (n = 3). The CHIP depletion significantly increased the cell surface half-life of rΔF508 CFTR, whereas c-Cbl depletion had little and Nedd4-2 depletion had no effect. (D) Cell surface half-life of rΔF508 CFTR was analyzed after CHIP depletion in combination with c-Cbl and/or Nedd4-2 as indicated. Depletion of c-Cbl and Nedd4-2 in combination with CHIP depletion did not have additional effect on the surface half-life of rΔF508 CFTR.

    Article Snippet: siRNA duplexes corresponding to non-conserved regions of human Dab2, μ2 and c-Cbl were purchased from Qiagen Inc. siRNA oligos for Nedd4-2 knock-down were purchased from Ambion® (Life technologies).

    Techniques: Chromatin Immunoprecipitation, Transfection, Cell Culture, Incubation

    Reduced endocytosis rates of rΔF508 CFTR following μ2 or Dab2 depletion. CFBE41o-ΔF cells were transfected with control, μ2 or Dab2 siRNA oligonucleotides as indicated. At 24 h after transfection, the cells were transferred to Transwell filters and incubated for an additional 4–5 days under an air-liquid interface. During the last 24 h, the cells were incubated at 27°C to promote ΔF508 CFTR rescue. The efficiency of μ2 and Dab2 depletion was > 90%. CFTR internalization assays were performed as described previously [ 28 ]. (A) Representative gels of CFTR internalization assays. The molecular mass in kDa is indicated on the right-hand side. (B) Quantitative analysis of rΔF508 CFTR internalization rates during a 7.5 min time period. The percentage of internalized CFTR was calculated from the loss of biotinylated CFTR during a 37°C incubation for time periods indicated after comparing to that at time 0 min under each condition (n = 3). (C) Quantitative analysis of CFTR internalization rates after 37°C warm-up for 2.5 min following μ2 or Dab2 depletion. Depletion of μ2 or Dab2 significantly reduced CFTR internalization rates in a 2.5 min time period (n = 3, *p

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: Reduced endocytosis rates of rΔF508 CFTR following μ2 or Dab2 depletion. CFBE41o-ΔF cells were transfected with control, μ2 or Dab2 siRNA oligonucleotides as indicated. At 24 h after transfection, the cells were transferred to Transwell filters and incubated for an additional 4–5 days under an air-liquid interface. During the last 24 h, the cells were incubated at 27°C to promote ΔF508 CFTR rescue. The efficiency of μ2 and Dab2 depletion was > 90%. CFTR internalization assays were performed as described previously [ 28 ]. (A) Representative gels of CFTR internalization assays. The molecular mass in kDa is indicated on the right-hand side. (B) Quantitative analysis of rΔF508 CFTR internalization rates during a 7.5 min time period. The percentage of internalized CFTR was calculated from the loss of biotinylated CFTR during a 37°C incubation for time periods indicated after comparing to that at time 0 min under each condition (n = 3). (C) Quantitative analysis of CFTR internalization rates after 37°C warm-up for 2.5 min following μ2 or Dab2 depletion. Depletion of μ2 or Dab2 significantly reduced CFTR internalization rates in a 2.5 min time period (n = 3, *p

    Article Snippet: siRNA duplexes corresponding to non-conserved regions of human Dab2, μ2 and c-Cbl were purchased from Qiagen Inc. siRNA oligos for Nedd4-2 knock-down were purchased from Ambion® (Life technologies).

    Techniques: Transfection, Incubation

    Increased cell surface half-life of rΔF508 CFTR in μ2- and Dab2-depleted cells. CFBE41o-ΔF cells were transfected with control, μ2, or Dab2 siRNA oligonucleotides. 48 h after transfection, the cells were cultured for 24 h at 27°C to allow cell-surface expression of rΔF508 CFTR. Cell surface rΔF508 CFTR was then monitored by biotinylation as described in the Material and Methods section after incubating with cycloheximide (CHX)-containing medium at 37°C for time periods indicated. Representative gels are shown ( A ) and quantitative analysis of the half-lives of rΔF508 CFTR under each experimental condition is shown ( B ). Dab2 depletion resulted in a ~2 fold increase in the half-life of cell surface rΔF508 CFTR (n = 3).

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: Increased cell surface half-life of rΔF508 CFTR in μ2- and Dab2-depleted cells. CFBE41o-ΔF cells were transfected with control, μ2, or Dab2 siRNA oligonucleotides. 48 h after transfection, the cells were cultured for 24 h at 27°C to allow cell-surface expression of rΔF508 CFTR. Cell surface rΔF508 CFTR was then monitored by biotinylation as described in the Material and Methods section after incubating with cycloheximide (CHX)-containing medium at 37°C for time periods indicated. Representative gels are shown ( A ) and quantitative analysis of the half-lives of rΔF508 CFTR under each experimental condition is shown ( B ). Dab2 depletion resulted in a ~2 fold increase in the half-life of cell surface rΔF508 CFTR (n = 3).

    Article Snippet: siRNA duplexes corresponding to non-conserved regions of human Dab2, μ2 and c-Cbl were purchased from Qiagen Inc. siRNA oligos for Nedd4-2 knock-down were purchased from Ambion® (Life technologies).

    Techniques: Transfection, Cell Culture, Expressing

    DOCK180 stabilizes pre-formed junctions. Keratinocytes were treated with siRNA oligos as labelled in the different panels and processed to determine actin recruitment to clustered E-cadherin (A–B) or aggregation assays (C–H). A–B, Following depletion of EGFR or DOCK180, cells were incubated with antibody-coated latex beads for 15 min and stained for F-actin. F-actin clusters are shown by arrows (A) and the proportion of attached beads containing F-actin clusters was quantified and expressed relative to controls (B). C–H, Keratinocytes depleted of DOCK180 (C–E) or SOS1 (F–H) were trypsinised and allowed to aggregate in suspension in the presence of calcium ions for 120 min, followed by disaggregation. C and F, Phase contrast images of initial samples, following aggregation for 120 min and after mechanical stress (disaggr). Black arrows point to aggregates. D and G, Relative sizes of all remaining aggregates were measured and shown in comparison to controls (arbitrarily set as 1). E and H, Confirmation of depletion efficiency following DOCK180 or SOS1 RNAi. Equal amounts of protein were loaded, actin used as a loading control. N = 3. Scale bar = 15 μm (A) or 200 μm (C, F). *, p

    Journal: Cellular Signalling

    Article Title: Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion

    doi: 10.1016/j.cellsig.2015.04.014

    Figure Lengend Snippet: DOCK180 stabilizes pre-formed junctions. Keratinocytes were treated with siRNA oligos as labelled in the different panels and processed to determine actin recruitment to clustered E-cadherin (A–B) or aggregation assays (C–H). A–B, Following depletion of EGFR or DOCK180, cells were incubated with antibody-coated latex beads for 15 min and stained for F-actin. F-actin clusters are shown by arrows (A) and the proportion of attached beads containing F-actin clusters was quantified and expressed relative to controls (B). C–H, Keratinocytes depleted of DOCK180 (C–E) or SOS1 (F–H) were trypsinised and allowed to aggregate in suspension in the presence of calcium ions for 120 min, followed by disaggregation. C and F, Phase contrast images of initial samples, following aggregation for 120 min and after mechanical stress (disaggr). Black arrows point to aggregates. D and G, Relative sizes of all remaining aggregates were measured and shown in comparison to controls (arbitrarily set as 1). E and H, Confirmation of depletion efficiency following DOCK180 or SOS1 RNAi. Equal amounts of protein were loaded, actin used as a loading control. N = 3. Scale bar = 15 μm (A) or 200 μm (C, F). *, p

    Article Snippet: Confluent cells were transfected with siRNA oligos and RNAifect (Qiagen) or INTERFERin (Peqlab) as per manufacturer's instructions.

    Techniques: Incubation, Staining

    EGFR, but not ErbB3, is required for cadherin-dependent Rac activation. A, Following induction of cell–cell contacts, keratinocyte lysates were immunoprecipitated with anti-E-cadherin antibodies, anti-α3-integrin (positive control) or no antibody (control) and probed with antibodies against proteins shown on the left of panels. B–C, Keratinocytes were treated with EGFR (oligo #1, oligo #2), ErbB3 (E3) or control (scr) siRNA oligos for 48 h. Equal amount of protein was separated on SDS–PAGE and probed with antibodies against proteins shown on the left of each panel. D–I, Keratinocytes were transfected with different siRNA oligos and junctions were initiated for 5 min by the addition of calcium ions and active Rac levels measured. D and G, Proteins were precipitated with GST–PAK-Crib beads (Rac∙GTP) and lysates (Total Rac) were probed with anti-Rac antibodies. The amount of GST fusion protein in each sample was evaluated by Amido Black staining (PAK-Crib). E and H, Depletion of EGFR or ErbB3 are shown. Beta-tubulin is shown as a loading control. F and I, Cell–cell-adhesion-dependent Rac activation was quantified and normalised to Rac·GTP levels at time 0 (no cell–cell contacts) for each siRNA group. Data is representative of 3 independent experiments (thereafter N = 3). *, p

    Journal: Cellular Signalling

    Article Title: Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion

    doi: 10.1016/j.cellsig.2015.04.014

    Figure Lengend Snippet: EGFR, but not ErbB3, is required for cadherin-dependent Rac activation. A, Following induction of cell–cell contacts, keratinocyte lysates were immunoprecipitated with anti-E-cadherin antibodies, anti-α3-integrin (positive control) or no antibody (control) and probed with antibodies against proteins shown on the left of panels. B–C, Keratinocytes were treated with EGFR (oligo #1, oligo #2), ErbB3 (E3) or control (scr) siRNA oligos for 48 h. Equal amount of protein was separated on SDS–PAGE and probed with antibodies against proteins shown on the left of each panel. D–I, Keratinocytes were transfected with different siRNA oligos and junctions were initiated for 5 min by the addition of calcium ions and active Rac levels measured. D and G, Proteins were precipitated with GST–PAK-Crib beads (Rac∙GTP) and lysates (Total Rac) were probed with anti-Rac antibodies. The amount of GST fusion protein in each sample was evaluated by Amido Black staining (PAK-Crib). E and H, Depletion of EGFR or ErbB3 are shown. Beta-tubulin is shown as a loading control. F and I, Cell–cell-adhesion-dependent Rac activation was quantified and normalised to Rac·GTP levels at time 0 (no cell–cell contacts) for each siRNA group. Data is representative of 3 independent experiments (thereafter N = 3). *, p

    Article Snippet: Confluent cells were transfected with siRNA oligos and RNAifect (Qiagen) or INTERFERin (Peqlab) as per manufacturer's instructions.

    Techniques: Activation Assay, Immunoprecipitation, Positive Control, SDS Page, Transfection, Staining

    DOCK180 is required for E-cadherin-mediated Rac activation. Cells were treated with DOCK180 (oligo #1, oligo #2), SOS1 (oligo #1) or control (scr) siRNA oligos. A–D, Lysates were prepared and used to determine efficiency of knockdown (A, B) and its specificity for DOCK180 or SOS1 depletion (C, D). Equal amounts of protein were separated on SDS–PAGE and probed with antibodies against proteins as shown on the left of each panel. E–H, Activation levels of Rac after 5 min of cell–cell contact formation. Pull down using GST–PAK-Crib was performed to precipitate active Rac (Rac∙GTP). E and G, Samples were probed with antibodies listed on the left of panels. Fusion proteins in each sample were evaluated by Amido black staining (PAK-Crib). Depletion of DOCK180 and SOS1 is shown and β-tubulin used as a loading control. F and H, Cell–cell adhesion-dependent Rac activation. Rac·GTP was quantified at different time points and normalised to Rac·GTP levels at time 0 (no cell–cell contacts) for each siRNA group (scr, DOCK180 or SOS1). N = 3; *, p

    Journal: Cellular Signalling

    Article Title: Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion

    doi: 10.1016/j.cellsig.2015.04.014

    Figure Lengend Snippet: DOCK180 is required for E-cadherin-mediated Rac activation. Cells were treated with DOCK180 (oligo #1, oligo #2), SOS1 (oligo #1) or control (scr) siRNA oligos. A–D, Lysates were prepared and used to determine efficiency of knockdown (A, B) and its specificity for DOCK180 or SOS1 depletion (C, D). Equal amounts of protein were separated on SDS–PAGE and probed with antibodies against proteins as shown on the left of each panel. E–H, Activation levels of Rac after 5 min of cell–cell contact formation. Pull down using GST–PAK-Crib was performed to precipitate active Rac (Rac∙GTP). E and G, Samples were probed with antibodies listed on the left of panels. Fusion proteins in each sample were evaluated by Amido black staining (PAK-Crib). Depletion of DOCK180 and SOS1 is shown and β-tubulin used as a loading control. F and H, Cell–cell adhesion-dependent Rac activation. Rac·GTP was quantified at different time points and normalised to Rac·GTP levels at time 0 (no cell–cell contacts) for each siRNA group (scr, DOCK180 or SOS1). N = 3; *, p

    Article Snippet: Confluent cells were transfected with siRNA oligos and RNAifect (Qiagen) or INTERFERin (Peqlab) as per manufacturer's instructions.

    Techniques: Activation Assay, SDS Page, Staining

    EGFR depletion perturbs cell–cell contacts. A, Keratinocytes treated with different siRNA oligos were induced to form cell–cell contacts for 30 min, fixed and stained for E-cadherin. Arrows show E-cadherin at junctions; arrowhead points to perturbed junction. B, Western blot shows knockdown of EGFR or ErbB3 (E3). C, Method for quantification of junction disruption. The length of cell–cell contacts (corner to corner) and the length of E-cadherin staining were obtained for each junction and expressed as a ratio (control junctions = 1). D, Disruption of E-cadherin localization by depletion of EGFR. ErbB3 (E3) and non-targeting oligos (con) were used as controls. N = 3 or N = 2 (ErbB3); about 150 junctions quantified in each replicate. Scale bar = 40 μm. *, p

    Journal: Cellular Signalling

    Article Title: Cooperation of distinct Rac-dependent pathways to stabilise E-cadherin adhesion

    doi: 10.1016/j.cellsig.2015.04.014

    Figure Lengend Snippet: EGFR depletion perturbs cell–cell contacts. A, Keratinocytes treated with different siRNA oligos were induced to form cell–cell contacts for 30 min, fixed and stained for E-cadherin. Arrows show E-cadherin at junctions; arrowhead points to perturbed junction. B, Western blot shows knockdown of EGFR or ErbB3 (E3). C, Method for quantification of junction disruption. The length of cell–cell contacts (corner to corner) and the length of E-cadherin staining were obtained for each junction and expressed as a ratio (control junctions = 1). D, Disruption of E-cadherin localization by depletion of EGFR. ErbB3 (E3) and non-targeting oligos (con) were used as controls. N = 3 or N = 2 (ErbB3); about 150 junctions quantified in each replicate. Scale bar = 40 μm. *, p

    Article Snippet: Confluent cells were transfected with siRNA oligos and RNAifect (Qiagen) or INTERFERin (Peqlab) as per manufacturer's instructions.

    Techniques: Staining, Western Blot

    ( A ) Schematic drawing of the HPV16 region around late 3′-splice site SA5639 and the 35-nucleotide biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA5639 is indicated. ( B ) Upper and lower panels show pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the region of HPV16 late 3′-splice site SA5639 followed by Western blot analysis using antibodies indicated to the right. (–) mock pull downs using streptavidin beads in the absence of oligo. ( C ) Quantitation of some of the Western blots of the pull downs in (B). ( D and E ) Western blots of indicated proteins on pull downs using biotinylated ssRNA oligos of a subset of the ssDNA oligos shown in (B).

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: ( A ) Schematic drawing of the HPV16 region around late 3′-splice site SA5639 and the 35-nucleotide biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA5639 is indicated. ( B ) Upper and lower panels show pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the region of HPV16 late 3′-splice site SA5639 followed by Western blot analysis using antibodies indicated to the right. (–) mock pull downs using streptavidin beads in the absence of oligo. ( C ) Quantitation of some of the Western blots of the pull downs in (B). ( D and E ) Western blots of indicated proteins on pull downs using biotinylated ssRNA oligos of a subset of the ssDNA oligos shown in (B).

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Western Blot, Quantitation Assay

    ( A ) Schematic representation of the HPV16 genome. Rectangles represent open reading frames, promoters p97 and p670 are indicated as arrows, filled and open triangles represent 5′- and 3′-splices sites respectively, HPV16 early and late polyA signals pAE and pAL are indicated. The regions on the HPV16 mRNAs from which the ssDNA and ssRNA oligos are derived are boxed. ( B ) Schematic drawing of the HPV16 region around early polyA signal pAE and the 35-nucleotide, biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 early polyA signal pAE is indicated as well as E5 and L2 coding regions. ( C ) Pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos spanning HPV16 pAE followed by Western blot for hnRNP L. (-); mock pull downs using streptavidine beads in the absence of oligo.

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: ( A ) Schematic representation of the HPV16 genome. Rectangles represent open reading frames, promoters p97 and p670 are indicated as arrows, filled and open triangles represent 5′- and 3′-splices sites respectively, HPV16 early and late polyA signals pAE and pAL are indicated. The regions on the HPV16 mRNAs from which the ssDNA and ssRNA oligos are derived are boxed. ( B ) Schematic drawing of the HPV16 region around early polyA signal pAE and the 35-nucleotide, biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 early polyA signal pAE is indicated as well as E5 and L2 coding regions. ( C ) Pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos spanning HPV16 pAE followed by Western blot for hnRNP L. (-); mock pull downs using streptavidine beads in the absence of oligo.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Derivative Assay, Western Blot

    ( A ) Upper panel: Schematic drawing of HPV16 exon 4 and the 35-nucleotide, biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA3358 and 5′-splice site SD3632 are indicated. Lower panel: Pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the E4 exon of HPV16 followed by Western blot analysis using antibodies to proteins indicated to the right. (-); mock pull downs using streptavidin beads in the absence of oligo. ( B ) Quantitation of some of the Western blots of the pull downs in (A). ( C ) Upper panel: Schematic drawing of shorter oligos (A-X) designed to better map binding sites for hnRNP L, hnRNP A1 and U2AF65. Lower panel: Western blots for hnRNP L, hnRNP A1 and U2AF65 of on proteins pulled down by the shorter biotinylated ssDNA oligos. ( D ) Western blots of indicated proteins on pull downs using biotinylated ssRNA oligos of a subset of the ssDNA oligos shown in (A). ( E ) Upper panel: Schematic drawing of shorter oligos (A–E) of the two original 35-nucleotide oligos 8 and 10 located near HPV16 late 5′-splice site SD3632. Lower panel: Western blots for hnRNP L, hnRNP A1 and U2AF65 of on proteins pulled down by the shorter biotinylated ssDNA oligos.

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: ( A ) Upper panel: Schematic drawing of HPV16 exon 4 and the 35-nucleotide, biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA3358 and 5′-splice site SD3632 are indicated. Lower panel: Pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the E4 exon of HPV16 followed by Western blot analysis using antibodies to proteins indicated to the right. (-); mock pull downs using streptavidin beads in the absence of oligo. ( B ) Quantitation of some of the Western blots of the pull downs in (A). ( C ) Upper panel: Schematic drawing of shorter oligos (A-X) designed to better map binding sites for hnRNP L, hnRNP A1 and U2AF65. Lower panel: Western blots for hnRNP L, hnRNP A1 and U2AF65 of on proteins pulled down by the shorter biotinylated ssDNA oligos. ( D ) Western blots of indicated proteins on pull downs using biotinylated ssRNA oligos of a subset of the ssDNA oligos shown in (A). ( E ) Upper panel: Schematic drawing of shorter oligos (A–E) of the two original 35-nucleotide oligos 8 and 10 located near HPV16 late 5′-splice site SD3632. Lower panel: Western blots for hnRNP L, hnRNP A1 and U2AF65 of on proteins pulled down by the shorter biotinylated ssDNA oligos.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Western Blot, Quantitation Assay, Binding Assay

    ( A ) Western blots of various RNA binding proteins in two independent preparations of nuclear extracts (Prep 1 and Prep 2) prepared from C33A2 cells treated with DMSO (D) or 100 uM Akt kinase inhibitor GDC0068 (G) for 3 h. Western blots of indicated proteins pulled down with biotinylated ssDNA oligos ( B ) or RNA oligos ( C ) representing sequences at the HPV16 3′-splice site SA3358 in the E4-coding exon. ( D ) Western blots of indicated proteins pulled down with biotinylated ssDNA oligos representing sequences at the HPV16 late 5′-splice site SD3632 in the E4-coding exon. ( E ) Western blots of indicated proteins pulled down with biotinylated ssRNA oligos spanning HPV16 5′-splice sites SD880 (BRnSD862) and SD3632 (BRnSD3632). ( F ) Western blots of hnRNP L, hnRNP A1, U2AF65 or U2AF35 pulled down from nuclear extracts using ssDNA oligos at HPV16 late splice site SA5639. For all pull downs in the figure, nuclear extracts prepared from C33A2 cells treated with DMSO (D) or Akt kinase inhibitor GDC0068 (G) were used.

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: ( A ) Western blots of various RNA binding proteins in two independent preparations of nuclear extracts (Prep 1 and Prep 2) prepared from C33A2 cells treated with DMSO (D) or 100 uM Akt kinase inhibitor GDC0068 (G) for 3 h. Western blots of indicated proteins pulled down with biotinylated ssDNA oligos ( B ) or RNA oligos ( C ) representing sequences at the HPV16 3′-splice site SA3358 in the E4-coding exon. ( D ) Western blots of indicated proteins pulled down with biotinylated ssDNA oligos representing sequences at the HPV16 late 5′-splice site SD3632 in the E4-coding exon. ( E ) Western blots of indicated proteins pulled down with biotinylated ssRNA oligos spanning HPV16 5′-splice sites SD880 (BRnSD862) and SD3632 (BRnSD3632). ( F ) Western blots of hnRNP L, hnRNP A1, U2AF65 or U2AF35 pulled down from nuclear extracts using ssDNA oligos at HPV16 late splice site SA5639. For all pull downs in the figure, nuclear extracts prepared from C33A2 cells treated with DMSO (D) or Akt kinase inhibitor GDC0068 (G) were used.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Western Blot, RNA Binding Assay

    Ubiquitination of rΔF508 CFTR by CHIP occurs in the post-endocytic compartments. (A) CFBE41o-ΔF cells were transfected with control or CHIP siRNA oligonucleotides at 37°C and then incubated for 48 h at 27°C to rescue the ΔF508 CFTR to the cell surface. During the last 16 h of low-temperature incubation, 150 μg/ml of cycloheximide was added to the culture to inhibit protein synthesis and eliminate the internal biosynthetic pool of ΔF508 CFTR. The cells were then switched to 37°C to promote endocytosis of the rΔF508 CFTR for the time periods indicated. The CFTR was then pulled-down by immunoprecipitation with specific antibody (24–1) and blotted with an ubiquitin antibody. The ubiquitin level is increased after 15 to 60 min warm-up at 37°C incubator in control cells (upper panel). The increase of ubiquitinated rΔF508 CFTR was attenuated under CHIP depletion (middle panel). The lower panel shows that the ubiquitination of rescued ΔF508 CFTR was reduced in the presence of 5 μM Dyngo 4a, which inhibits endocytosis. The relative ubiquitin level of rΔF508 CFTR after 37°C warm-up was analyzed in control, CHIP depleted and Dyngo 4a-treated cells (n = 3). (B) The ubiquitinated pool of rΔF508 CFTR was analyzed after Dab2 or CHIP depletion as described above. Dab2 depletion increases the CHIP-mediated ubiquitination of rΔF508 CFTR by blocking the post-endocytic trafficking.

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: Ubiquitination of rΔF508 CFTR by CHIP occurs in the post-endocytic compartments. (A) CFBE41o-ΔF cells were transfected with control or CHIP siRNA oligonucleotides at 37°C and then incubated for 48 h at 27°C to rescue the ΔF508 CFTR to the cell surface. During the last 16 h of low-temperature incubation, 150 μg/ml of cycloheximide was added to the culture to inhibit protein synthesis and eliminate the internal biosynthetic pool of ΔF508 CFTR. The cells were then switched to 37°C to promote endocytosis of the rΔF508 CFTR for the time periods indicated. The CFTR was then pulled-down by immunoprecipitation with specific antibody (24–1) and blotted with an ubiquitin antibody. The ubiquitin level is increased after 15 to 60 min warm-up at 37°C incubator in control cells (upper panel). The increase of ubiquitinated rΔF508 CFTR was attenuated under CHIP depletion (middle panel). The lower panel shows that the ubiquitination of rescued ΔF508 CFTR was reduced in the presence of 5 μM Dyngo 4a, which inhibits endocytosis. The relative ubiquitin level of rΔF508 CFTR after 37°C warm-up was analyzed in control, CHIP depleted and Dyngo 4a-treated cells (n = 3). (B) The ubiquitinated pool of rΔF508 CFTR was analyzed after Dab2 or CHIP depletion as described above. Dab2 depletion increases the CHIP-mediated ubiquitination of rΔF508 CFTR by blocking the post-endocytic trafficking.

    Article Snippet: Human STUB1 (CHIP) was depleted by using siRNA oligos from Dharmacon as described previously [ ].

    Techniques: Chromatin Immunoprecipitation, Transfection, Incubation, Immunoprecipitation, Blocking Assay

    c-Cbl, CHIP or Nedd4-2 depletion do not affect rΔF508 CFTR endocytosis. CFBE41o-ΔF cells were transfected with control or c-Cbl, CHIP, or Nedd4-2 siRNA oligonucleotides. At 24 h after transfection, cells were transferred to Transwell filters and cultured for an additional 4–5 days. The last 24 h, the cells were cultured at 27°C to promote ΔF508 CFTR delivery to the cell surface. (A) Cell surface expression of rΔF508 CFTR after siRNA transfection of control (C), c-Cbl, CHIP or Nedd4-2 as indicated. The knockdown efficiency of c-Cbl, CHIP, and Nedd4-2 was > 95%. β-actin was blotted as a loading control. (B) Representative blots showing the remaining surface rΔF508 CFTR after 2.5 min internalization at 37°C. (C) Quantitative analysis of rΔF508 CFTR internalization after 2.5 min warm-up following c-Cbl, CHIP, or Nedd4-2 siRNA depletion and low-temperature rescue. The rate of rΔF508 CFTR internalization was measured after 2.5 min warm-up as described in the Material and Methods section. Depletion of c-Cbl, CHIP, or Nedd4-2 had no significant (N.S.) effect on rΔF508 CFTR internalization (n = 3).

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: c-Cbl, CHIP or Nedd4-2 depletion do not affect rΔF508 CFTR endocytosis. CFBE41o-ΔF cells were transfected with control or c-Cbl, CHIP, or Nedd4-2 siRNA oligonucleotides. At 24 h after transfection, cells were transferred to Transwell filters and cultured for an additional 4–5 days. The last 24 h, the cells were cultured at 27°C to promote ΔF508 CFTR delivery to the cell surface. (A) Cell surface expression of rΔF508 CFTR after siRNA transfection of control (C), c-Cbl, CHIP or Nedd4-2 as indicated. The knockdown efficiency of c-Cbl, CHIP, and Nedd4-2 was > 95%. β-actin was blotted as a loading control. (B) Representative blots showing the remaining surface rΔF508 CFTR after 2.5 min internalization at 37°C. (C) Quantitative analysis of rΔF508 CFTR internalization after 2.5 min warm-up following c-Cbl, CHIP, or Nedd4-2 siRNA depletion and low-temperature rescue. The rate of rΔF508 CFTR internalization was measured after 2.5 min warm-up as described in the Material and Methods section. Depletion of c-Cbl, CHIP, or Nedd4-2 had no significant (N.S.) effect on rΔF508 CFTR internalization (n = 3).

    Article Snippet: Human STUB1 (CHIP) was depleted by using siRNA oligos from Dharmacon as described previously [ ].

    Techniques: Chromatin Immunoprecipitation, Transfection, Cell Culture, Expressing

    CHIP Depletion increases the function of rΔF508 CFTR. CFBE41o-ΔF cells were transfected with control or CHIP siRNA oligonucleotides, seeded on to Transwell filters and the I SC across the monolayers was measured in Ussing chambers as described in Fig 4 legend. (A) Representative tracings from control and CHIP-depleted monolayers. After a stable baseline was attained, 20 μM forskolin (F), 50 μM genistein (G) and 10 μM CFTR inh -172 were added at the indicated arrows. (B) Forskolin and genistein activated I SC . ΔI SC was calculated as an increase in I SC after forskolin and genistein addition over the base-line currents. C. CFTR inh -172 inhibited I SC . ΔI SC was calculated as a decrease in I SC after CFTR inh -172 addition. (n = 6)

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: CHIP Depletion increases the function of rΔF508 CFTR. CFBE41o-ΔF cells were transfected with control or CHIP siRNA oligonucleotides, seeded on to Transwell filters and the I SC across the monolayers was measured in Ussing chambers as described in Fig 4 legend. (A) Representative tracings from control and CHIP-depleted monolayers. After a stable baseline was attained, 20 μM forskolin (F), 50 μM genistein (G) and 10 μM CFTR inh -172 were added at the indicated arrows. (B) Forskolin and genistein activated I SC . ΔI SC was calculated as an increase in I SC after forskolin and genistein addition over the base-line currents. C. CFTR inh -172 inhibited I SC . ΔI SC was calculated as a decrease in I SC after CFTR inh -172 addition. (n = 6)

    Article Snippet: Human STUB1 (CHIP) was depleted by using siRNA oligos from Dharmacon as described previously [ ].

    Techniques: Chromatin Immunoprecipitation, Transfection

    Increased transepithelial chloride transport following Dab2 depletion in CFBE41o-ΔF monolayers. CFBE41o-ΔF cells were transfected with control or Dab2 siRNA oligonucleotides. At 24 h after transfection, cells were lifted, seeded on to Transwell filters and cultured for an additional 4–5 days. The last 24 h, the cells were cultured at 27°C to promote ΔF508 CFTR delivery to the cell surface. The I SC across the monolayers was measured in Ussing chambers as described in the Experimental Section. (A) Representative tracings from control and Dab2-depleted monolayers. After a stable baseline was attained, 20 μM forskolin (F), 50 μM genistein (G) and 10 μM CFTR inh -172 were added at the indicated arrows. (B) Forskolin and genistein activated I SC . ΔI SC was calculated as an increase in I SC after forskolin and genistein addition over the base-line currents (n = 4). C. CFTR inh -172 inhibited I SC . ΔI SC was calculated as a decrease in I SC after CFTR inh -172 addition.

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: Increased transepithelial chloride transport following Dab2 depletion in CFBE41o-ΔF monolayers. CFBE41o-ΔF cells were transfected with control or Dab2 siRNA oligonucleotides. At 24 h after transfection, cells were lifted, seeded on to Transwell filters and cultured for an additional 4–5 days. The last 24 h, the cells were cultured at 27°C to promote ΔF508 CFTR delivery to the cell surface. The I SC across the monolayers was measured in Ussing chambers as described in the Experimental Section. (A) Representative tracings from control and Dab2-depleted monolayers. After a stable baseline was attained, 20 μM forskolin (F), 50 μM genistein (G) and 10 μM CFTR inh -172 were added at the indicated arrows. (B) Forskolin and genistein activated I SC . ΔI SC was calculated as an increase in I SC after forskolin and genistein addition over the base-line currents (n = 4). C. CFTR inh -172 inhibited I SC . ΔI SC was calculated as a decrease in I SC after CFTR inh -172 addition.

    Article Snippet: Human STUB1 (CHIP) was depleted by using siRNA oligos from Dharmacon as described previously [ ].

    Techniques: Transfection, Cell Culture

    rΔF508 CFTR delivery to the late endosomes is inhibited in Dab2-depleted cells. CFBE41o-ΔF cells were treated with control (A and B) or Dab2-specific (C) siRNA oligonucleotides. At 24 h after transfection, the cells were cultured at 27°C for an additional 48 h to facilitate cell surface delivery of ΔF508 CFTR. After the low-temperature rescue, one set of the control cells was transferred to 37°C for 1 h (A), another set of the control (B) and the Dab2-depleted cells (C) were treated with 5 mM ammonium chloride for 1 h at 37°C followed by immunofluorescent staining of CFTR and M6PR. (A) r ΔF508 CFTR and mannose-6-phosphate receptor (M6PR) do not co-localize in control untreated cells. (B) Ammonium chloride treatment (inhibition of lysosomal degradation) resulted in rΔF508 CFTR and M6PR co-localization (right-hand panel, arrowheads) . (C) Dab2 depletion and ammonium chloride treatment together enhanced rΔF508 CFTR staining; however, no co-localization of rΔF508 CFTR and M6PR is apparent.

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: rΔF508 CFTR delivery to the late endosomes is inhibited in Dab2-depleted cells. CFBE41o-ΔF cells were treated with control (A and B) or Dab2-specific (C) siRNA oligonucleotides. At 24 h after transfection, the cells were cultured at 27°C for an additional 48 h to facilitate cell surface delivery of ΔF508 CFTR. After the low-temperature rescue, one set of the control cells was transferred to 37°C for 1 h (A), another set of the control (B) and the Dab2-depleted cells (C) were treated with 5 mM ammonium chloride for 1 h at 37°C followed by immunofluorescent staining of CFTR and M6PR. (A) r ΔF508 CFTR and mannose-6-phosphate receptor (M6PR) do not co-localize in control untreated cells. (B) Ammonium chloride treatment (inhibition of lysosomal degradation) resulted in rΔF508 CFTR and M6PR co-localization (right-hand panel, arrowheads) . (C) Dab2 depletion and ammonium chloride treatment together enhanced rΔF508 CFTR staining; however, no co-localization of rΔF508 CFTR and M6PR is apparent.

    Article Snippet: Human STUB1 (CHIP) was depleted by using siRNA oligos from Dharmacon as described previously [ ].

    Techniques: Transfection, Cell Culture, Staining, Inhibition

    CHIP depletion increases the surface half-life. CFBE41o-ΔF cells were transfected with control or c-Cbl (A) , CHIP (B), or Nedd4-2 (C) siRNA oligonucleotides and cultured for 48 h at 37°C. The cells were then transferred to 27°C and incubated for an additional 48 h. Protein synthesis was stopped by preincubation with cycloheximide during the last 2 h at 27°C. Fresh media containing cycloheximide (CHX) was added and the cells were incubated for 0 to 6 h at 37°C and the surface pool of rΔF508 CFTR was detected using biotinylation as described in the Material and Methods section. Quantitative analysis of the blots is shown at the bottom (n = 3). The CHIP depletion significantly increased the cell surface half-life of rΔF508 CFTR, whereas c-Cbl depletion had little and Nedd4-2 depletion had no effect. (D) Cell surface half-life of rΔF508 CFTR was analyzed after CHIP depletion in combination with c-Cbl and/or Nedd4-2 as indicated. Depletion of c-Cbl and Nedd4-2 in combination with CHIP depletion did not have additional effect on the surface half-life of rΔF508 CFTR.

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: CHIP depletion increases the surface half-life. CFBE41o-ΔF cells were transfected with control or c-Cbl (A) , CHIP (B), or Nedd4-2 (C) siRNA oligonucleotides and cultured for 48 h at 37°C. The cells were then transferred to 27°C and incubated for an additional 48 h. Protein synthesis was stopped by preincubation with cycloheximide during the last 2 h at 27°C. Fresh media containing cycloheximide (CHX) was added and the cells were incubated for 0 to 6 h at 37°C and the surface pool of rΔF508 CFTR was detected using biotinylation as described in the Material and Methods section. Quantitative analysis of the blots is shown at the bottom (n = 3). The CHIP depletion significantly increased the cell surface half-life of rΔF508 CFTR, whereas c-Cbl depletion had little and Nedd4-2 depletion had no effect. (D) Cell surface half-life of rΔF508 CFTR was analyzed after CHIP depletion in combination with c-Cbl and/or Nedd4-2 as indicated. Depletion of c-Cbl and Nedd4-2 in combination with CHIP depletion did not have additional effect on the surface half-life of rΔF508 CFTR.

    Article Snippet: Human STUB1 (CHIP) was depleted by using siRNA oligos from Dharmacon as described previously [ ].

    Techniques: Chromatin Immunoprecipitation, Transfection, Cell Culture, Incubation

    Reduced endocytosis rates of rΔF508 CFTR following μ2 or Dab2 depletion. CFBE41o-ΔF cells were transfected with control, μ2 or Dab2 siRNA oligonucleotides as indicated. At 24 h after transfection, the cells were transferred to Transwell filters and incubated for an additional 4–5 days under an air-liquid interface. During the last 24 h, the cells were incubated at 27°C to promote ΔF508 CFTR rescue. The efficiency of μ2 and Dab2 depletion was > 90%. CFTR internalization assays were performed as described previously [ 28 ]. (A) Representative gels of CFTR internalization assays. The molecular mass in kDa is indicated on the right-hand side. (B) Quantitative analysis of rΔF508 CFTR internalization rates during a 7.5 min time period. The percentage of internalized CFTR was calculated from the loss of biotinylated CFTR during a 37°C incubation for time periods indicated after comparing to that at time 0 min under each condition (n = 3). (C) Quantitative analysis of CFTR internalization rates after 37°C warm-up for 2.5 min following μ2 or Dab2 depletion. Depletion of μ2 or Dab2 significantly reduced CFTR internalization rates in a 2.5 min time period (n = 3, *p

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: Reduced endocytosis rates of rΔF508 CFTR following μ2 or Dab2 depletion. CFBE41o-ΔF cells were transfected with control, μ2 or Dab2 siRNA oligonucleotides as indicated. At 24 h after transfection, the cells were transferred to Transwell filters and incubated for an additional 4–5 days under an air-liquid interface. During the last 24 h, the cells were incubated at 27°C to promote ΔF508 CFTR rescue. The efficiency of μ2 and Dab2 depletion was > 90%. CFTR internalization assays were performed as described previously [ 28 ]. (A) Representative gels of CFTR internalization assays. The molecular mass in kDa is indicated on the right-hand side. (B) Quantitative analysis of rΔF508 CFTR internalization rates during a 7.5 min time period. The percentage of internalized CFTR was calculated from the loss of biotinylated CFTR during a 37°C incubation for time periods indicated after comparing to that at time 0 min under each condition (n = 3). (C) Quantitative analysis of CFTR internalization rates after 37°C warm-up for 2.5 min following μ2 or Dab2 depletion. Depletion of μ2 or Dab2 significantly reduced CFTR internalization rates in a 2.5 min time period (n = 3, *p

    Article Snippet: Human STUB1 (CHIP) was depleted by using siRNA oligos from Dharmacon as described previously [ ].

    Techniques: Transfection, Incubation

    Increased cell surface half-life of rΔF508 CFTR in μ2- and Dab2-depleted cells. CFBE41o-ΔF cells were transfected with control, μ2, or Dab2 siRNA oligonucleotides. 48 h after transfection, the cells were cultured for 24 h at 27°C to allow cell-surface expression of rΔF508 CFTR. Cell surface rΔF508 CFTR was then monitored by biotinylation as described in the Material and Methods section after incubating with cycloheximide (CHX)-containing medium at 37°C for time periods indicated. Representative gels are shown ( A ) and quantitative analysis of the half-lives of rΔF508 CFTR under each experimental condition is shown ( B ). Dab2 depletion resulted in a ~2 fold increase in the half-life of cell surface rΔF508 CFTR (n = 3).

    Journal: PLoS ONE

    Article Title: ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    doi: 10.1371/journal.pone.0123131

    Figure Lengend Snippet: Increased cell surface half-life of rΔF508 CFTR in μ2- and Dab2-depleted cells. CFBE41o-ΔF cells were transfected with control, μ2, or Dab2 siRNA oligonucleotides. 48 h after transfection, the cells were cultured for 24 h at 27°C to allow cell-surface expression of rΔF508 CFTR. Cell surface rΔF508 CFTR was then monitored by biotinylation as described in the Material and Methods section after incubating with cycloheximide (CHX)-containing medium at 37°C for time periods indicated. Representative gels are shown ( A ) and quantitative analysis of the half-lives of rΔF508 CFTR under each experimental condition is shown ( B ). Dab2 depletion resulted in a ~2 fold increase in the half-life of cell surface rΔF508 CFTR (n = 3).

    Article Snippet: Human STUB1 (CHIP) was depleted by using siRNA oligos from Dharmacon as described previously [ ].

    Techniques: Transfection, Cell Culture, Expressing

    Larval seizure was aggravated by downregulation of stx1b transcription in the PTZ-induced seizure model. Levels of stx1b gene transcription ( n = 20) (A) and STX1B protein ( n = 3) (B) were reduced and c-fos gene transcription was increased in the larval (7 dpf) brain ( n = 20) (C) by stx1b morpholino oligos injection in the PTZ model, as compared with in the PTZ-only and the morpholino oligos-only injection models. The larval swimming experiment ( n = 24) (D) showed that average speed and total distance were not changed, but that the abnormal pathway and overspeed were increased following 20 min in the dark condition and that photosensitive seizure was aggravated under the condition of light–dark transition with 5 min in the dark and 10 s in the light for three cycles in the PTZ plus stx1b morpholino oligos larvae, as compared with the two groups of the PTZ-only and the stx1b morpholino oligos-only injection models. The data show average speeds during the 20 min in the dark and the 10 s in the dark–light transformation; the boxes indicate the difference of locomotion distances and speeds between the light–dark transitions. Swimming tracks were recorded at 2 min in the dark condition and the red trajectory indicates overactive movement and the green trajectory indicates active movement. stx1b -MO1 and stx1b -MO2 were two morpholino oligos that bound to the stx1b messenger RNA initiate sequence with a different sequence; by using two target oligos, their inhibition effect was confirmed with each other. ∗∗∗ P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Syntaxin 1B Mediates Berberine’s Roles in Epilepsy-Like Behavior in a Pentylenetetrazole-Induced Seizure Zebrafish Model

    doi: 10.3389/fnmol.2018.00378

    Figure Lengend Snippet: Larval seizure was aggravated by downregulation of stx1b transcription in the PTZ-induced seizure model. Levels of stx1b gene transcription ( n = 20) (A) and STX1B protein ( n = 3) (B) were reduced and c-fos gene transcription was increased in the larval (7 dpf) brain ( n = 20) (C) by stx1b morpholino oligos injection in the PTZ model, as compared with in the PTZ-only and the morpholino oligos-only injection models. The larval swimming experiment ( n = 24) (D) showed that average speed and total distance were not changed, but that the abnormal pathway and overspeed were increased following 20 min in the dark condition and that photosensitive seizure was aggravated under the condition of light–dark transition with 5 min in the dark and 10 s in the light for three cycles in the PTZ plus stx1b morpholino oligos larvae, as compared with the two groups of the PTZ-only and the stx1b morpholino oligos-only injection models. The data show average speeds during the 20 min in the dark and the 10 s in the dark–light transformation; the boxes indicate the difference of locomotion distances and speeds between the light–dark transitions. Swimming tracks were recorded at 2 min in the dark condition and the red trajectory indicates overactive movement and the green trajectory indicates active movement. stx1b -MO1 and stx1b -MO2 were two morpholino oligos that bound to the stx1b messenger RNA initiate sequence with a different sequence; by using two target oligos, their inhibition effect was confirmed with each other. ∗∗∗ P

    Article Snippet: Two stx1b morpholino oligos and a scrambled morpholino oligo were purchased from Gene Tools, LLC (Philomath, OR, United States).

    Techniques: Injection, Transformation Assay, Sequencing, Inhibition

    Comparative analysis of epilepsy-like seizure and STX1B protein levels between BBR with and without stx1b morpholino oligos injection in the PTZ-induced seizure zebrafish. (A) Behavioral comparison indicates that the BBR effect of antiseizure was weakened by stx1b morpholino oligo injection. (B) Comparison of STX1B protein levels induced by BBR between stx1b gene knockdown and non-knockdown. Western blotting showed that levels of STX1B protein were significantly decreased by stx1b morpholino oligo injection under BBR existence. The histograms are generated from data in Figure 5 of behavior and western blotting, Figure 6 of behavior, and Figure 7 of western blotting. # P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Syntaxin 1B Mediates Berberine’s Roles in Epilepsy-Like Behavior in a Pentylenetetrazole-Induced Seizure Zebrafish Model

    doi: 10.3389/fnmol.2018.00378

    Figure Lengend Snippet: Comparative analysis of epilepsy-like seizure and STX1B protein levels between BBR with and without stx1b morpholino oligos injection in the PTZ-induced seizure zebrafish. (A) Behavioral comparison indicates that the BBR effect of antiseizure was weakened by stx1b morpholino oligo injection. (B) Comparison of STX1B protein levels induced by BBR between stx1b gene knockdown and non-knockdown. Western blotting showed that levels of STX1B protein were significantly decreased by stx1b morpholino oligo injection under BBR existence. The histograms are generated from data in Figure 5 of behavior and western blotting, Figure 6 of behavior, and Figure 7 of western blotting. # P

    Article Snippet: Two stx1b morpholino oligos and a scrambled morpholino oligo were purchased from Gene Tools, LLC (Philomath, OR, United States).

    Techniques: Injection, Western Blot, Generated

    Downregulation of STX1B weakened the effects of BBR on anticonvulsant in the PTZ-induced seizure zebrafish model. (A) Hybridization in situ showed that there was a change in the c-fos messenger RNA level in the larval (7 dpf) brain that was induced by BBR in the PTZ plus stx1b morpholino oligos group versus in the three control groups of wild-type, stx1b morpholino oligos injection, and PTZ plus stx1b morpholino oligos ( n = 20). (B,C) STX1B downregulation attenuated the efficiency of BBR inhibition on larval overexcited locomotion in terms of speed and distance under non-stimulation conditions and eliminated the action of BBR under dark–light transitions. Swimming trajectories are presented in 2 min recording charts; red tracks indicate over locomotion, while the rectangles indicate the difference between light–dark transitions ( n = 24). P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Syntaxin 1B Mediates Berberine’s Roles in Epilepsy-Like Behavior in a Pentylenetetrazole-Induced Seizure Zebrafish Model

    doi: 10.3389/fnmol.2018.00378

    Figure Lengend Snippet: Downregulation of STX1B weakened the effects of BBR on anticonvulsant in the PTZ-induced seizure zebrafish model. (A) Hybridization in situ showed that there was a change in the c-fos messenger RNA level in the larval (7 dpf) brain that was induced by BBR in the PTZ plus stx1b morpholino oligos group versus in the three control groups of wild-type, stx1b morpholino oligos injection, and PTZ plus stx1b morpholino oligos ( n = 20). (B,C) STX1B downregulation attenuated the efficiency of BBR inhibition on larval overexcited locomotion in terms of speed and distance under non-stimulation conditions and eliminated the action of BBR under dark–light transitions. Swimming trajectories are presented in 2 min recording charts; red tracks indicate over locomotion, while the rectangles indicate the difference between light–dark transitions ( n = 24). P

    Article Snippet: Two stx1b morpholino oligos and a scrambled morpholino oligo were purchased from Gene Tools, LLC (Philomath, OR, United States).

    Techniques: Hybridization, In Situ, Injection, Inhibition

    Stx1b morpholino oligos injection suppressed BBR activation on STX1B expression in the PTZ-model larvae. (A) Hybridization in situ results show that a change of the stx1b messenger RNA level in the larval (7 dpf) brain was induced by BBR with stx1b morpholino oligos injection in the PTZ-model zebrafish, as compared with in the wild-type, stx1b morpholino oligos injection, and PTZ plus stx1b morpholino oligos groups ( n = 20). (B) Western blotting results indicated a change of the STX1B protein level similar to the change of the stx1b messenger RNA level under the same treatments ( n = 3). P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Syntaxin 1B Mediates Berberine’s Roles in Epilepsy-Like Behavior in a Pentylenetetrazole-Induced Seizure Zebrafish Model

    doi: 10.3389/fnmol.2018.00378

    Figure Lengend Snippet: Stx1b morpholino oligos injection suppressed BBR activation on STX1B expression in the PTZ-model larvae. (A) Hybridization in situ results show that a change of the stx1b messenger RNA level in the larval (7 dpf) brain was induced by BBR with stx1b morpholino oligos injection in the PTZ-model zebrafish, as compared with in the wild-type, stx1b morpholino oligos injection, and PTZ plus stx1b morpholino oligos groups ( n = 20). (B) Western blotting results indicated a change of the STX1B protein level similar to the change of the stx1b messenger RNA level under the same treatments ( n = 3). P

    Article Snippet: Two stx1b morpholino oligos and a scrambled morpholino oligo were purchased from Gene Tools, LLC (Philomath, OR, United States).

    Techniques: Injection, Activation Assay, Expressing, Hybridization, In Situ, Western Blot

    Summary of ICP4 domains and sites of DNA interaction and homo-dimerization. ( A ) The domain composition of HSV1 ICP4 with predicted secondary structure (SSpred) and ordered regions (ORDERpred) from PSIPRED ( 37 ). Predicted α-helices and β-sheets are shown as red and yellow respectively, with ordered regions colored green. ( B ) Sequence alignment of ICP4 DBDs from HSV1, HSV2 and VZV (Uniprot codes: P08392, P90493, Q8AZM1 respectively) from Clustal omega ( 36 ). Secondary structure elements determined here are labeled on the HSV-1 sequence. Residues previously probed by mutagenesis are highlighted blue with black arrows pointing to those with lower affinity for DNA ( 13 , 14 , 16 ). Below the sequences, blocks colored gray or purple indicate residues which form homo-dimer or DNA contacts respectively as observed in the crystallography data. Purple circles indicate protein–DNA contacts derived from NMR chemical shift perturbations in intrinsically disordered regions (IDRs). ( C ) Cartoon representation of the crystal structure with helices, sheets and loops shown as cylinders, arrows and coil respectively: (i) ICP4N · IE3_19mer with DNA colored purple and pink for sense and anti-sense strands respectively, one protein chain is colored gray and other blue through red from N-to-C termini. (ii) As with panel i with view rotated 90°.

    Journal: Nucleic Acids Research

    Article Title: The herpes viral transcription factor ICP4 forms a novel DNA recognition complex

    doi: 10.1093/nar/gkx419

    Figure Lengend Snippet: Summary of ICP4 domains and sites of DNA interaction and homo-dimerization. ( A ) The domain composition of HSV1 ICP4 with predicted secondary structure (SSpred) and ordered regions (ORDERpred) from PSIPRED ( 37 ). Predicted α-helices and β-sheets are shown as red and yellow respectively, with ordered regions colored green. ( B ) Sequence alignment of ICP4 DBDs from HSV1, HSV2 and VZV (Uniprot codes: P08392, P90493, Q8AZM1 respectively) from Clustal omega ( 36 ). Secondary structure elements determined here are labeled on the HSV-1 sequence. Residues previously probed by mutagenesis are highlighted blue with black arrows pointing to those with lower affinity for DNA ( 13 , 14 , 16 ). Below the sequences, blocks colored gray or purple indicate residues which form homo-dimer or DNA contacts respectively as observed in the crystallography data. Purple circles indicate protein–DNA contacts derived from NMR chemical shift perturbations in intrinsically disordered regions (IDRs). ( C ) Cartoon representation of the crystal structure with helices, sheets and loops shown as cylinders, arrows and coil respectively: (i) ICP4N · IE3_19mer with DNA colored purple and pink for sense and anti-sense strands respectively, one protein chain is colored gray and other blue through red from N-to-C termini. (ii) As with panel i with view rotated 90°.

    Article Snippet: In order to study the DNA interactions of ICP4N constructs, synthetic IE3 DNA oligos were purchased (Invitrogen), namely IE3_19mer forward: CCGATCGTCCACACGGAGC and reverse-complement: GCTCCGTGTGGACGATCGG, IE3_19merMUT forward: CCGATCGTCCAAGATTAGC and reverse complement: GCTAATCTTGGACGATCGG, plus IE3_12mer forward: CCGATCGTCCAC and reverse-complement: GTGGACGATCGG.

    Techniques: Sequencing, Labeling, Mutagenesis, Derivative Assay, Nuclear Magnetic Resonance

    SAXS and NMR analysis of the ICP4N · IE3_19mer complex. ( A ) Dimensionless Kratky plot of the ICP4N bound (red) and unbound (blue) to DNA. Cross-hairs denote the Guinier–Kratky point (1.732, 1.104), the peak position for an ideal, globular particle. As indicated by the upward-right shift of the peaks in the dimensionless Kratky plot, ICP4N is more globular in the presence of DNA. ( B ) The calculated solution-state SAXS profile for the crystal structures of ICP4N·IE3_19mer complex (black line) compared to the measured scatter data (red circles). ( C ) Multi-phase ab initio model generated from SAXS data using MONSA show the presence of DNA (orange) and protein (teal) and their arrangement. ( D ) The crystal structure of the complex docked into the ab initio model revealing unoccupied volume around the DNA as well as above and below the protein dimer, assigned to the N-terminal IDRs. ( E ) NMR characterization of IDRs of the ICP4N dimer upon addition of equimolar amount of IE3_19mer duplex. 1 H- 15 N TROSY spectrum of ICP4N showing sharp peaks assigned to residues within the unstructured N- and C-terminal regions in free and IE3 DNA bound forms, colored blue and red respectively. Peaks are labeled with assignments; when an unambiguous assignment was not possible the peaks are labeled with their amino acid type.

    Journal: Nucleic Acids Research

    Article Title: The herpes viral transcription factor ICP4 forms a novel DNA recognition complex

    doi: 10.1093/nar/gkx419

    Figure Lengend Snippet: SAXS and NMR analysis of the ICP4N · IE3_19mer complex. ( A ) Dimensionless Kratky plot of the ICP4N bound (red) and unbound (blue) to DNA. Cross-hairs denote the Guinier–Kratky point (1.732, 1.104), the peak position for an ideal, globular particle. As indicated by the upward-right shift of the peaks in the dimensionless Kratky plot, ICP4N is more globular in the presence of DNA. ( B ) The calculated solution-state SAXS profile for the crystal structures of ICP4N·IE3_19mer complex (black line) compared to the measured scatter data (red circles). ( C ) Multi-phase ab initio model generated from SAXS data using MONSA show the presence of DNA (orange) and protein (teal) and their arrangement. ( D ) The crystal structure of the complex docked into the ab initio model revealing unoccupied volume around the DNA as well as above and below the protein dimer, assigned to the N-terminal IDRs. ( E ) NMR characterization of IDRs of the ICP4N dimer upon addition of equimolar amount of IE3_19mer duplex. 1 H- 15 N TROSY spectrum of ICP4N showing sharp peaks assigned to residues within the unstructured N- and C-terminal regions in free and IE3 DNA bound forms, colored blue and red respectively. Peaks are labeled with assignments; when an unambiguous assignment was not possible the peaks are labeled with their amino acid type.

    Article Snippet: In order to study the DNA interactions of ICP4N constructs, synthetic IE3 DNA oligos were purchased (Invitrogen), namely IE3_19mer forward: CCGATCGTCCACACGGAGC and reverse-complement: GCTCCGTGTGGACGATCGG, IE3_19merMUT forward: CCGATCGTCCAAGATTAGC and reverse complement: GCTAATCTTGGACGATCGG, plus IE3_12mer forward: CCGATCGTCCAC and reverse-complement: GTGGACGATCGG.

    Techniques: Nuclear Magnetic Resonance, Generated, Labeling

    Binding of ICP4N and ICP4ΔIDR to biotinylated DNA duplexes measured by SPR. ( A ) Sensorgrams of different concentrations (nano-molar concentrations indicated on each plot) of ICP4 proteins binding to IE3 DNA duplexes. ( B ) Equilibrium analysis of SPR. ( C ) Mean dissociation constants (±SD) measured for each interaction, non-WT bases are underlined.

    Journal: Nucleic Acids Research

    Article Title: The herpes viral transcription factor ICP4 forms a novel DNA recognition complex

    doi: 10.1093/nar/gkx419

    Figure Lengend Snippet: Binding of ICP4N and ICP4ΔIDR to biotinylated DNA duplexes measured by SPR. ( A ) Sensorgrams of different concentrations (nano-molar concentrations indicated on each plot) of ICP4 proteins binding to IE3 DNA duplexes. ( B ) Equilibrium analysis of SPR. ( C ) Mean dissociation constants (±SD) measured for each interaction, non-WT bases are underlined.

    Article Snippet: In order to study the DNA interactions of ICP4N constructs, synthetic IE3 DNA oligos were purchased (Invitrogen), namely IE3_19mer forward: CCGATCGTCCACACGGAGC and reverse-complement: GCTCCGTGTGGACGATCGG, IE3_19merMUT forward: CCGATCGTCCAAGATTAGC and reverse complement: GCTAATCTTGGACGATCGG, plus IE3_12mer forward: CCGATCGTCCAC and reverse-complement: GTGGACGATCGG.

    Techniques: Binding Assay, SPR Assay

    Details of the protein–DNA interface in the ICP4N · IE3_19mer structure. ( A ) Schematic of ICP4N-IE3 DNA interaction model. Protein–DNA hydrogen bonds identified from the crystal structure are indicated by dashed lines with locations of other contacts indicated with dark gray lines. Protein residues are colored blue or green when corresponding to chain C or D respectively. The vertical dashed arrow marks the DNA region that NMR, SAXS and SPR data suggested is bound by an IDR of the protein (residues 258–289). ICP4 consensus is sequence shown to the left side. ( B ) Overall ICP4N · IE3_19mer structure, with protein chains C and colored blue and green respectively and DNA space fill surface is shown colored dark and light orange for the sense (chain H) and antisense strands (chain G) respectively. Hydrogen bonds are indicated by dashes. Details of base-pair interactions, shown for (i) major groove bound by the ICP4N globular homo-dimer residues 416–419 and 456–457, (ii) DNA kink intercalated by F283 and (iii) minor groove bound by residues 416–419 and 456–457. ( C ) Surface of the ICP4N dimer colored by electrostatic potential (red through blue for acidic to basic charge) calculated by Adaptive Poisson-Boltzmann Solver module in Chimera ( 53 ). DNA is shown in cartoon form with the ICP4 consensus sequence labeled on sense strand. ( D ) Plot of DNA major- and minor-groove widths measured in both models in the ICP4N · IE3_19mer asymmetric unit ( 54 ).

    Journal: Nucleic Acids Research

    Article Title: The herpes viral transcription factor ICP4 forms a novel DNA recognition complex

    doi: 10.1093/nar/gkx419

    Figure Lengend Snippet: Details of the protein–DNA interface in the ICP4N · IE3_19mer structure. ( A ) Schematic of ICP4N-IE3 DNA interaction model. Protein–DNA hydrogen bonds identified from the crystal structure are indicated by dashed lines with locations of other contacts indicated with dark gray lines. Protein residues are colored blue or green when corresponding to chain C or D respectively. The vertical dashed arrow marks the DNA region that NMR, SAXS and SPR data suggested is bound by an IDR of the protein (residues 258–289). ICP4 consensus is sequence shown to the left side. ( B ) Overall ICP4N · IE3_19mer structure, with protein chains C and colored blue and green respectively and DNA space fill surface is shown colored dark and light orange for the sense (chain H) and antisense strands (chain G) respectively. Hydrogen bonds are indicated by dashes. Details of base-pair interactions, shown for (i) major groove bound by the ICP4N globular homo-dimer residues 416–419 and 456–457, (ii) DNA kink intercalated by F283 and (iii) minor groove bound by residues 416–419 and 456–457. ( C ) Surface of the ICP4N dimer colored by electrostatic potential (red through blue for acidic to basic charge) calculated by Adaptive Poisson-Boltzmann Solver module in Chimera ( 53 ). DNA is shown in cartoon form with the ICP4 consensus sequence labeled on sense strand. ( D ) Plot of DNA major- and minor-groove widths measured in both models in the ICP4N · IE3_19mer asymmetric unit ( 54 ).

    Article Snippet: In order to study the DNA interactions of ICP4N constructs, synthetic IE3 DNA oligos were purchased (Invitrogen), namely IE3_19mer forward: CCGATCGTCCACACGGAGC and reverse-complement: GCTCCGTGTGGACGATCGG, IE3_19merMUT forward: CCGATCGTCCAAGATTAGC and reverse complement: GCTAATCTTGGACGATCGG, plus IE3_12mer forward: CCGATCGTCCAC and reverse-complement: GTGGACGATCGG.

    Techniques: Nuclear Magnetic Resonance, SPR Assay, Sequencing, Labeling

    Structural details of and biophysical characterization of ICP4N dimerization. ( A ) Cartoon of protein chains C and D (colored blue and green respectively) from the ICP4N·IE3_19mer structure with α-helices 5, 6 and 7 highlighted which form the major hydrophobic homo-dimer interface. ( B ) Details of the residues within the major homo-dimer interface, hydrophobic contacts are indicated by orange lines and hydrogen bonds by pink dashes. ( C ) SEC-MALS profile of ICP4N with and without IE3_19mer DNA, shown as dashed or solid lines respectively and refractive index (black lines) and molecular mass (green lines), plotted against elution volume. ( D ) Velocity AUC analysis of ICP4N with and without IE3_19mer DNA, shown as dashed or solid lines respectively. For each sample a major peak was observed corresponding to a dimeric protein, free or in complex with DNA.

    Journal: Nucleic Acids Research

    Article Title: The herpes viral transcription factor ICP4 forms a novel DNA recognition complex

    doi: 10.1093/nar/gkx419

    Figure Lengend Snippet: Structural details of and biophysical characterization of ICP4N dimerization. ( A ) Cartoon of protein chains C and D (colored blue and green respectively) from the ICP4N·IE3_19mer structure with α-helices 5, 6 and 7 highlighted which form the major hydrophobic homo-dimer interface. ( B ) Details of the residues within the major homo-dimer interface, hydrophobic contacts are indicated by orange lines and hydrogen bonds by pink dashes. ( C ) SEC-MALS profile of ICP4N with and without IE3_19mer DNA, shown as dashed or solid lines respectively and refractive index (black lines) and molecular mass (green lines), plotted against elution volume. ( D ) Velocity AUC analysis of ICP4N with and without IE3_19mer DNA, shown as dashed or solid lines respectively. For each sample a major peak was observed corresponding to a dimeric protein, free or in complex with DNA.

    Article Snippet: In order to study the DNA interactions of ICP4N constructs, synthetic IE3 DNA oligos were purchased (Invitrogen), namely IE3_19mer forward: CCGATCGTCCACACGGAGC and reverse-complement: GCTCCGTGTGGACGATCGG, IE3_19merMUT forward: CCGATCGTCCAAGATTAGC and reverse complement: GCTAATCTTGGACGATCGG, plus IE3_12mer forward: CCGATCGTCCAC and reverse-complement: GTGGACGATCGG.

    Techniques: Size-exclusion Chromatography

    Model of action of the ICP4 DNA binding domain (DBD). The globular homo-dimer is represented by gray and black ovals and N-terminal IDRs by lines. ( A ) Free protein adopts an expanded conformation with the IDRs extended. ( B ) When not in contact with a DNA consensus site, ICP4-DBD and particularly the IDRs search DNA strands for sequence motifs. ( C ) Binding to the IE3 consensus site which overlaps with the transcription initiation site, ICP4 forms an asymmetric complex by the synergistic action of the globular region to the RTCGTC motif and an IDR with the downstream YnYSG motif. One IDR is not involved in specific DNA recognition and points upstream toward the TATA box, which is compatible with tripartite complex formation by the TATA binding protein, TFIIB and ICP4.

    Journal: Nucleic Acids Research

    Article Title: The herpes viral transcription factor ICP4 forms a novel DNA recognition complex

    doi: 10.1093/nar/gkx419

    Figure Lengend Snippet: Model of action of the ICP4 DNA binding domain (DBD). The globular homo-dimer is represented by gray and black ovals and N-terminal IDRs by lines. ( A ) Free protein adopts an expanded conformation with the IDRs extended. ( B ) When not in contact with a DNA consensus site, ICP4-DBD and particularly the IDRs search DNA strands for sequence motifs. ( C ) Binding to the IE3 consensus site which overlaps with the transcription initiation site, ICP4 forms an asymmetric complex by the synergistic action of the globular region to the RTCGTC motif and an IDR with the downstream YnYSG motif. One IDR is not involved in specific DNA recognition and points upstream toward the TATA box, which is compatible with tripartite complex formation by the TATA binding protein, TFIIB and ICP4.

    Article Snippet: In order to study the DNA interactions of ICP4N constructs, synthetic IE3 DNA oligos were purchased (Invitrogen), namely IE3_19mer forward: CCGATCGTCCACACGGAGC and reverse-complement: GCTCCGTGTGGACGATCGG, IE3_19merMUT forward: CCGATCGTCCAAGATTAGC and reverse complement: GCTAATCTTGGACGATCGG, plus IE3_12mer forward: CCGATCGTCCAC and reverse-complement: GTGGACGATCGG.

    Techniques: Binding Assay, Sequencing

    ( A ) Schematic representation of HPV16 plasmid pHPV16ANE2fs that is integrated in the genome of the transfected and immortalized human keratinocyte cell line 3310 ( 42 ). Open reading frames are represented as rectangles, the early and late promoters p97 and p670 are indicated as arrows, and splice sites as triangles. The long control region (LCR) and the early and late polyA signals pAE and pAL are indicated. Two loxP sites and two SphI restriction sites flanking the HPV16 genome are indicated, as is the RSV-neoR-SV40pA cassette. The E2 frame-shift mutation is indicated. ( B ) Western blot analysis of differentiation marker involucrin, phosphorylated Akt kinase (p-Akt S473) and the Akt phosphorylation substrate p-GSK3b and b-actin in the absence (DMSO) or presence of Akt inhibitor GDC0068, and in the absence (–) or presence (+) of Ca-induced differentiation. ( C ) RT-PCR of HPV16 L1, L1i, E4 and GAPDH mRNAs in the absence (DMSO) or presence of Akt inhibitor GDC0068, and in the absence (–) or presence (+) of Ca-induced differentiation. ( D ) Upper left panel: RT-PCR of HPV16 E4 mRNAs spliced from SD880 to SA3358 in 3310 cells grown in the absence (DMSO) or presence of Akt inhibitor GDC0068, and in the absence (–) or presence (+) of Ca-induced differentiation. Upper right panel: extracts from UV irradiated 3310 cells grown in the absence (DMSO) or presence of Akt inhibitor GDC0068, and in the absence (–) or presence (+) of Ca-induced differentiation were subjected to immunoprecipitation with IgG or anti-hnRNP L antibody followed by RNA extraction and RT-PCR with primers that detect E4 mRNAs spliced from SD880 to SA3358. ( E ) RT-qPCR on the HPV16 E4 mRNAs spliced from SD880 to SA3358 shown in (D).

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: ( A ) Schematic representation of HPV16 plasmid pHPV16ANE2fs that is integrated in the genome of the transfected and immortalized human keratinocyte cell line 3310 ( 42 ). Open reading frames are represented as rectangles, the early and late promoters p97 and p670 are indicated as arrows, and splice sites as triangles. The long control region (LCR) and the early and late polyA signals pAE and pAL are indicated. Two loxP sites and two SphI restriction sites flanking the HPV16 genome are indicated, as is the RSV-neoR-SV40pA cassette. The E2 frame-shift mutation is indicated. ( B ) Western blot analysis of differentiation marker involucrin, phosphorylated Akt kinase (p-Akt S473) and the Akt phosphorylation substrate p-GSK3b and b-actin in the absence (DMSO) or presence of Akt inhibitor GDC0068, and in the absence (–) or presence (+) of Ca-induced differentiation. ( C ) RT-PCR of HPV16 L1, L1i, E4 and GAPDH mRNAs in the absence (DMSO) or presence of Akt inhibitor GDC0068, and in the absence (–) or presence (+) of Ca-induced differentiation. ( D ) Upper left panel: RT-PCR of HPV16 E4 mRNAs spliced from SD880 to SA3358 in 3310 cells grown in the absence (DMSO) or presence of Akt inhibitor GDC0068, and in the absence (–) or presence (+) of Ca-induced differentiation. Upper right panel: extracts from UV irradiated 3310 cells grown in the absence (DMSO) or presence of Akt inhibitor GDC0068, and in the absence (–) or presence (+) of Ca-induced differentiation were subjected to immunoprecipitation with IgG or anti-hnRNP L antibody followed by RNA extraction and RT-PCR with primers that detect E4 mRNAs spliced from SD880 to SA3358. ( E ) RT-qPCR on the HPV16 E4 mRNAs spliced from SD880 to SA3358 shown in (D).

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Plasmid Preparation, Transfection, Mutagenesis, Western Blot, Marker, Reverse Transcription Polymerase Chain Reaction, Irradiation, Immunoprecipitation, RNA Extraction, Quantitative RT-PCR

    ( A ) Schematic representation of the pBELsLuc reporter plasmid stably integrated in the genome of the C33A2 reporter cell line. Transcription of the HPV16 sequences in the pBELsLuc plasmid is driven by the human cytomegalovirus promoter (CMV). The sLuc gene inserted into the L1 region is indicated and is preceded by the poliovirus 2A internal ribosome entry site (IRES). HPV16 E2 and E4 mRNAs mRNAs produced by C33A2 cells are indicated in light grey (E2 and E4 mRNAs) and HPV16 late mRNAs encoding sLuc that can be induced in this reporter cell line are indicated in black. The location of RT-PCR primers is indicated below. ( B ) Secreted luciferase enzyme activity (sLuc) in the cell culture medium of reporter cell line C33A2 treated with various concentrations of the Akt kinase inhibitor GDC0068. sLuc activity was monitored at the indicated time points. sLuc activity is displayed as fold over DMSO-treated C33A2 cells at the various time points. ( C ) RT-PCR on total RNA extracted from the C33A2 reporter cell line treated with DMSO alone or Akti kinase inhibitor GDC0068 for 4.5hrs or 9hrs. The RT-PCR primers detected HPV16 L1/L1i-, E4- or GAPDH-mRNAs. ( D ) RT-PCR on total RNA extracted from the C33A2 reporter cell line treated with DMSO alone or various concentrations of Akt kinase inhibitor GDC0068 (50-, 100- or 200uM) for 9hrs. The RT-PCR primers detected HPV16 L1/L1i-, L2-, total late (L2 + L1 +L1i), E4-, E2- or GAPDH-mRNAs. ( E ) A 3′-RACE assay on total RNA extracted from C33A2 cells. Primers were F-Set2 and (dT)17-P3 for pAE and sLuc-S-inner and (dT)17-P3 for pAL (for primers see Supplementary Table S1 ). Spliced HPV16 E4 mRNAs and cellular GAPDH mRNAs are also shown. ( F ) RT-qPCR on the cDNA samples used for RT-PCR in Figure 2D .

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: ( A ) Schematic representation of the pBELsLuc reporter plasmid stably integrated in the genome of the C33A2 reporter cell line. Transcription of the HPV16 sequences in the pBELsLuc plasmid is driven by the human cytomegalovirus promoter (CMV). The sLuc gene inserted into the L1 region is indicated and is preceded by the poliovirus 2A internal ribosome entry site (IRES). HPV16 E2 and E4 mRNAs mRNAs produced by C33A2 cells are indicated in light grey (E2 and E4 mRNAs) and HPV16 late mRNAs encoding sLuc that can be induced in this reporter cell line are indicated in black. The location of RT-PCR primers is indicated below. ( B ) Secreted luciferase enzyme activity (sLuc) in the cell culture medium of reporter cell line C33A2 treated with various concentrations of the Akt kinase inhibitor GDC0068. sLuc activity was monitored at the indicated time points. sLuc activity is displayed as fold over DMSO-treated C33A2 cells at the various time points. ( C ) RT-PCR on total RNA extracted from the C33A2 reporter cell line treated with DMSO alone or Akti kinase inhibitor GDC0068 for 4.5hrs or 9hrs. The RT-PCR primers detected HPV16 L1/L1i-, E4- or GAPDH-mRNAs. ( D ) RT-PCR on total RNA extracted from the C33A2 reporter cell line treated with DMSO alone or various concentrations of Akt kinase inhibitor GDC0068 (50-, 100- or 200uM) for 9hrs. The RT-PCR primers detected HPV16 L1/L1i-, L2-, total late (L2 + L1 +L1i), E4-, E2- or GAPDH-mRNAs. ( E ) A 3′-RACE assay on total RNA extracted from C33A2 cells. Primers were F-Set2 and (dT)17-P3 for pAE and sLuc-S-inner and (dT)17-P3 for pAL (for primers see Supplementary Table S1 ). Spliced HPV16 E4 mRNAs and cellular GAPDH mRNAs are also shown. ( F ) RT-qPCR on the cDNA samples used for RT-PCR in Figure 2D .

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Plasmid Preparation, Stable Transfection, Produced, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, Cell Culture, Quantitative RT-PCR

    ( A ) Schematic representation of the HPV16 subgenomic expression plasmid pBEL. Transcription of the HPV16 sequences in the pBEL plasmid is driven by the human cytomegalovirus promoter (CMV). Rectangles represent open reading frames. Filled and open triangles represent 5′- and 3′-splices sites respectively, and HPV16 early and late polyA signals are indicated as pAE and pAL respectively. The HPV16 E4 and E2 mRNAs produced by pBEL upon transfection of mammalian cells is indicated in light grey. HPV16 late mRNAs L1, L1i and L2 that can be induced from the pBEL reporter plasmid are indicated in black. Positions of HPV16 RT-PCR primers are indicated. ( B ) Left panel: RT-PCR of HPV16 E4 mRNAs spliced from SD880 to SA3358 in 293T cells transfected with or without HPV16 subgenomic plasmid pBEL in the absence or presence of hnRNP L expressing plasmid pCMV-hnRNP L. The transfected cells were subjected to UV irradiation before harvest as detailed in Materials and methods for CLIP assay. Right panel: The extracts from the transfected cells were subjected to immunoprecipitation with IgG or anti-hnRNP L antibody followed by RNA extraction and RT-PCR with primers that detect E4 mRNAs spliced from SD880 to SA3358. ( C ) RT-qPCR on the RNA analysed in (B). ( D and E ) RT-PCR of HPV16 E4 mRNAs spliced from SD880 to SA3358 in 293T cells transfected with HPV16 subgenomic plasmid pBEL in the absence or presence of hnRNP L expressing plasmid pCMV-hnRNP L and in the absence or presence of 100uM Akt kinase inhibitor GDC0068 (GDC0068). The transfected cells were subjected to UV irradiation and extracts from the transfected cells were prepared and subjected to immunoprecipitation with IgG or anti-hnRNP L antibody followed by RNA extraction and RT-PCR with primers that detect HPV16 E4 mRNAs (D) or all HPV16 late mRNAs (Total late: HPV16 L2, L1 and L1i mRNAs).

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: ( A ) Schematic representation of the HPV16 subgenomic expression plasmid pBEL. Transcription of the HPV16 sequences in the pBEL plasmid is driven by the human cytomegalovirus promoter (CMV). Rectangles represent open reading frames. Filled and open triangles represent 5′- and 3′-splices sites respectively, and HPV16 early and late polyA signals are indicated as pAE and pAL respectively. The HPV16 E4 and E2 mRNAs produced by pBEL upon transfection of mammalian cells is indicated in light grey. HPV16 late mRNAs L1, L1i and L2 that can be induced from the pBEL reporter plasmid are indicated in black. Positions of HPV16 RT-PCR primers are indicated. ( B ) Left panel: RT-PCR of HPV16 E4 mRNAs spliced from SD880 to SA3358 in 293T cells transfected with or without HPV16 subgenomic plasmid pBEL in the absence or presence of hnRNP L expressing plasmid pCMV-hnRNP L. The transfected cells were subjected to UV irradiation before harvest as detailed in Materials and methods for CLIP assay. Right panel: The extracts from the transfected cells were subjected to immunoprecipitation with IgG or anti-hnRNP L antibody followed by RNA extraction and RT-PCR with primers that detect E4 mRNAs spliced from SD880 to SA3358. ( C ) RT-qPCR on the RNA analysed in (B). ( D and E ) RT-PCR of HPV16 E4 mRNAs spliced from SD880 to SA3358 in 293T cells transfected with HPV16 subgenomic plasmid pBEL in the absence or presence of hnRNP L expressing plasmid pCMV-hnRNP L and in the absence or presence of 100uM Akt kinase inhibitor GDC0068 (GDC0068). The transfected cells were subjected to UV irradiation and extracts from the transfected cells were prepared and subjected to immunoprecipitation with IgG or anti-hnRNP L antibody followed by RNA extraction and RT-PCR with primers that detect HPV16 E4 mRNAs (D) or all HPV16 late mRNAs (Total late: HPV16 L2, L1 and L1i mRNAs).

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Expressing, Plasmid Preparation, Produced, Transfection, Reverse Transcription Polymerase Chain Reaction, Irradiation, Cross-linking Immunoprecipitation, Immunoprecipitation, RNA Extraction, Quantitative RT-PCR

    ( A ) Schematic representation of the HPV16 genome. Rectangles represent open reading frames, promoters p97 and p670 are indicated as arrows, filled and open triangles represent 5′- and 3′-splices sites respectively, HPV16 early and late polyA signals pAE and pAL are indicated. ( B ) Schematic representations of reporter plasmids pE4EL1M, pE4EL1, pE4D and pE4DL1D. Lines indicate segments from the HPV16 genome that have been inserted into the ‘HPV16 mini-constructs’. The plasmids encode the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter followed by HPV16 sequences that encompass HPV16 splice sites SD880, SA3358, SD3632 and SA5639 and the simian virus 40 (SV40) polyadenylation signal (SV40 pA). The hnRNP L binding sites mapped to oligos G, M, T, 8B, 10B, -3C, -1A, and 2 are indicated. Binding site ‘2’ consist of the overlapping binding sites 2 2A, 2B and 2C). The bindings sites refer to the mapping of hnRNP L binding sites in Figures 3 and 4 and supplementary figures S2 and S3 . C33A cells were transfected with the various plasmids and cytoplasmic RNA was extracted and subjected to cDNA synthesis and RT-PCR with the oligonucleotides shown in (B) and indicated below the gels. ( C ) RT-PCR with primers 773S and E42s detecting mRNAs spliced between SD880 and SA3358. ( D ) RT-PCR with primers 773S and L1as detecting all spliced HPV16 mRNAs. ( E ) RT-PCR with primers 773S and L1AM specifically detecting mRNAs spliced to SA5639 in plasmid pE4EL1M that contains a mutant L1 sequence in which previously described splicing silencer elements have been inactivated ( 49 ). ( F ) RT-PCR with primers gapdhs and gapdha detecting spliced gapdh mRNAs. gap; gapdh mRNAs.

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: ( A ) Schematic representation of the HPV16 genome. Rectangles represent open reading frames, promoters p97 and p670 are indicated as arrows, filled and open triangles represent 5′- and 3′-splices sites respectively, HPV16 early and late polyA signals pAE and pAL are indicated. ( B ) Schematic representations of reporter plasmids pE4EL1M, pE4EL1, pE4D and pE4DL1D. Lines indicate segments from the HPV16 genome that have been inserted into the ‘HPV16 mini-constructs’. The plasmids encode the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter followed by HPV16 sequences that encompass HPV16 splice sites SD880, SA3358, SD3632 and SA5639 and the simian virus 40 (SV40) polyadenylation signal (SV40 pA). The hnRNP L binding sites mapped to oligos G, M, T, 8B, 10B, -3C, -1A, and 2 are indicated. Binding site ‘2’ consist of the overlapping binding sites 2 2A, 2B and 2C). The bindings sites refer to the mapping of hnRNP L binding sites in Figures 3 and 4 and supplementary figures S2 and S3 . C33A cells were transfected with the various plasmids and cytoplasmic RNA was extracted and subjected to cDNA synthesis and RT-PCR with the oligonucleotides shown in (B) and indicated below the gels. ( C ) RT-PCR with primers 773S and E42s detecting mRNAs spliced between SD880 and SA3358. ( D ) RT-PCR with primers 773S and L1as detecting all spliced HPV16 mRNAs. ( E ) RT-PCR with primers 773S and L1AM specifically detecting mRNAs spliced to SA5639 in plasmid pE4EL1M that contains a mutant L1 sequence in which previously described splicing silencer elements have been inactivated ( 49 ). ( F ) RT-PCR with primers gapdhs and gapdha detecting spliced gapdh mRNAs. gap; gapdh mRNAs.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Construct, Binding Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Mutagenesis, Sequencing

    ( A ) Western blots of various RNA binding proteins in two independent preparations of nuclear extracts (Prep 1 and Prep 2) prepared from C33A2 cells treated with DMSO (D) or 100 uM Akt kinase inhibitor GDC0068 (G) for 3 h. Western blots of indicated proteins pulled down with biotinylated ssDNA oligos ( B ) or RNA oligos ( C ) representing sequences at the HPV16 3′-splice site SA3358 in the E4-coding exon. ( D ) Western blots of indicated proteins pulled down with biotinylated ssDNA oligos representing sequences at the HPV16 late 5′-splice site SD3632 in the E4-coding exon. ( E ) Western blots of indicated proteins pulled down with biotinylated ssRNA oligos spanning HPV16 5′-splice sites SD880 (BRnSD862) and SD3632 (BRnSD3632). ( F ) Western blots of hnRNP L, hnRNP A1, U2AF65 or U2AF35 pulled down from nuclear extracts using ssDNA oligos at HPV16 late splice site SA5639. For all pull downs in the figure, nuclear extracts prepared from C33A2 cells treated with DMSO (D) or Akt kinase inhibitor GDC0068 (G) were used.

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: ( A ) Western blots of various RNA binding proteins in two independent preparations of nuclear extracts (Prep 1 and Prep 2) prepared from C33A2 cells treated with DMSO (D) or 100 uM Akt kinase inhibitor GDC0068 (G) for 3 h. Western blots of indicated proteins pulled down with biotinylated ssDNA oligos ( B ) or RNA oligos ( C ) representing sequences at the HPV16 3′-splice site SA3358 in the E4-coding exon. ( D ) Western blots of indicated proteins pulled down with biotinylated ssDNA oligos representing sequences at the HPV16 late 5′-splice site SD3632 in the E4-coding exon. ( E ) Western blots of indicated proteins pulled down with biotinylated ssRNA oligos spanning HPV16 5′-splice sites SD880 (BRnSD862) and SD3632 (BRnSD3632). ( F ) Western blots of hnRNP L, hnRNP A1, U2AF65 or U2AF35 pulled down from nuclear extracts using ssDNA oligos at HPV16 late splice site SA5639. For all pull downs in the figure, nuclear extracts prepared from C33A2 cells treated with DMSO (D) or Akt kinase inhibitor GDC0068 (G) were used.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Western Blot, RNA Binding Assay

    Model for the control of HPV16 late gene expression by Akt-kinase regulated RNA binding hnRNP-proteins. ( A ) At efficiently used HPV16 3′-splice site SA3358, hnRNP L (L) binds primarily downstream of the splice site in the exonic sequences and does not interfere with U2AF65/35 binding upstream of SA3358. This binding pattern suggest that hnRNP L does not inhibit SA3358, and is consistent with a positive role for hnRNP L on SA3358. At the same time, hnRNP L binds to splicing inhibitory sequences upstream of SD3632, adjacent to hnRNP D (D) binding sites that have been shown previously to suppress SD3632, which suggest a splicing inhibitory role for hnRNP L at SD3632. In addition, hnRNP L is pulled down by oligos that also bind to hnRNP C (C1) in the HPV16 early UTR, and hnRNP L interacts with hnRNP C. We suggest a model in which Akt-phosphorylated hnRNP L binds at HPV16 SA3358, SD3632 and pAE and that this prevents utilisation of HPV16 late 5′-splice site SD3632 and favour utilisation of HPV16 3′-splice site SA3358 and the HPV16 early polyadenylation signal pAE. At the other suppressed HPV16 late splice site SA5639, hnRNP L binds at both upstream and downstream sequences. The interactions of Sam68 ( 68 ), hnRNP A1 (A1), hnRNP A2 (A2) and hnRNP L (L) with sequences at HPV16 SA5639 is consistent with an inhibitory function of these proteins on splicing, most likely by inhibiting binding of U2AF65 at SA5639. This scenario represents the early state of the HPV16 life cycle. ( B ) As the HPV16 infected cells differentiate and Akt kinase activity is reduced, hnRNP L is dephosphorylated and binding to HPV16 mRNAs is reduced. We suggest a model for activation of HPV16 late gene expression in which interactions between hnRNP L and splicing silencer sequences at SD3632, the early UTR and with the hnRNP C are reduced or lost. Inhibition of Akt kinase dephosphorylates hnRNP L, thereby freeing hnRNP C at the early UTR, allowing it to interfere with hnRNP D at SD3632, which results in recognition of HPV16 SD3632 by U1snRNP activation of HPV16 L1 mRNA production. We have previously shown that overexpression of hnRNP C activates SD3632 in an HPV16 early UTR-dependent manner ( 41 ). Similarly, lost binding of hnRNP L at HPV16 late 3′-splice site SA5639 allows this splice site to interact with splicing factor U2AF65, thereby activating SA5639. Since each hnRNP L molecule can interact simultaneously with at least two RNA binding sites, and two RNA-bound hnRNP L proteins can interact with each other ( 68 ), one may speculate that multiple binding sites for hnRNP L enhances the inhibitory effect on HPV16 late splice site SA5639. The overall result of Akt inhibition and hnRNP L dephosphorylation, is that binding of hnRNP L to HPV16 mRNAs decreases and U2AF65 binding increases. This scenario represents the late state of the HPV16 life cycle. A1, hnRNP A1; A2, hnRNP A2; C1, hnRNP C; D, hnRNP D; L, hnRNP L; 68, Sam68; pAE, HPV16 early polyadenylation signal; pAL, HPV16 late polyadenylation signal.

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: Model for the control of HPV16 late gene expression by Akt-kinase regulated RNA binding hnRNP-proteins. ( A ) At efficiently used HPV16 3′-splice site SA3358, hnRNP L (L) binds primarily downstream of the splice site in the exonic sequences and does not interfere with U2AF65/35 binding upstream of SA3358. This binding pattern suggest that hnRNP L does not inhibit SA3358, and is consistent with a positive role for hnRNP L on SA3358. At the same time, hnRNP L binds to splicing inhibitory sequences upstream of SD3632, adjacent to hnRNP D (D) binding sites that have been shown previously to suppress SD3632, which suggest a splicing inhibitory role for hnRNP L at SD3632. In addition, hnRNP L is pulled down by oligos that also bind to hnRNP C (C1) in the HPV16 early UTR, and hnRNP L interacts with hnRNP C. We suggest a model in which Akt-phosphorylated hnRNP L binds at HPV16 SA3358, SD3632 and pAE and that this prevents utilisation of HPV16 late 5′-splice site SD3632 and favour utilisation of HPV16 3′-splice site SA3358 and the HPV16 early polyadenylation signal pAE. At the other suppressed HPV16 late splice site SA5639, hnRNP L binds at both upstream and downstream sequences. The interactions of Sam68 ( 68 ), hnRNP A1 (A1), hnRNP A2 (A2) and hnRNP L (L) with sequences at HPV16 SA5639 is consistent with an inhibitory function of these proteins on splicing, most likely by inhibiting binding of U2AF65 at SA5639. This scenario represents the early state of the HPV16 life cycle. ( B ) As the HPV16 infected cells differentiate and Akt kinase activity is reduced, hnRNP L is dephosphorylated and binding to HPV16 mRNAs is reduced. We suggest a model for activation of HPV16 late gene expression in which interactions between hnRNP L and splicing silencer sequences at SD3632, the early UTR and with the hnRNP C are reduced or lost. Inhibition of Akt kinase dephosphorylates hnRNP L, thereby freeing hnRNP C at the early UTR, allowing it to interfere with hnRNP D at SD3632, which results in recognition of HPV16 SD3632 by U1snRNP activation of HPV16 L1 mRNA production. We have previously shown that overexpression of hnRNP C activates SD3632 in an HPV16 early UTR-dependent manner ( 41 ). Similarly, lost binding of hnRNP L at HPV16 late 3′-splice site SA5639 allows this splice site to interact with splicing factor U2AF65, thereby activating SA5639. Since each hnRNP L molecule can interact simultaneously with at least two RNA binding sites, and two RNA-bound hnRNP L proteins can interact with each other ( 68 ), one may speculate that multiple binding sites for hnRNP L enhances the inhibitory effect on HPV16 late splice site SA5639. The overall result of Akt inhibition and hnRNP L dephosphorylation, is that binding of hnRNP L to HPV16 mRNAs decreases and U2AF65 binding increases. This scenario represents the late state of the HPV16 life cycle. A1, hnRNP A1; A2, hnRNP A2; C1, hnRNP C; D, hnRNP D; L, hnRNP L; 68, Sam68; pAE, HPV16 early polyadenylation signal; pAL, HPV16 late polyadenylation signal.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Expressing, RNA Binding Assay, Binding Assay, Infection, Activity Assay, Activation Assay, Inhibition, Over Expression, De-Phosphorylation Assay

    ( A ) sLuc activity induced from pBELsLuc in HeLa cells co-transfected with pBELsLuc and the indicated plasmids. ( B ) 3′-RACE assay on total RNA extracted from HeLa cells transfected with pBELsLuc reporter plasmid and empty vector or a plasmid expressing hnRNP C1. The primers specifically detect mRNAs polyadenylated at HPV16 pAE or HPV16 pAL. The pAE- or pAL-3′-RACE products were quantified and the levels detected in cells transfected with hnRNP C plasmid were divided by the levels detected in cells transfected with empty plasmid (vector). The ratios are indicated below the gels. ( C ) RT-PCR on HPV16 E4 mRNAs or L1 and L1i mRNAs ( D ) produced from pBELsLuc transiently transfected into HeLa cells in the presence of empty vector or plasmid expressing hnRNP C1. The RT-PCR products were quantified and the levels detected in cells transfected with hnRNP C plasmid were divided by the levels detected in cells transfected with empty plasmid (vector). In case of the L1 mRNAs, the ratios between the L1 and L1i splice variants of the L1 mRNAs in cells transfected with hnRNP C plasmid or empty plasmid (vector) are shown. The ratios are indicated below the gels. GAP, GAPDH mRNAs. ( E ) Schematic representation of genomic HPV16 plasmid pHPV16ANSL ( 32 , 59 ). LoxP sites and HPV16 early (p97) and late (p670) promoters and early (pAE) and late (pAL) poly(A) signals are indicated. The effect of the cre recombinase on these plasmids is illustrated ( 59 ). ( F ) sLuc activity produced in HeLa cells transfected with pHPV16ANSL in the absence or presence of various concentrations of hnRNP C-expression plasmid. ( G ) 3′-RACE assay on total RNA extracted from HeLa cells transfected with pHPV16ANSL and empty vector or hnRNP C1- or Fip1- expression plasmid. The 3′-RACE primers specifically detect mRNAs polyadenylated at HPV16 pAE or HPV16 pAL. The pAE- or pAL-3′-RACE products were quantified and the levels detected in cells transfected with hnRNP C plasmid or Fip1 plasmid were divided by the levels detected in cells transfected with empty plasmid (vector). The ratios are indicated below the gels.

    Journal: Nucleic Acids Research

    Article Title: The DNA damage response activates HPV16 late gene expression at the level of RNA processing

    doi: 10.1093/nar/gky227

    Figure Lengend Snippet: ( A ) sLuc activity induced from pBELsLuc in HeLa cells co-transfected with pBELsLuc and the indicated plasmids. ( B ) 3′-RACE assay on total RNA extracted from HeLa cells transfected with pBELsLuc reporter plasmid and empty vector or a plasmid expressing hnRNP C1. The primers specifically detect mRNAs polyadenylated at HPV16 pAE or HPV16 pAL. The pAE- or pAL-3′-RACE products were quantified and the levels detected in cells transfected with hnRNP C plasmid were divided by the levels detected in cells transfected with empty plasmid (vector). The ratios are indicated below the gels. ( C ) RT-PCR on HPV16 E4 mRNAs or L1 and L1i mRNAs ( D ) produced from pBELsLuc transiently transfected into HeLa cells in the presence of empty vector or plasmid expressing hnRNP C1. The RT-PCR products were quantified and the levels detected in cells transfected with hnRNP C plasmid were divided by the levels detected in cells transfected with empty plasmid (vector). In case of the L1 mRNAs, the ratios between the L1 and L1i splice variants of the L1 mRNAs in cells transfected with hnRNP C plasmid or empty plasmid (vector) are shown. The ratios are indicated below the gels. GAP, GAPDH mRNAs. ( E ) Schematic representation of genomic HPV16 plasmid pHPV16ANSL ( 32 , 59 ). LoxP sites and HPV16 early (p97) and late (p670) promoters and early (pAE) and late (pAL) poly(A) signals are indicated. The effect of the cre recombinase on these plasmids is illustrated ( 59 ). ( F ) sLuc activity produced in HeLa cells transfected with pHPV16ANSL in the absence or presence of various concentrations of hnRNP C-expression plasmid. ( G ) 3′-RACE assay on total RNA extracted from HeLa cells transfected with pHPV16ANSL and empty vector or hnRNP C1- or Fip1- expression plasmid. The 3′-RACE primers specifically detect mRNAs polyadenylated at HPV16 pAE or HPV16 pAL. The pAE- or pAL-3′-RACE products were quantified and the levels detected in cells transfected with hnRNP C plasmid or Fip1 plasmid were divided by the levels detected in cells transfected with empty plasmid (vector). The ratios are indicated below the gels.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O-Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich.

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Produced

    ChIP analyses on HPV16-positive tonsillar cancer cell line HN26 that contains episomal HPV16 DNA (unpublished results). Melphalan treated HN26 cells were subjected to ChIP analysis using antibodies to DDR factors BRCA1 and BLAF1 ( A ) and RNA binding proteins U2A65 and hnRNP C ( B ). Primers and antibodies are listed in Supplementary Tables S3 and 4 , respectively, and the location in the HPV16 genome of the PCR primers is shown in Supplementary Figure S5 . Mean values with standard deviations of the amount of immunoprecipitated DNA compared to DNA from DMSO-treated cells are displayed. The q-PCR values obtained for each primer pair with DNA extracted from DMSO-treated HN26 cells were set to 1 to correct for differences between different ChIP extracts. Chip extracts were prepared from C33A2 cells treated with melphalan for the indicated time-periods.

    Journal: Nucleic Acids Research

    Article Title: The DNA damage response activates HPV16 late gene expression at the level of RNA processing

    doi: 10.1093/nar/gky227

    Figure Lengend Snippet: ChIP analyses on HPV16-positive tonsillar cancer cell line HN26 that contains episomal HPV16 DNA (unpublished results). Melphalan treated HN26 cells were subjected to ChIP analysis using antibodies to DDR factors BRCA1 and BLAF1 ( A ) and RNA binding proteins U2A65 and hnRNP C ( B ). Primers and antibodies are listed in Supplementary Tables S3 and 4 , respectively, and the location in the HPV16 genome of the PCR primers is shown in Supplementary Figure S5 . Mean values with standard deviations of the amount of immunoprecipitated DNA compared to DNA from DMSO-treated cells are displayed. The q-PCR values obtained for each primer pair with DNA extracted from DMSO-treated HN26 cells were set to 1 to correct for differences between different ChIP extracts. Chip extracts were prepared from C33A2 cells treated with melphalan for the indicated time-periods.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O-Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich.

    Techniques: Chromatin Immunoprecipitation, RNA Binding Assay, Polymerase Chain Reaction, Immunoprecipitation

    ( A ) 3′-RACE assays on total RNA extracted from C33A2 cells treated with DMSO (D) or 100 µM melphalan (M) for 22 h. The primers specifically detect mRNAs polyadenylated HPV16 pAE or HPV16 pAL. The left and right panels show 3′-RACE with two different primer pairs. ( B ) A 3′-RACE experiment on total RNA extracted from C33A2 cells treated with DMSO or 100 µM melphalan for various indicated time-points. The 3′-RACE reactions were performed in the absence (−) or presence (+) of RT-enzyme. pAE, mRNAs polyadenylated at HPV16 early polyadenylation signal pAE; pAL, mRNAs polyadenylated at HPV16 late polyadenylation signal pAL. ( C ) Quantiations of the pAE levels (left panel) or the pAL levels (right panel) shown in (C). pAE levels at 0 h of melphalan treatment were set as 100% (left panel) and pAL levels at 22 h of melphalan treatment were set as 100% (right panel). ( D ) Ct values of total mRNA levels produced from pBELsLuc or gapdh in C33A2 cells at different time points after addition of melphalan. Location of primers in pBELsLuc are shown in the schematic representation of pBELsLuc in Supplementary Figure S6 .

    Journal: Nucleic Acids Research

    Article Title: The DNA damage response activates HPV16 late gene expression at the level of RNA processing

    doi: 10.1093/nar/gky227

    Figure Lengend Snippet: ( A ) 3′-RACE assays on total RNA extracted from C33A2 cells treated with DMSO (D) or 100 µM melphalan (M) for 22 h. The primers specifically detect mRNAs polyadenylated HPV16 pAE or HPV16 pAL. The left and right panels show 3′-RACE with two different primer pairs. ( B ) A 3′-RACE experiment on total RNA extracted from C33A2 cells treated with DMSO or 100 µM melphalan for various indicated time-points. The 3′-RACE reactions were performed in the absence (−) or presence (+) of RT-enzyme. pAE, mRNAs polyadenylated at HPV16 early polyadenylation signal pAE; pAL, mRNAs polyadenylated at HPV16 late polyadenylation signal pAL. ( C ) Quantiations of the pAE levels (left panel) or the pAL levels (right panel) shown in (C). pAE levels at 0 h of melphalan treatment were set as 100% (left panel) and pAL levels at 22 h of melphalan treatment were set as 100% (right panel). ( D ) Ct values of total mRNA levels produced from pBELsLuc or gapdh in C33A2 cells at different time points after addition of melphalan. Location of primers in pBELsLuc are shown in the schematic representation of pBELsLuc in Supplementary Figure S6 .

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O-Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich.

    Techniques: Produced

    ( A ) Schematic drawing of the HPV16 genome with a blow-up of the region around the HPV16 early polyadenylation signal pAE. The 35-nt biotinylated ssDNA oligos (overlapping by 5-nt) used in pull down assays are indicated. ( B ) Western blot with antibodies to the indicated polyadenylation factors and RNA binding proteins pulled down with the indicated biotinylated oligos using cellular extracts prepared from C33A2 cells treated with DMSO (−) or melphalan (+) for 6 h. Ratios of quantified levels of the various pulled down proteins in melphalan treated cells (+) over levels of pulled down protein in DMSO-treated cells (−) are shown below the gel. ( C ) Western blot on pull-down with biotinylated RNA oligos. Ratios of quantified levels of hnRNP C or CstF64 pulled down in melphalan treated cells (+) over levels of pulled down proteins in DMSO-treated cells (−) are shown below the gel. ( D ) Western blot with antibody to hnRNP C on proteins pulled down with the biotinylated oligos using cellular extracts prepared from C33A2 cells treated with melphalan for the indicated time periods. Ratios of hnRNP C levels in melphalan treated cells at various time points over levels of pulled down hnRNP C in C33A2 cells treated for 0 h are shown below the gels.

    Journal: Nucleic Acids Research

    Article Title: The DNA damage response activates HPV16 late gene expression at the level of RNA processing

    doi: 10.1093/nar/gky227

    Figure Lengend Snippet: ( A ) Schematic drawing of the HPV16 genome with a blow-up of the region around the HPV16 early polyadenylation signal pAE. The 35-nt biotinylated ssDNA oligos (overlapping by 5-nt) used in pull down assays are indicated. ( B ) Western blot with antibodies to the indicated polyadenylation factors and RNA binding proteins pulled down with the indicated biotinylated oligos using cellular extracts prepared from C33A2 cells treated with DMSO (−) or melphalan (+) for 6 h. Ratios of quantified levels of the various pulled down proteins in melphalan treated cells (+) over levels of pulled down protein in DMSO-treated cells (−) are shown below the gel. ( C ) Western blot on pull-down with biotinylated RNA oligos. Ratios of quantified levels of hnRNP C or CstF64 pulled down in melphalan treated cells (+) over levels of pulled down proteins in DMSO-treated cells (−) are shown below the gel. ( D ) Western blot with antibody to hnRNP C on proteins pulled down with the biotinylated oligos using cellular extracts prepared from C33A2 cells treated with melphalan for the indicated time periods. Ratios of hnRNP C levels in melphalan treated cells at various time points over levels of pulled down hnRNP C in C33A2 cells treated for 0 h are shown below the gels.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O-Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich.

    Techniques: Western Blot, RNA Binding Assay

    ( A – E ) Cell extracts from DMSO or melphalan-treated C33A2 cells were subjected to immunoprecipitation with the antibodies to phosphorylated BRCA1 (p-BRCA1) (A), hnRNP C (B) (C), Fip1 (D) or CPSF30 (E) followed by western blotting with antibodies to hnRNP C, CstF64 or Fip1 as indicated in the figure. The hours of DMSO or melphalan incubation of C33A2 cells are indicated on top of the gels. The levels of co-immunoprecipitated protein and the levels of each protein in the input extracts were quantified. Percent of input protein co-immunoprecipitated by each antibody in extracts from DMSO or melphalan treated cells are shown below each gel. For hnRNP C co-immunoprecipitations with anti-Fip1 or anti-CPSF30 antibody, the levels of co-immunoprecipiated hnRNP C in melphalan treated cells were divided by the levels of hnRNP C co-immunoprecipitated with anti Fip1 antibody or anti-CPSF30 in DMSO treated cells (D and E). ( F ) ChIP assays on DNA from C33A2 cells using antibody to hnRNP C and qPCR of the indicated HPV16 amplicons. Mean values with standard deviations of the amount of immunoprecipitated DNA compared to DNA from DMSO-treated cells are displayed. The qPCR values obtained for each primer pair with DNA extracted from DMSO-treated C33A2 cells were set to 1 to correct for differences between different ChIP extracts. Chip extracts were prepared from C33A2 cells treated with melphalan for the indicated time-periods. ( G ) C33A2 cells treated with DMSO or melphalan for the indicated time points were UV irradiated and subjected to CLIP assay as detailed in ‘Materials and Methods’ section. The RNA–protein complexes were immunoprecipitated with antibody to hnRNP C1 and the RNA extracted from the immunoprecipitated complexes was subjected to RT-PCR with primers that detect HPV16 E4 mRNAs spliced from SD880 to SA3358. ( H ) Western blot on extracts from C33A2 cells transfected with scrambled siRNAs (scr) or siRNAs to hnRNP C. hnRNP C levels were quantified in extracts prepared from cells transfected with the indicated siRNAs and divided by hnRNP C levels in extracts from cells transfected with scrambled siRNAs (scr). ( I ) sLuc activity produced by C33A2 cells transfected with scrambled siRNAs (scr) or siRNAs to hnRNP C followed by addition of 100 µM melphalan.

    Journal: Nucleic Acids Research

    Article Title: The DNA damage response activates HPV16 late gene expression at the level of RNA processing

    doi: 10.1093/nar/gky227

    Figure Lengend Snippet: ( A – E ) Cell extracts from DMSO or melphalan-treated C33A2 cells were subjected to immunoprecipitation with the antibodies to phosphorylated BRCA1 (p-BRCA1) (A), hnRNP C (B) (C), Fip1 (D) or CPSF30 (E) followed by western blotting with antibodies to hnRNP C, CstF64 or Fip1 as indicated in the figure. The hours of DMSO or melphalan incubation of C33A2 cells are indicated on top of the gels. The levels of co-immunoprecipitated protein and the levels of each protein in the input extracts were quantified. Percent of input protein co-immunoprecipitated by each antibody in extracts from DMSO or melphalan treated cells are shown below each gel. For hnRNP C co-immunoprecipitations with anti-Fip1 or anti-CPSF30 antibody, the levels of co-immunoprecipiated hnRNP C in melphalan treated cells were divided by the levels of hnRNP C co-immunoprecipitated with anti Fip1 antibody or anti-CPSF30 in DMSO treated cells (D and E). ( F ) ChIP assays on DNA from C33A2 cells using antibody to hnRNP C and qPCR of the indicated HPV16 amplicons. Mean values with standard deviations of the amount of immunoprecipitated DNA compared to DNA from DMSO-treated cells are displayed. The qPCR values obtained for each primer pair with DNA extracted from DMSO-treated C33A2 cells were set to 1 to correct for differences between different ChIP extracts. Chip extracts were prepared from C33A2 cells treated with melphalan for the indicated time-periods. ( G ) C33A2 cells treated with DMSO or melphalan for the indicated time points were UV irradiated and subjected to CLIP assay as detailed in ‘Materials and Methods’ section. The RNA–protein complexes were immunoprecipitated with antibody to hnRNP C1 and the RNA extracted from the immunoprecipitated complexes was subjected to RT-PCR with primers that detect HPV16 E4 mRNAs spliced from SD880 to SA3358. ( H ) Western blot on extracts from C33A2 cells transfected with scrambled siRNAs (scr) or siRNAs to hnRNP C. hnRNP C levels were quantified in extracts prepared from cells transfected with the indicated siRNAs and divided by hnRNP C levels in extracts from cells transfected with scrambled siRNAs (scr). ( I ) sLuc activity produced by C33A2 cells transfected with scrambled siRNAs (scr) or siRNAs to hnRNP C followed by addition of 100 µM melphalan.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O-Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich.

    Techniques: Immunoprecipitation, Western Blot, Incubation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Irradiation, Cross-linking Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Transfection, Activity Assay, Produced

    ( A ) Western blot on TRAP150 in C33A2 cells treated with DMSO (D) or with 100 µM melphalan (M) for the indicated time periods. TRAP150 levels were normalized to actin and TRAP150 over actin in untreated cells was set as 1. ( B and C ) Cell extracts from DMSO or melphalan treated C33A2 cells were subjected to immunoprecipitation with the antibodies to phosphorylated BRCA1 (p-BRCA1) (B) or TRAP150 (C), followed by western blotting with antibodies to TRAP150 or U2AF65, respectively. The hours of DMSO or melphalan incubation of C33A2 cells are indicated on top of the gels. The levels of immunoprecipitated protein and the levels of each protein in the input extracts were quantified. Percent of input protein immunoprecipitated by each antibody in extracts from DMSO or melphalan-treated cells are shown below each gel. ( D ) ChIP analyses on C33A2 cells using antibody to TRAP150 and qPCR of the indicated HPV16 amplicons. Primers and antibodies are listed in Supplementary Tables S3 and 4 , respectively, and the location in the HPV16 genome of the PCR primers is shown in Supplementary Figure S5 . Mean values with standard deviations of the amount of immunoprecipitated DNA compared to DNA from DMSO-treated cells are displayed. The qPCR values obtained for each primer pair with DNA extracted from DMSO-treated C33A2 cells were set to 1 to correct for differences between different ChIP extracts. Chip extracts were prepared from C33A2 cells treated with melphalan for the indicated time-periods. ( E ) C33A2 cells treated with DMSO or melphalan for the indicated time points were UV irradiated and subjected to CLIP assay as detailed in ‘Materials and Methods’ section. The RNA–protein complexes were immunoprecipitated with antibodies to U2AF65, BARD1, BCLAF1 or phosphorylated BRCA1 (p-BRCA1) and the RNA extracted from the immunoprecipitated complexes were subjected to RT-PCR with primers that detect HPV16 E4 mRNAs spliced from SD880 to SA3358. ( F ) Western blot on extracts from C33A2 cells transfected with scrambled siRNAs (scr) or siRNAs to BCLAF1, TRAP150 or U2AF65. Protein levels were quantified in extracts prepared from cells transfected with the indicated siRNAs and divided by protein levels in extracts from cells transfected with scrambled siRNAs (scr). ( G ) sLuc activity produced by C33A2 cells transfected with scrambled siRNAs (scr) or siRNAs to BCLAF1, TRAP150 or U2AF65 followed by addition of 100 µM melphalan.

    Journal: Nucleic Acids Research

    Article Title: The DNA damage response activates HPV16 late gene expression at the level of RNA processing

    doi: 10.1093/nar/gky227

    Figure Lengend Snippet: ( A ) Western blot on TRAP150 in C33A2 cells treated with DMSO (D) or with 100 µM melphalan (M) for the indicated time periods. TRAP150 levels were normalized to actin and TRAP150 over actin in untreated cells was set as 1. ( B and C ) Cell extracts from DMSO or melphalan treated C33A2 cells were subjected to immunoprecipitation with the antibodies to phosphorylated BRCA1 (p-BRCA1) (B) or TRAP150 (C), followed by western blotting with antibodies to TRAP150 or U2AF65, respectively. The hours of DMSO or melphalan incubation of C33A2 cells are indicated on top of the gels. The levels of immunoprecipitated protein and the levels of each protein in the input extracts were quantified. Percent of input protein immunoprecipitated by each antibody in extracts from DMSO or melphalan-treated cells are shown below each gel. ( D ) ChIP analyses on C33A2 cells using antibody to TRAP150 and qPCR of the indicated HPV16 amplicons. Primers and antibodies are listed in Supplementary Tables S3 and 4 , respectively, and the location in the HPV16 genome of the PCR primers is shown in Supplementary Figure S5 . Mean values with standard deviations of the amount of immunoprecipitated DNA compared to DNA from DMSO-treated cells are displayed. The qPCR values obtained for each primer pair with DNA extracted from DMSO-treated C33A2 cells were set to 1 to correct for differences between different ChIP extracts. Chip extracts were prepared from C33A2 cells treated with melphalan for the indicated time-periods. ( E ) C33A2 cells treated with DMSO or melphalan for the indicated time points were UV irradiated and subjected to CLIP assay as detailed in ‘Materials and Methods’ section. The RNA–protein complexes were immunoprecipitated with antibodies to U2AF65, BARD1, BCLAF1 or phosphorylated BRCA1 (p-BRCA1) and the RNA extracted from the immunoprecipitated complexes were subjected to RT-PCR with primers that detect HPV16 E4 mRNAs spliced from SD880 to SA3358. ( F ) Western blot on extracts from C33A2 cells transfected with scrambled siRNAs (scr) or siRNAs to BCLAF1, TRAP150 or U2AF65. Protein levels were quantified in extracts prepared from cells transfected with the indicated siRNAs and divided by protein levels in extracts from cells transfected with scrambled siRNAs (scr). ( G ) sLuc activity produced by C33A2 cells transfected with scrambled siRNAs (scr) or siRNAs to BCLAF1, TRAP150 or U2AF65 followed by addition of 100 µM melphalan.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O-Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich.

    Techniques: Western Blot, Immunoprecipitation, Incubation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Irradiation, Cross-linking Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Transfection, Activity Assay, Produced

    ( A ) Schematic drawing of the HPV16 region around late 3′-splice site SA5639 and the 35-nucleotide biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA5639 is indicated. ( B ) Upper and lower panels show pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the region of HPV16 late 3′-splice site SA5639 followed by Western blot analysis using antibodies indicated to the right. (–) mock pull downs using streptavidin beads in the absence of oligo. ( C ) Quantitation of some of the Western blots of the pull downs in (B). ( D and E ) Western blots of indicated proteins on pull downs using biotinylated ssRNA oligos of a subset of the ssDNA oligos shown in (B).

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: ( A ) Schematic drawing of the HPV16 region around late 3′-splice site SA5639 and the 35-nucleotide biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA5639 is indicated. ( B ) Upper and lower panels show pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the region of HPV16 late 3′-splice site SA5639 followed by Western blot analysis using antibodies indicated to the right. (–) mock pull downs using streptavidin beads in the absence of oligo. ( C ) Quantitation of some of the Western blots of the pull downs in (B). ( D and E ) Western blots of indicated proteins on pull downs using biotinylated ssRNA oligos of a subset of the ssDNA oligos shown in (B).

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Western Blot, Quantitation Assay

    ( A ) Schematic representation of the HPV16 genome. Rectangles represent open reading frames, promoters p97 and p670 are indicated as arrows, filled and open triangles represent 5′- and 3′-splices sites respectively, HPV16 early and late polyA signals pAE and pAL are indicated. The regions on the HPV16 mRNAs from which the ssDNA and ssRNA oligos are derived are boxed. ( B ) Schematic drawing of the HPV16 region around early polyA signal pAE and the 35-nucleotide, biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 early polyA signal pAE is indicated as well as E5 and L2 coding regions. ( C ) Pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos spanning HPV16 pAE followed by Western blot for hnRNP L. (-); mock pull downs using streptavidine beads in the absence of oligo.

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: ( A ) Schematic representation of the HPV16 genome. Rectangles represent open reading frames, promoters p97 and p670 are indicated as arrows, filled and open triangles represent 5′- and 3′-splices sites respectively, HPV16 early and late polyA signals pAE and pAL are indicated. The regions on the HPV16 mRNAs from which the ssDNA and ssRNA oligos are derived are boxed. ( B ) Schematic drawing of the HPV16 region around early polyA signal pAE and the 35-nucleotide, biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 early polyA signal pAE is indicated as well as E5 and L2 coding regions. ( C ) Pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos spanning HPV16 pAE followed by Western blot for hnRNP L. (-); mock pull downs using streptavidine beads in the absence of oligo.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Derivative Assay, Western Blot

    ( A ) Upper panel: Schematic drawing of HPV16 exon 4 and the 35-nucleotide, biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA3358 and 5′-splice site SD3632 are indicated. Lower panel: Pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the E4 exon of HPV16 followed by Western blot analysis using antibodies to proteins indicated to the right. (-); mock pull downs using streptavidin beads in the absence of oligo. ( B ) Quantitation of some of the Western blots of the pull downs in (A). ( C ) Upper panel: Schematic drawing of shorter oligos (A-X) designed to better map binding sites for hnRNP L, hnRNP A1 and U2AF65. Lower panel: Western blots for hnRNP L, hnRNP A1 and U2AF65 of on proteins pulled down by the shorter biotinylated ssDNA oligos. ( D ) Western blots of indicated proteins on pull downs using biotinylated ssRNA oligos of a subset of the ssDNA oligos shown in (A). ( E ) Upper panel: Schematic drawing of shorter oligos (A–E) of the two original 35-nucleotide oligos 8 and 10 located near HPV16 late 5′-splice site SD3632. Lower panel: Western blots for hnRNP L, hnRNP A1 and U2AF65 of on proteins pulled down by the shorter biotinylated ssDNA oligos.

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: ( A ) Upper panel: Schematic drawing of HPV16 exon 4 and the 35-nucleotide, biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA3358 and 5′-splice site SD3632 are indicated. Lower panel: Pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the E4 exon of HPV16 followed by Western blot analysis using antibodies to proteins indicated to the right. (-); mock pull downs using streptavidin beads in the absence of oligo. ( B ) Quantitation of some of the Western blots of the pull downs in (A). ( C ) Upper panel: Schematic drawing of shorter oligos (A-X) designed to better map binding sites for hnRNP L, hnRNP A1 and U2AF65. Lower panel: Western blots for hnRNP L, hnRNP A1 and U2AF65 of on proteins pulled down by the shorter biotinylated ssDNA oligos. ( D ) Western blots of indicated proteins on pull downs using biotinylated ssRNA oligos of a subset of the ssDNA oligos shown in (A). ( E ) Upper panel: Schematic drawing of shorter oligos (A–E) of the two original 35-nucleotide oligos 8 and 10 located near HPV16 late 5′-splice site SD3632. Lower panel: Western blots for hnRNP L, hnRNP A1 and U2AF65 of on proteins pulled down by the shorter biotinylated ssDNA oligos.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Western Blot, Quantitation Assay, Binding Assay

    ( A ) Western blots of various RNA binding proteins in two independent preparations of nuclear extracts (Prep 1 and Prep 2) prepared from C33A2 cells treated with DMSO (D) or 100 uM Akt kinase inhibitor GDC0068 (G) for 3 h. Western blots of indicated proteins pulled down with biotinylated ssDNA oligos ( B ) or RNA oligos ( C ) representing sequences at the HPV16 3′-splice site SA3358 in the E4-coding exon. ( D ) Western blots of indicated proteins pulled down with biotinylated ssDNA oligos representing sequences at the HPV16 late 5′-splice site SD3632 in the E4-coding exon. ( E ) Western blots of indicated proteins pulled down with biotinylated ssRNA oligos spanning HPV16 5′-splice sites SD880 (BRnSD862) and SD3632 (BRnSD3632). ( F ) Western blots of hnRNP L, hnRNP A1, U2AF65 or U2AF35 pulled down from nuclear extracts using ssDNA oligos at HPV16 late splice site SA5639. For all pull downs in the figure, nuclear extracts prepared from C33A2 cells treated with DMSO (D) or Akt kinase inhibitor GDC0068 (G) were used.

    Journal: Nucleic Acids Research

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    doi: 10.1093/nar/gkx606

    Figure Lengend Snippet: ( A ) Western blots of various RNA binding proteins in two independent preparations of nuclear extracts (Prep 1 and Prep 2) prepared from C33A2 cells treated with DMSO (D) or 100 uM Akt kinase inhibitor GDC0068 (G) for 3 h. Western blots of indicated proteins pulled down with biotinylated ssDNA oligos ( B ) or RNA oligos ( C ) representing sequences at the HPV16 3′-splice site SA3358 in the E4-coding exon. ( D ) Western blots of indicated proteins pulled down with biotinylated ssDNA oligos representing sequences at the HPV16 late 5′-splice site SD3632 in the E4-coding exon. ( E ) Western blots of indicated proteins pulled down with biotinylated ssRNA oligos spanning HPV16 5′-splice sites SD880 (BRnSD862) and SD3632 (BRnSD3632). ( F ) Western blots of hnRNP L, hnRNP A1, U2AF65 or U2AF35 pulled down from nuclear extracts using ssDNA oligos at HPV16 late splice site SA5639. For all pull downs in the figure, nuclear extracts prepared from C33A2 cells treated with DMSO (D) or Akt kinase inhibitor GDC0068 (G) were used.

    Article Snippet: Biotin-labeled ssDNA oligos and 2′-O -Me-RNA oligos were purchased from Eurofins Genomics and biotin-labeled ssRNA oligos were purchased from SIGMA Aldrich ( ).

    Techniques: Western Blot, RNA Binding Assay

    Increase of sub-G1 and reduction of G1 to S phase cells via TPX2 silencing. Notes: ( A ) PPC1 cells were synchronized by double thymidine and nocodazol (Thy–Thy–Noc) block or double thymidine (Thy–Thy) block and then released for the indicated time period. The phase of cell fracture during cell cycle progression was monitored through protein expression evolution. Cyclin B1 indicated G2/M phase progression, and cyclin A indicated S to G2/M phase progression. TPX2 protein levels were measured using a Western blot assay. ( B ) Scrambled siRNA (20 nM, si-CTL) or the siRNA oligos pool against TPX2 (20 nM, si-TPX2) was transfected into PPC1 cells for 72 h, followed by cell harvesting. The knockdown cells were fixed and stained with DAPI to examine the proportions of the cell cycle by using an image-flow cytometry assay. ( C ) Representative data for cell cycle proportions were analyzed and quantified using Nucleoview NC-3000 software. The results are expressed as mean ± SD from three individual experiments (Student’s t -test, P

    Journal: OncoTargets and therapy

    Article Title: Targeted TPX2 increases chromosome missegregation and suppresses tumor cell growth in human prostate cancer

    doi: 10.2147/OTT.S136491

    Figure Lengend Snippet: Increase of sub-G1 and reduction of G1 to S phase cells via TPX2 silencing. Notes: ( A ) PPC1 cells were synchronized by double thymidine and nocodazol (Thy–Thy–Noc) block or double thymidine (Thy–Thy) block and then released for the indicated time period. The phase of cell fracture during cell cycle progression was monitored through protein expression evolution. Cyclin B1 indicated G2/M phase progression, and cyclin A indicated S to G2/M phase progression. TPX2 protein levels were measured using a Western blot assay. ( B ) Scrambled siRNA (20 nM, si-CTL) or the siRNA oligos pool against TPX2 (20 nM, si-TPX2) was transfected into PPC1 cells for 72 h, followed by cell harvesting. The knockdown cells were fixed and stained with DAPI to examine the proportions of the cell cycle by using an image-flow cytometry assay. ( C ) Representative data for cell cycle proportions were analyzed and quantified using Nucleoview NC-3000 software. The results are expressed as mean ± SD from three individual experiments (Student’s t -test, P

    Article Snippet: The TPX2 siRNA oligos pool (1: 5′-GGACGAACCGGUAGUGAU-3′; 2: 5′-GCAUAAGGCAAAUCCAAUA-3′; 3: 5′-GUACCAUUGUUAAGCCUUU-3′; 4: 5′-GAAAUU CUACCCUCUAAGA-3′) was synthesized by Sigma-Aldrich Co. All transient transfections of the TPX2 siRNA oligos pool at a final concentration of 20 nM were accomplished with LipofectAMINE RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s protocols.

    Techniques: Blocking Assay, Expressing, Western Blot, CTL Assay, Transfection, Cell Harvesting, Staining, Flow Cytometry, Cytometry, Software

    TPX2 depletion changes the cell fitness in LNCap prostate cancer cell line. Notes: Scrambled siRNA (20 nM, si-CTL) or the siRNA oligos pool against TPX2 (20 nM, si-TPX2) was transfected into LNCap human prostate cancer cells for 96 h. ( A ) RT-PCR was used to detect the mRNA levels of TPX2 in LNCap cells. siRNA-transfected cells were harvested and lysed for immunoblotting to determine the protein levels of TPX2. β-actin was used as a loading control. Protein expression was quantified by densitometric analysis. ( B ) Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay, and the luminescence units indicating cell growth were measured and plotted as the growth curve. ( * The cell growth was inhibition in si-TPX2 treated cells compared with si-CTL treated cells). ( C ) Representative data and ( D ) quantitative results of siRNA-transfected cells that were seeded in ultralow attachment 96-well microplates for spheroid formation assays by using ImageJ software. ( E ) LNCap spheroids presented irregular morphology; hence, viable cells were measured using the CellTiter-Glo Luminescent Cell Viability Assay, and the luminescence units indicating cell growth were measured and plotted as the bar plot. ( F ) Scrambled siRNA (20 nM, si-CTL) or the siRNA oligos pool against TPX2 (20 nM, si-TPX2) was transfected into LNCap cells for 72 h, followed by cell harvesting. The knockdown cells were fixed and stained with DAPI to examine the proportions of the cell cycle by using an image-flow cytometry assay. ( G ) Representative data for cell cycle proportions were analyzed and quantified using Nucleoview NC-3000 software. ( H ) Western blot analysis of the levels of TPX2, CDK1, cyclin B1, CDK2, cyclin A, and cyclin E proteins in TPX2-targeted cells. β-actin was used as a loading control. ( I ) Representative images of TPX2 and Phalloidin (a high-affinity F-actin probe conjugated to the red-orange fluorescent dye, tetramethylrhodamine) immunofluorescence in TPX2-depleted LNCap cells. TPX2 expression was decreased in si-TPX2 treated cells, and morphologic examination of the cell nucleus (DAPI stain) revealed that transfection with the TPX2 RNAi oligo pool resulted in increased multinucleated cells. Scale bar =25 mm; magnification 400×. ( J ) Histograms of multinucleation in LNCap cells. All representative graphs are from two independent experiments. Values are presented as mean ± SD (Student’s t -test, P

    Journal: OncoTargets and therapy

    Article Title: Targeted TPX2 increases chromosome missegregation and suppresses tumor cell growth in human prostate cancer

    doi: 10.2147/OTT.S136491

    Figure Lengend Snippet: TPX2 depletion changes the cell fitness in LNCap prostate cancer cell line. Notes: Scrambled siRNA (20 nM, si-CTL) or the siRNA oligos pool against TPX2 (20 nM, si-TPX2) was transfected into LNCap human prostate cancer cells for 96 h. ( A ) RT-PCR was used to detect the mRNA levels of TPX2 in LNCap cells. siRNA-transfected cells were harvested and lysed for immunoblotting to determine the protein levels of TPX2. β-actin was used as a loading control. Protein expression was quantified by densitometric analysis. ( B ) Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay, and the luminescence units indicating cell growth were measured and plotted as the growth curve. ( * The cell growth was inhibition in si-TPX2 treated cells compared with si-CTL treated cells). ( C ) Representative data and ( D ) quantitative results of siRNA-transfected cells that were seeded in ultralow attachment 96-well microplates for spheroid formation assays by using ImageJ software. ( E ) LNCap spheroids presented irregular morphology; hence, viable cells were measured using the CellTiter-Glo Luminescent Cell Viability Assay, and the luminescence units indicating cell growth were measured and plotted as the bar plot. ( F ) Scrambled siRNA (20 nM, si-CTL) or the siRNA oligos pool against TPX2 (20 nM, si-TPX2) was transfected into LNCap cells for 72 h, followed by cell harvesting. The knockdown cells were fixed and stained with DAPI to examine the proportions of the cell cycle by using an image-flow cytometry assay. ( G ) Representative data for cell cycle proportions were analyzed and quantified using Nucleoview NC-3000 software. ( H ) Western blot analysis of the levels of TPX2, CDK1, cyclin B1, CDK2, cyclin A, and cyclin E proteins in TPX2-targeted cells. β-actin was used as a loading control. ( I ) Representative images of TPX2 and Phalloidin (a high-affinity F-actin probe conjugated to the red-orange fluorescent dye, tetramethylrhodamine) immunofluorescence in TPX2-depleted LNCap cells. TPX2 expression was decreased in si-TPX2 treated cells, and morphologic examination of the cell nucleus (DAPI stain) revealed that transfection with the TPX2 RNAi oligo pool resulted in increased multinucleated cells. Scale bar =25 mm; magnification 400×. ( J ) Histograms of multinucleation in LNCap cells. All representative graphs are from two independent experiments. Values are presented as mean ± SD (Student’s t -test, P

    Article Snippet: The TPX2 siRNA oligos pool (1: 5′-GGACGAACCGGUAGUGAU-3′; 2: 5′-GCAUAAGGCAAAUCCAAUA-3′; 3: 5′-GUACCAUUGUUAAGCCUUU-3′; 4: 5′-GAAAUU CUACCCUCUAAGA-3′) was synthesized by Sigma-Aldrich Co. All transient transfections of the TPX2 siRNA oligos pool at a final concentration of 20 nM were accomplished with LipofectAMINE RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s protocols.

    Techniques: CTL Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Viability Assay, Inhibition, Software, Cell Harvesting, Staining, Flow Cytometry, Cytometry, Western Blot, Immunofluorescence

    PPC1 cell apoptosis caused by TPX2 silencing. Notes: Annexin V expression analysis was used for apoptosis cell detection. ( A ) Representative images of scrambled siRNA (20 nM, si-CTL) or the siRNA oligos pool against TPX2 (20 nM, si-TPX2) that was transfected into PPC1 cells for 72 h, followed by cell staining with annexin V (green) and Hoechst 33342 (nucleus; blue) for the immunofluorescence assay. Notably, annexin V was increased in the si-TPX2–treated cells. Scale bar =50 mm; magnification 200×. ( B ) Quantitative results of annexin V-positive staining cell. Values are presented as mean ± SD (Student’s t -test, P

    Journal: OncoTargets and therapy

    Article Title: Targeted TPX2 increases chromosome missegregation and suppresses tumor cell growth in human prostate cancer

    doi: 10.2147/OTT.S136491

    Figure Lengend Snippet: PPC1 cell apoptosis caused by TPX2 silencing. Notes: Annexin V expression analysis was used for apoptosis cell detection. ( A ) Representative images of scrambled siRNA (20 nM, si-CTL) or the siRNA oligos pool against TPX2 (20 nM, si-TPX2) that was transfected into PPC1 cells for 72 h, followed by cell staining with annexin V (green) and Hoechst 33342 (nucleus; blue) for the immunofluorescence assay. Notably, annexin V was increased in the si-TPX2–treated cells. Scale bar =50 mm; magnification 200×. ( B ) Quantitative results of annexin V-positive staining cell. Values are presented as mean ± SD (Student’s t -test, P

    Article Snippet: The TPX2 siRNA oligos pool (1: 5′-GGACGAACCGGUAGUGAU-3′; 2: 5′-GCAUAAGGCAAAUCCAAUA-3′; 3: 5′-GUACCAUUGUUAAGCCUUU-3′; 4: 5′-GAAAUU CUACCCUCUAAGA-3′) was synthesized by Sigma-Aldrich Co. All transient transfections of the TPX2 siRNA oligos pool at a final concentration of 20 nM were accomplished with LipofectAMINE RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s protocols.

    Techniques: Expressing, CTL Assay, Transfection, Staining, Immunofluorescence

    Depletion of TPX2, as reflected by changes in cell cycle regulation. Notes: ( A ) Graphical representation of a heat map showing fourfold gene expression changes in TPX2-depleted PPC1 cells. ( B ) Scatter plot analysis of gene expression changes; specifically, 1 gene was upregulated (red circle) and 15 genes were downregulated (green circles). ( C ) Fold change of normalized expression between scrambled siRNA and the siRNA oligo pool-transfected PPC1 cells that were analyzed using pathway-focused validated qPCR arrays; notably, downregulation of genes is associated with cell cycle progression and proliferation in conjunction with the upregulation of growth inhibitory genes. Abbreviations: qPCR, quantitative polymerase chain reaction; si-CTL, small interference-negative control.

    Journal: OncoTargets and therapy

    Article Title: Targeted TPX2 increases chromosome missegregation and suppresses tumor cell growth in human prostate cancer

    doi: 10.2147/OTT.S136491

    Figure Lengend Snippet: Depletion of TPX2, as reflected by changes in cell cycle regulation. Notes: ( A ) Graphical representation of a heat map showing fourfold gene expression changes in TPX2-depleted PPC1 cells. ( B ) Scatter plot analysis of gene expression changes; specifically, 1 gene was upregulated (red circle) and 15 genes were downregulated (green circles). ( C ) Fold change of normalized expression between scrambled siRNA and the siRNA oligo pool-transfected PPC1 cells that were analyzed using pathway-focused validated qPCR arrays; notably, downregulation of genes is associated with cell cycle progression and proliferation in conjunction with the upregulation of growth inhibitory genes. Abbreviations: qPCR, quantitative polymerase chain reaction; si-CTL, small interference-negative control.

    Article Snippet: The TPX2 siRNA oligos pool (1: 5′-GGACGAACCGGUAGUGAU-3′; 2: 5′-GCAUAAGGCAAAUCCAAUA-3′; 3: 5′-GUACCAUUGUUAAGCCUUU-3′; 4: 5′-GAAAUU CUACCCUCUAAGA-3′) was synthesized by Sigma-Aldrich Co. All transient transfections of the TPX2 siRNA oligos pool at a final concentration of 20 nM were accomplished with LipofectAMINE RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s protocols.

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, CTL Assay, Negative Control

    Inhibition of cell growth and reduction of tumorigenesis in human prostate cancer cell lines via TPX2 silencing. Notes: Scrambled siRNA (20 nM, si-CTL) or the siRNA oligos pool against TPX2 (20 nM, si-TPX2) was transfected into human prostate cancer cells (PPC1) for 72 h. ( A ) Cells were harvested and lysed for immunoblotting to determine the protein levels of TPX2. β-actin was used as a loading control. Protein expression was quantified by densitometric analysis. The ratios (TPX2/β-actin) of band intensities are shown. ( B ) Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay, and the luminescence units indicating cell growth were measured and plotted as the growth curve. ( * The cell growth was inhibition in si-TPX2 treated cells compared with si-CTL treated cells). ( C ) Representative data and quantitative results of siRNA-transfected cells that were seeded in six-well plates for the clonogenic assay. ( D ) Representative data and quantitative results of siRNA-transfected cells that were seeded in ultralow attachment 96-well microplates for spheroid formation assays. All representative graphs are from three independent experiments. Values are presented as mean ± SD (Student’s t -test, P

    Journal: OncoTargets and therapy

    Article Title: Targeted TPX2 increases chromosome missegregation and suppresses tumor cell growth in human prostate cancer

    doi: 10.2147/OTT.S136491

    Figure Lengend Snippet: Inhibition of cell growth and reduction of tumorigenesis in human prostate cancer cell lines via TPX2 silencing. Notes: Scrambled siRNA (20 nM, si-CTL) or the siRNA oligos pool against TPX2 (20 nM, si-TPX2) was transfected into human prostate cancer cells (PPC1) for 72 h. ( A ) Cells were harvested and lysed for immunoblotting to determine the protein levels of TPX2. β-actin was used as a loading control. Protein expression was quantified by densitometric analysis. The ratios (TPX2/β-actin) of band intensities are shown. ( B ) Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay, and the luminescence units indicating cell growth were measured and plotted as the growth curve. ( * The cell growth was inhibition in si-TPX2 treated cells compared with si-CTL treated cells). ( C ) Representative data and quantitative results of siRNA-transfected cells that were seeded in six-well plates for the clonogenic assay. ( D ) Representative data and quantitative results of siRNA-transfected cells that were seeded in ultralow attachment 96-well microplates for spheroid formation assays. All representative graphs are from three independent experiments. Values are presented as mean ± SD (Student’s t -test, P

    Article Snippet: The TPX2 siRNA oligos pool (1: 5′-GGACGAACCGGUAGUGAU-3′; 2: 5′-GCAUAAGGCAAAUCCAAUA-3′; 3: 5′-GUACCAUUGUUAAGCCUUU-3′; 4: 5′-GAAAUU CUACCCUCUAAGA-3′) was synthesized by Sigma-Aldrich Co. All transient transfections of the TPX2 siRNA oligos pool at a final concentration of 20 nM were accomplished with LipofectAMINE RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s protocols.

    Techniques: Inhibition, CTL Assay, Transfection, Expressing, Cell Viability Assay, Clonogenic Assay

    AMP-activated protein kinase (AMPK)-phosphoprotein enriched in astrocytes 15 kDa (PEA15) axis is critical for the anchorage-independent growth of breast cancer cells. (A) Michigan Cancer Foundation 7 breast cancer cell line (MCF7), BT-474, and primary breast cancer-derived cells cultured in adherent (ADH) condition or as cancer spheres (CS) for a week were harvested and subjected to immunoblotting; n = 3. (B) MCF7 cells cultured in methylcellulose for a week in the presence of 10 μM AMPK inhibitor Compound C or dimethyl sulphoxide (DMSO) (vehicle control) were subjected to immunoblotting. Graph represents number of spheres formed/20 fields. Error bars represent standard error of the mean (SEM); n = 3. (C-F) Adherent MCF7 breast cancer cells were transfected with specified small interfering RNA (siRNA) oligos/plasmids (see below). Two days posttransfection, 1 × 10 5 cells/35 mm dish were seeded in methylcellulose. After 48 hrs, cells were retrieved from some dishes and subjected to immunoblot analyses for the specified proteins. Parallel dishes were allowed to form spheres; graph represents sphere formation at the end of a week, error bar represents SEM: (C) control siRNA or siRNA targeting AMPK α2, n = 4; (D) control siRNA or siRNA targeting PEA15, (n = 4); (E) control siRNA or siRNA targeting PEA15 and seeded in methylcellulose in the presence of 100 μM AMPK activator, A769662; DMSO served as vehicle control, n = 4; (F) transfected with CSCG constructs expressing flag-tagged wild-type (WT) or S116A mutant of PEA15, n = 4. (G) MDAMB231 cells were treated with DMSO or 100 μM AMPK activator, A769662, and immunoprecipitated with anti-Fas-associated death domain protein (FADD) antibody. The immunoprecipates were resolved by SDS-PAGE followed by immunoblotting for specified proteins, n = 3. (H) BT 474 cells stably expressing flag-tagged WT-PEA15 or PEA15-S116A mutant were seeded in methyl cellulose for two days, retrieved and immunoprecipitated with anti-flag antibody. The immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting for specified proteins, n = 3.

    Journal: Breast Cancer Research : BCR

    Article Title: Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells

    doi: 10.1186/s13058-014-0420-z

    Figure Lengend Snippet: AMP-activated protein kinase (AMPK)-phosphoprotein enriched in astrocytes 15 kDa (PEA15) axis is critical for the anchorage-independent growth of breast cancer cells. (A) Michigan Cancer Foundation 7 breast cancer cell line (MCF7), BT-474, and primary breast cancer-derived cells cultured in adherent (ADH) condition or as cancer spheres (CS) for a week were harvested and subjected to immunoblotting; n = 3. (B) MCF7 cells cultured in methylcellulose for a week in the presence of 10 μM AMPK inhibitor Compound C or dimethyl sulphoxide (DMSO) (vehicle control) were subjected to immunoblotting. Graph represents number of spheres formed/20 fields. Error bars represent standard error of the mean (SEM); n = 3. (C-F) Adherent MCF7 breast cancer cells were transfected with specified small interfering RNA (siRNA) oligos/plasmids (see below). Two days posttransfection, 1 × 10 5 cells/35 mm dish were seeded in methylcellulose. After 48 hrs, cells were retrieved from some dishes and subjected to immunoblot analyses for the specified proteins. Parallel dishes were allowed to form spheres; graph represents sphere formation at the end of a week, error bar represents SEM: (C) control siRNA or siRNA targeting AMPK α2, n = 4; (D) control siRNA or siRNA targeting PEA15, (n = 4); (E) control siRNA or siRNA targeting PEA15 and seeded in methylcellulose in the presence of 100 μM AMPK activator, A769662; DMSO served as vehicle control, n = 4; (F) transfected with CSCG constructs expressing flag-tagged wild-type (WT) or S116A mutant of PEA15, n = 4. (G) MDAMB231 cells were treated with DMSO or 100 μM AMPK activator, A769662, and immunoprecipitated with anti-Fas-associated death domain protein (FADD) antibody. The immunoprecipates were resolved by SDS-PAGE followed by immunoblotting for specified proteins, n = 3. (H) BT 474 cells stably expressing flag-tagged WT-PEA15 or PEA15-S116A mutant were seeded in methyl cellulose for two days, retrieved and immunoprecipitated with anti-flag antibody. The immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting for specified proteins, n = 3.

    Article Snippet: Breast cancer cell lines were transfected with specific siRNA oligos targeting AMPK α2 and PEA15 (Cell Signaling Technology); universal negative siRNA (Sigma-Aldrich) was used as control.

    Techniques: Derivative Assay, Cell Culture, Transfection, Small Interfering RNA, Construct, Expressing, Mutagenesis, Immunoprecipitation, SDS Page, Stable Transfection

    AMP-activated protein kinase (AMPK) inhibits apoptosis through phosphoprotein enriched in astrocytes 15 kDa (PEA15) phosphorylation. Primary human mammary epithelial cells (HMECs) cultured under following conditions were harvested and subjected to immunoblot analysis for the specified proteins: (A) HMECs seeded in adherent (ADH) condition for six days or in suspension (in ultra-low (UL) plates) for two, four or six days; n = 3. (B) HMECs seeded in suspension in the presence of 100 μM AMPK activator (A769662), 10 μM AMPK inhibitor (Compound C) or dimethyl sulphoxide (DMSO) (vehicle control) for a week; n = 3. (C) HMECs seeded in UL plates and transfected with control small interfering RNA (siRNA) or siRNA targeting AMPK α2 (Dharmacon) were harvested after one week; n = 4. (D) HMECs seeded in ADH condition or in suspension in UL plates for a week. Graph represents densitometric analysis of western blots to quantify phospho PEA15 Ser 116 relative to total PEA15. Error bars represent standard error of the mean (SEM); (n = 6). (E) HMECs seeded in UL plates for 24 hrs and treated with DMSO (vehicle control), 100 μM AMPK activator (A769662), or 10 μM AMPK inhibitor (Compound C) for a period of 2 hrs; n = 3. (F) HMECs seeded in UL plates and treated with increasing amounts of AMPK inhibitor Compound C, in the presence of a constant amount of AMPK activator A769662, for 2 hrs; n = 3. (G) HMECs seeded in UL plates and transfected with control siRNA or siRNA targeting AMPK α2 and harvested two days following transfection; n = 4. (H) HMECs seeded in UL plates and transfected with control siRNA or siRNA targeting AMPK α2 were treated with 100 μM AMPK activator (A769662) after two days following transfection for 2 hrs and harvested, n = 3.

    Journal: Breast Cancer Research : BCR

    Article Title: Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells

    doi: 10.1186/s13058-014-0420-z

    Figure Lengend Snippet: AMP-activated protein kinase (AMPK) inhibits apoptosis through phosphoprotein enriched in astrocytes 15 kDa (PEA15) phosphorylation. Primary human mammary epithelial cells (HMECs) cultured under following conditions were harvested and subjected to immunoblot analysis for the specified proteins: (A) HMECs seeded in adherent (ADH) condition for six days or in suspension (in ultra-low (UL) plates) for two, four or six days; n = 3. (B) HMECs seeded in suspension in the presence of 100 μM AMPK activator (A769662), 10 μM AMPK inhibitor (Compound C) or dimethyl sulphoxide (DMSO) (vehicle control) for a week; n = 3. (C) HMECs seeded in UL plates and transfected with control small interfering RNA (siRNA) or siRNA targeting AMPK α2 (Dharmacon) were harvested after one week; n = 4. (D) HMECs seeded in ADH condition or in suspension in UL plates for a week. Graph represents densitometric analysis of western blots to quantify phospho PEA15 Ser 116 relative to total PEA15. Error bars represent standard error of the mean (SEM); (n = 6). (E) HMECs seeded in UL plates for 24 hrs and treated with DMSO (vehicle control), 100 μM AMPK activator (A769662), or 10 μM AMPK inhibitor (Compound C) for a period of 2 hrs; n = 3. (F) HMECs seeded in UL plates and treated with increasing amounts of AMPK inhibitor Compound C, in the presence of a constant amount of AMPK activator A769662, for 2 hrs; n = 3. (G) HMECs seeded in UL plates and transfected with control siRNA or siRNA targeting AMPK α2 and harvested two days following transfection; n = 4. (H) HMECs seeded in UL plates and transfected with control siRNA or siRNA targeting AMPK α2 were treated with 100 μM AMPK activator (A769662) after two days following transfection for 2 hrs and harvested, n = 3.

    Article Snippet: Breast cancer cell lines were transfected with specific siRNA oligos targeting AMPK α2 and PEA15 (Cell Signaling Technology); universal negative siRNA (Sigma-Aldrich) was used as control.

    Techniques: Cell Culture, Transfection, Small Interfering RNA, Western Blot

    AMP-activated protein kinase (AMPK)-phosphoprotein enriched in astrocytes 15 kDa (PEA15) axis is critical for tumorigenicity of breast cancer cells. (A) Breast cancer cell line derived from metastatic site (pleural effusion) (MDAMB231) cells were transfected with control small interfering RNA (siRNA) or siRNA targeting AMPK α2 and injected subcutaneously (1 × 10 6 cells/injection) into six nude mice (control siRNA transfected cells in the left flank, and AMPK α2 siRNA-transfected cells in the right flank) and monitored for tumor formation for seven weeks. ( B and C ) MDAMB231 cells (B) and BT 474 cells (C) stably expressing short hairpin RNA (shRNA) pool against AMPK α2 or scrambled shRNA were introduced subcutaneously (1 × 10 6 cells/injection) into three nude mice and tumor formation was monitored for the indicated time period. Control shRNA-expressing cells were injected in the left flank, and AMPK α2 shRNA-expressing cells in the right flank. Tumors were resected and tumor-derived cells subjected to immunoblotting for specified proteins. ( D and E ) MDAMB231 cells (D) and BT 474 cells (E) stably expressing CSCG-WT-Flag PEA15 or CSCG-S116A-Flag-PEA15 were injected subcutaneously (1 × 10 6 cells/injection) into six nude mice and tumor formation was monitored for the indicated time period. Cells stably expressing wild-type (WT)-PEA15 were injected in the left flank, while cells stably expressing S116A-PEA15 were injected in the right flank. Tumors were resected and tumor-derived cells subjected to immunoblotting for specified proteins.

    Journal: Breast Cancer Research : BCR

    Article Title: Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells

    doi: 10.1186/s13058-014-0420-z

    Figure Lengend Snippet: AMP-activated protein kinase (AMPK)-phosphoprotein enriched in astrocytes 15 kDa (PEA15) axis is critical for tumorigenicity of breast cancer cells. (A) Breast cancer cell line derived from metastatic site (pleural effusion) (MDAMB231) cells were transfected with control small interfering RNA (siRNA) or siRNA targeting AMPK α2 and injected subcutaneously (1 × 10 6 cells/injection) into six nude mice (control siRNA transfected cells in the left flank, and AMPK α2 siRNA-transfected cells in the right flank) and monitored for tumor formation for seven weeks. ( B and C ) MDAMB231 cells (B) and BT 474 cells (C) stably expressing short hairpin RNA (shRNA) pool against AMPK α2 or scrambled shRNA were introduced subcutaneously (1 × 10 6 cells/injection) into three nude mice and tumor formation was monitored for the indicated time period. Control shRNA-expressing cells were injected in the left flank, and AMPK α2 shRNA-expressing cells in the right flank. Tumors were resected and tumor-derived cells subjected to immunoblotting for specified proteins. ( D and E ) MDAMB231 cells (D) and BT 474 cells (E) stably expressing CSCG-WT-Flag PEA15 or CSCG-S116A-Flag-PEA15 were injected subcutaneously (1 × 10 6 cells/injection) into six nude mice and tumor formation was monitored for the indicated time period. Cells stably expressing wild-type (WT)-PEA15 were injected in the left flank, while cells stably expressing S116A-PEA15 were injected in the right flank. Tumors were resected and tumor-derived cells subjected to immunoblotting for specified proteins.

    Article Snippet: Breast cancer cell lines were transfected with specific siRNA oligos targeting AMPK α2 and PEA15 (Cell Signaling Technology); universal negative siRNA (Sigma-Aldrich) was used as control.

    Techniques: Derivative Assay, Transfection, Small Interfering RNA, Injection, Mouse Assay, Stable Transfection, Expressing, shRNA

    Mammospheres show elevated AMP-activated protein kinase (AMPK) activity. (A) Phase-contrast microscopic images (left) of freshly isolated primary human mammary epithelial cells (HMECs) cultured for a week as monolayers in adherent (ADH) condition in regular tissue culture (TC) dishes, or as mammospheres (MS) in suspension in ultra-low attachment plates (UL plates); magnification ×200. Experimental strategies (right) involving HMECs seeded in suspension (also see Methods ). (B and C) Primary HMECs cultured in ADH condition or in suspension as MS were harvested at the end of a week and subjected to immunoblot analyses for the specified proteins. Graphs represent densitometry analyses of western blots to quantify phosphoprotein relative to total protein. Error bars represent standard error of the mean (SEM), (n = 6). The phosphorylated (p)AMPK antibody recognizes both AMPK α1 and α2 isoforms; antibodies against total AMPK recognize both α1/2 together, or only individual isoforms α1 or α2; α-tubulin served as loading control for all western blot experiments. (D) Primary HMECs were cultured in UL plates in the presence of 10 μM AMPK inhibitor, Compound C (CC); dimethyl sulfoxide (DMSO) served as vehicle control. Two days after treatment, a quarter of the cells were harvested and subjected to immunoblot analyses for specified proteins, while the rest were scored for total number of MS formed at the end of a week (graph). Error bar represents SEM, (n = 4). (E) . Primary HMECs seeded in UL plates were transfected with control small interfering RNA (siRNA) or siRNA targeting AMPK α2 (Dharmacon, Thermo Fisher Scientific, Waltham, MA, USA). Two days posttransfection, a quarter of the cells were harvested and subjected to immunoblot analyses for the specified proteins, while the rest were scored for total number of MS formed at the end of a week (graph). Error bars represent SEM; n = 4.

    Journal: Breast Cancer Research : BCR

    Article Title: Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells

    doi: 10.1186/s13058-014-0420-z

    Figure Lengend Snippet: Mammospheres show elevated AMP-activated protein kinase (AMPK) activity. (A) Phase-contrast microscopic images (left) of freshly isolated primary human mammary epithelial cells (HMECs) cultured for a week as monolayers in adherent (ADH) condition in regular tissue culture (TC) dishes, or as mammospheres (MS) in suspension in ultra-low attachment plates (UL plates); magnification ×200. Experimental strategies (right) involving HMECs seeded in suspension (also see Methods ). (B and C) Primary HMECs cultured in ADH condition or in suspension as MS were harvested at the end of a week and subjected to immunoblot analyses for the specified proteins. Graphs represent densitometry analyses of western blots to quantify phosphoprotein relative to total protein. Error bars represent standard error of the mean (SEM), (n = 6). The phosphorylated (p)AMPK antibody recognizes both AMPK α1 and α2 isoforms; antibodies against total AMPK recognize both α1/2 together, or only individual isoforms α1 or α2; α-tubulin served as loading control for all western blot experiments. (D) Primary HMECs were cultured in UL plates in the presence of 10 μM AMPK inhibitor, Compound C (CC); dimethyl sulfoxide (DMSO) served as vehicle control. Two days after treatment, a quarter of the cells were harvested and subjected to immunoblot analyses for specified proteins, while the rest were scored for total number of MS formed at the end of a week (graph). Error bar represents SEM, (n = 4). (E) . Primary HMECs seeded in UL plates were transfected with control small interfering RNA (siRNA) or siRNA targeting AMPK α2 (Dharmacon, Thermo Fisher Scientific, Waltham, MA, USA). Two days posttransfection, a quarter of the cells were harvested and subjected to immunoblot analyses for the specified proteins, while the rest were scored for total number of MS formed at the end of a week (graph). Error bars represent SEM; n = 4.

    Article Snippet: Breast cancer cell lines were transfected with specific siRNA oligos targeting AMPK α2 and PEA15 (Cell Signaling Technology); universal negative siRNA (Sigma-Aldrich) was used as control.

    Techniques: Activity Assay, Isolation, Cell Culture, Mass Spectrometry, Western Blot, Transfection, Small Interfering RNA

    The Rab10-EHBP1-EHD2 complex mediates the engulfment of LDs by autophagic organelles. ( A ) Live-cell confocal fluorescence microscopy of two distinct LDs (stained with MDH, blue) from Hep3B cells expressing either mCherry-Rab10 (top series) or GFP-Rab10 (lower series). Imaging reveals the association of LD-bound Rab10 at early time points with phagophore/autophagosome-associated Rab10 (0 s) extending to nearly surround the perimeter of LDs at later time points. Dashed outlines provide fiducial points of reference as the envelopment of the LD by the phagophore progresses. These events are representative of data from more than 30 individual cells examined by live-cell imaging. ( B and C ) Quantification of the percentage of LDs associated with LC3- or Atg16L1-positive structures in Hep3B hepatoma cells after 48-hour siRNA treatment with the indicated siRNAs (Tri-siRNA, triple knockdown). Cells were preloaded with 150 μM oleic acid overnight. ( D and E ) Quantification of the percentage of LDs associated with LC3- or Atg16L1-positive structures after culture in low-serum conditions in WT or Rab10 KO MEFs. Cells were preloaded with 400 μM oleic acid overnight. ( F ) Quantification of the percentage of LDs associated with LAMP1-positive structures after 48-hour siRNA treatment followed by 48-hour starvation from n = 3 independent experiments, measuring 20 cells per condition. Cells were preloaded with 150 μM oleic acid overnight. ( G and H ) LDs visualized from WT or Rab10 KO MEFs were divided into three groups on the basis of their association with LAMP-1: “none,” “attached,” or “engulfed” (G). Manual counting of LDs (H) in each group from WT and Rab10 KO MEFs. The graphs represent observations from n = 3 independent experiments, measuring 20 cells per condition. Cells were preloaded with 400 μM oleic acid overnight. Data are represented as means ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Scale bars, 1 μm.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: The Rab10-EHBP1-EHD2 complex mediates the engulfment of LDs by autophagic organelles. ( A ) Live-cell confocal fluorescence microscopy of two distinct LDs (stained with MDH, blue) from Hep3B cells expressing either mCherry-Rab10 (top series) or GFP-Rab10 (lower series). Imaging reveals the association of LD-bound Rab10 at early time points with phagophore/autophagosome-associated Rab10 (0 s) extending to nearly surround the perimeter of LDs at later time points. Dashed outlines provide fiducial points of reference as the envelopment of the LD by the phagophore progresses. These events are representative of data from more than 30 individual cells examined by live-cell imaging. ( B and C ) Quantification of the percentage of LDs associated with LC3- or Atg16L1-positive structures in Hep3B hepatoma cells after 48-hour siRNA treatment with the indicated siRNAs (Tri-siRNA, triple knockdown). Cells were preloaded with 150 μM oleic acid overnight. ( D and E ) Quantification of the percentage of LDs associated with LC3- or Atg16L1-positive structures after culture in low-serum conditions in WT or Rab10 KO MEFs. Cells were preloaded with 400 μM oleic acid overnight. ( F ) Quantification of the percentage of LDs associated with LAMP1-positive structures after 48-hour siRNA treatment followed by 48-hour starvation from n = 3 independent experiments, measuring 20 cells per condition. Cells were preloaded with 150 μM oleic acid overnight. ( G and H ) LDs visualized from WT or Rab10 KO MEFs were divided into three groups on the basis of their association with LAMP-1: “none,” “attached,” or “engulfed” (G). Manual counting of LDs (H) in each group from WT and Rab10 KO MEFs. The graphs represent observations from n = 3 independent experiments, measuring 20 cells per condition. Cells were preloaded with 400 μM oleic acid overnight. Data are represented as means ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Scale bars, 1 μm.

    Article Snippet: The siRNA oligos targeting Rab10, EHBP1, EHD2, Rab7, and Atg7 were purchased from Thermo Fisher Scientific, and cells were transfected with the RNAiMAX reagent (Invitrogen).

    Techniques: Fluorescence, Microscopy, Staining, Expressing, Imaging, Live Cell Imaging, Gene Knockout

    Rab10 acts downstream of Rab7 to facilitate the autophagic degradation of LDs. ( A and B ) Immunoblot of GST-EHBP1– or GST-RILP–mediated pulldowns for Rab7 (A) or Rab10 (B) in Hep3B hepatoma cells. Rab7 exhibits a specific affinity for RILP, whereas Rab10 interacts with both EHBP1 and RILP. ( C ) Representative immunoblotting of the results for GST or GST-RILP pulldowns of Rab7 in WT or Rab10 KO MEFs, showing that Rab10 KO does not affect the binding of Rab7 to RILP. ( D ) Immunoblotting of subcellular density gradient fractions of Hep3B cells following serum starvation in HBSS for 2 hours, followed by flotation of a CLF through an 8 to 27% discontinuous OptiPrep gradient. Boxes indicate distinct peak density fractions for either Rab10 (blue box, fraction 2) or Rab7 (red box, fraction 4). ( E ) Live-cell confocal fluorescence microscopy of a HuH-7 hepatoma cell cotransfected with both GFP-Rab7 and mCherry-Rab10 and subjected to serum starvation. LDs are stained with MDH (blue). Arrows indicate the extension of a Rab7-positive membrane away from the LD and the subsequent recruitment of a Rab10-positive structure (arrowheads) to the LD. ( F ) Quantification of the average percentage of LDs positive for the presence of Rab10 alone, Rab7 alone, or both Rab10 and Rab7 from n = 10 cells. ( G ) Quantification of the average number of Rab10-positive LDs in resting or serum-starved HuH-7 hepatoma cells treated with NT siRNA or Rab7 siRNA. ( H ) Quantification of the average number of Rab7-positive LDs in resting or serum-starved HuH-7 cells treated with NT siRNA or Rab10 siRNA. Data represent the means from n = 3 independent experiments, measuring > 50 cells per condition per repeat. *** P ≤ 0.001. Scale bars, 1 μm.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: Rab10 acts downstream of Rab7 to facilitate the autophagic degradation of LDs. ( A and B ) Immunoblot of GST-EHBP1– or GST-RILP–mediated pulldowns for Rab7 (A) or Rab10 (B) in Hep3B hepatoma cells. Rab7 exhibits a specific affinity for RILP, whereas Rab10 interacts with both EHBP1 and RILP. ( C ) Representative immunoblotting of the results for GST or GST-RILP pulldowns of Rab7 in WT or Rab10 KO MEFs, showing that Rab10 KO does not affect the binding of Rab7 to RILP. ( D ) Immunoblotting of subcellular density gradient fractions of Hep3B cells following serum starvation in HBSS for 2 hours, followed by flotation of a CLF through an 8 to 27% discontinuous OptiPrep gradient. Boxes indicate distinct peak density fractions for either Rab10 (blue box, fraction 2) or Rab7 (red box, fraction 4). ( E ) Live-cell confocal fluorescence microscopy of a HuH-7 hepatoma cell cotransfected with both GFP-Rab7 and mCherry-Rab10 and subjected to serum starvation. LDs are stained with MDH (blue). Arrows indicate the extension of a Rab7-positive membrane away from the LD and the subsequent recruitment of a Rab10-positive structure (arrowheads) to the LD. ( F ) Quantification of the average percentage of LDs positive for the presence of Rab10 alone, Rab7 alone, or both Rab10 and Rab7 from n = 10 cells. ( G ) Quantification of the average number of Rab10-positive LDs in resting or serum-starved HuH-7 hepatoma cells treated with NT siRNA or Rab7 siRNA. ( H ) Quantification of the average number of Rab7-positive LDs in resting or serum-starved HuH-7 cells treated with NT siRNA or Rab10 siRNA. Data represent the means from n = 3 independent experiments, measuring > 50 cells per condition per repeat. *** P ≤ 0.001. Scale bars, 1 μm.

    Article Snippet: The siRNA oligos targeting Rab10, EHBP1, EHD2, Rab7, and Atg7 were purchased from Thermo Fisher Scientific, and cells were transfected with the RNAiMAX reagent (Invitrogen).

    Techniques: Gene Knockout, Binding Assay, Fluorescence, Microscopy, Staining

    Serum starvation potentiates the formation of a Rab10 complex together with the membrane trafficking proteins EHBP1 and EHD2 at the lipophagic junction. ( A ) Immunoblot analysis of Hep3B cells transfected to express HA-EHBP1, lysed, and subjected to a GST-Rab10 pulldown. ( B ) Immunoblot analysis of a GST pulldown experiment in Hep3B cells coexpressing a GST-tagged form of the Rab10-interacting domain of EHBP1 (residues 600 to 902) and HA-tagged Rab10-WT, Rab10-T23N, or Rab10-Q68L. ( C and D ) Representative immunoblots from pulldown experiments using the same GST-EHBP1 fragment to examine the effect of EHBP1 interactions with endogenous Rab10 after serum starvation (C) or treatment with Torin1 (D) ( n = 3 independent experiments for each condition). Numbers below the pulldown blot represent the mean fold enrichment in protein levels. ( E and F ) Representative immunoblots from GST-Rab10 pulldown experiments in Hep3B cells, probing for interactions between Rab10 and EHD2 under serum-starved (E) or Torin1-treated conditions (F) ( n = 3 independent experiments for each condition). Numbers below the pulldown blot represent the mean fold enrichment in protein levels. ( G ) Representative immunoblot of a GST-Rab10 pulldown of EHD2 in Hep3B cells previously treated with either siNT or siEHBP1 and subjected to HBSS starvation for 1 hour ( n = 3 independent experiments), quantified in ( H ). ( I ) Fluorescence images of EHBP1-positive LDs in HuH-7 cells expressing HA-EHBP1 and starved in HBSS for 1 hour before fixation and immunostaining with anti-HA antibody (green). Cells were preloaded with 150 μM oleic acid overnight. LDs are stained with ORO (red). ( J ) Fluorescence image of a HuH-7 cell transfected to express GFP-EHD2 (green) and mCherry-Rab10 (red) before serum starvation in HBSS for 1 hour and fixation. The boxed region shows a higher-magnification image of an example of an MDH-stained LD (blue) that is also positive for GFP-EHD2 and mCherry-Rab10. Scale bars, 10 μm. ( K and L ) Quantification of the appearance of GFP-Rab10–positive LDs (K) or GFP-EHD2–positive LDs (L) in HuH-7 cells following siRNA-mediated knockdown of EHBP1 and EHD2 (K) or EHBP1 and Rab10 (L) for 48 hours, followed by serum starvation in HBSS for 1 hour. Results are from n = 3 independent experiments each and are represented as mean ± SD. ** P ≤ 0.01; *** P ≤ 0.001. A total of 80 cells were quantified per condition. ( M ) Live-cell confocal fluorescence imaging of a starved Hep3B hepatoma cell coexpressing GFP-EHD2 and mCherry-Rab10, depicting the sequential recruitment of Rab10 and EHD2 to MDH-labeled LDs (blue). Note the presence of the mCherry-Rab10–positive structure at the periphery of the LD (arrowhead) before the recruitment of GFP-EHD2, resulting in the emergence of signal overlap by 35 min. Scale bars, 5 μm.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: Serum starvation potentiates the formation of a Rab10 complex together with the membrane trafficking proteins EHBP1 and EHD2 at the lipophagic junction. ( A ) Immunoblot analysis of Hep3B cells transfected to express HA-EHBP1, lysed, and subjected to a GST-Rab10 pulldown. ( B ) Immunoblot analysis of a GST pulldown experiment in Hep3B cells coexpressing a GST-tagged form of the Rab10-interacting domain of EHBP1 (residues 600 to 902) and HA-tagged Rab10-WT, Rab10-T23N, or Rab10-Q68L. ( C and D ) Representative immunoblots from pulldown experiments using the same GST-EHBP1 fragment to examine the effect of EHBP1 interactions with endogenous Rab10 after serum starvation (C) or treatment with Torin1 (D) ( n = 3 independent experiments for each condition). Numbers below the pulldown blot represent the mean fold enrichment in protein levels. ( E and F ) Representative immunoblots from GST-Rab10 pulldown experiments in Hep3B cells, probing for interactions between Rab10 and EHD2 under serum-starved (E) or Torin1-treated conditions (F) ( n = 3 independent experiments for each condition). Numbers below the pulldown blot represent the mean fold enrichment in protein levels. ( G ) Representative immunoblot of a GST-Rab10 pulldown of EHD2 in Hep3B cells previously treated with either siNT or siEHBP1 and subjected to HBSS starvation for 1 hour ( n = 3 independent experiments), quantified in ( H ). ( I ) Fluorescence images of EHBP1-positive LDs in HuH-7 cells expressing HA-EHBP1 and starved in HBSS for 1 hour before fixation and immunostaining with anti-HA antibody (green). Cells were preloaded with 150 μM oleic acid overnight. LDs are stained with ORO (red). ( J ) Fluorescence image of a HuH-7 cell transfected to express GFP-EHD2 (green) and mCherry-Rab10 (red) before serum starvation in HBSS for 1 hour and fixation. The boxed region shows a higher-magnification image of an example of an MDH-stained LD (blue) that is also positive for GFP-EHD2 and mCherry-Rab10. Scale bars, 10 μm. ( K and L ) Quantification of the appearance of GFP-Rab10–positive LDs (K) or GFP-EHD2–positive LDs (L) in HuH-7 cells following siRNA-mediated knockdown of EHBP1 and EHD2 (K) or EHBP1 and Rab10 (L) for 48 hours, followed by serum starvation in HBSS for 1 hour. Results are from n = 3 independent experiments each and are represented as mean ± SD. ** P ≤ 0.01; *** P ≤ 0.001. A total of 80 cells were quantified per condition. ( M ) Live-cell confocal fluorescence imaging of a starved Hep3B hepatoma cell coexpressing GFP-EHD2 and mCherry-Rab10, depicting the sequential recruitment of Rab10 and EHD2 to MDH-labeled LDs (blue). Note the presence of the mCherry-Rab10–positive structure at the periphery of the LD (arrowhead) before the recruitment of GFP-EHD2, resulting in the emergence of signal overlap by 35 min. Scale bars, 5 μm.

    Article Snippet: The siRNA oligos targeting Rab10, EHBP1, EHD2, Rab7, and Atg7 were purchased from Thermo Fisher Scientific, and cells were transfected with the RNAiMAX reagent (Invitrogen).

    Techniques: Transfection, Hemagglutination Assay, Western Blot, Fluorescence, Expressing, Immunostaining, Staining, Imaging, Labeling

    Rab10 function is required for LD catabolism. ( A ) Representative immunoblot from n = 3 independent experiments showing the efficiency ( > 90%) of Rab10 siRNA knockdown in HuH-7 hepatoma cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ( B and C ) Fluorescence imaging of HuH-7 cells subjected to 48-hour treatment with either control nontargeting (NT) siRNA or Rab10-directed siRNA and subsequently starved for an additional 48 hours. LDs are stained with ORO, and nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). Total ORO-stained area per cell was quantified in (C) from n = 3 independent experiments ( > 350 cells measured per experiment). ( D ) HuH-7 cells were treated with control NT siRNA or Rab10 siRNA for 48 hours before re-expression of GFP-tagged forms of wild-type (WT), active (−Q68L), or inactive (−T23N) forms of Rab10. Cells were then serum-starved for a period of 48 hours to look for rescue of the LD breakdown phenotype (total ORO-stained area quantified in n = 3 independent experiments, 30 cells per repeat). ( E ) Quantification of a similar knockdown/re-expression experiment performed in (D), with the exception that the lysosomal protease inhibitor CQ was included in the medium during the starvation period ( n = 3 independent experiments, 25 cells measured per repeat). ( F to I ) LDs accumulate in cells isolated from Rab10 KO mice. Cells were preloaded with 400 μM oleic acid overnight. (F) Representative immunoblot of MEFs isolated from WT or Rab10 KO embryos. (G) Measurement of total triglyceride content in WT or Rab10 KO MEFs. (H) Representative fluorescence images of oleate-loaded WT or Rab10 KO MEFs, stained with ORO and DAPI. (I) Digital quantification of average LD content per cell in WT or Rab10 KO MEFs ( n = 3 independent experiments, 300 cells per repeat). ( J ) Quantification of the total LD area per cell in Rab10 KO MEFs transfected with either GFP vector alone or GFP-Rab10 ( n = 3 independent experiments, 400 cells per repeat). Cells were preloaded with 400 μM oleic acid overnight. ( K ) Quantification of the release of [ 3 H]H 2 O into the medium as a functional readout of mitochondrial β-oxidation in either WT or Rab10 MEFs subjected to 6 hours of growth under full-serum or serum-starved conditions. Cells were pulse-labeled with [9,10- 3 H]oleic acid. Data are represented as means ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; N.S., not significant. Scale bars, 10 μm.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: Rab10 function is required for LD catabolism. ( A ) Representative immunoblot from n = 3 independent experiments showing the efficiency ( > 90%) of Rab10 siRNA knockdown in HuH-7 hepatoma cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ( B and C ) Fluorescence imaging of HuH-7 cells subjected to 48-hour treatment with either control nontargeting (NT) siRNA or Rab10-directed siRNA and subsequently starved for an additional 48 hours. LDs are stained with ORO, and nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). Total ORO-stained area per cell was quantified in (C) from n = 3 independent experiments ( > 350 cells measured per experiment). ( D ) HuH-7 cells were treated with control NT siRNA or Rab10 siRNA for 48 hours before re-expression of GFP-tagged forms of wild-type (WT), active (−Q68L), or inactive (−T23N) forms of Rab10. Cells were then serum-starved for a period of 48 hours to look for rescue of the LD breakdown phenotype (total ORO-stained area quantified in n = 3 independent experiments, 30 cells per repeat). ( E ) Quantification of a similar knockdown/re-expression experiment performed in (D), with the exception that the lysosomal protease inhibitor CQ was included in the medium during the starvation period ( n = 3 independent experiments, 25 cells measured per repeat). ( F to I ) LDs accumulate in cells isolated from Rab10 KO mice. Cells were preloaded with 400 μM oleic acid overnight. (F) Representative immunoblot of MEFs isolated from WT or Rab10 KO embryos. (G) Measurement of total triglyceride content in WT or Rab10 KO MEFs. (H) Representative fluorescence images of oleate-loaded WT or Rab10 KO MEFs, stained with ORO and DAPI. (I) Digital quantification of average LD content per cell in WT or Rab10 KO MEFs ( n = 3 independent experiments, 300 cells per repeat). ( J ) Quantification of the total LD area per cell in Rab10 KO MEFs transfected with either GFP vector alone or GFP-Rab10 ( n = 3 independent experiments, 400 cells per repeat). Cells were preloaded with 400 μM oleic acid overnight. ( K ) Quantification of the release of [ 3 H]H 2 O into the medium as a functional readout of mitochondrial β-oxidation in either WT or Rab10 MEFs subjected to 6 hours of growth under full-serum or serum-starved conditions. Cells were pulse-labeled with [9,10- 3 H]oleic acid. Data are represented as means ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; N.S., not significant. Scale bars, 10 μm.

    Article Snippet: The siRNA oligos targeting Rab10, EHBP1, EHD2, Rab7, and Atg7 were purchased from Thermo Fisher Scientific, and cells were transfected with the RNAiMAX reagent (Invitrogen).

    Techniques: Fluorescence, Imaging, Staining, Expressing, Protease Inhibitor, Isolation, Gene Knockout, Mouse Assay, Transfection, Plasmid Preparation, Functional Assay, Labeling

    EHD2 and EHBP1 are involved in Rab10-mediated LD breakdown. ( A and B ) Representative immunoblots showing the efficiency of a 48-hour EHD2 (A) or EHBP1 (B) siRNA–mediated knockdown in the Hep3B hepatoma cell line. ( C ) Quantification of the effect of EHD2 or EHBP1 knockdown on LD breakdown in Hep3B cells following 48-hour knockdown and 48-hour serum starvation. Average ORO-stained LD area (in pixels) per cell was calculated from n = 3 independent experiments in 100 cells per condition. Cells were preloaded with 150 μM oleic acid overnight. ( D ) The dependence of LD catabolism on EHD2 activity was examined by depleting Hep3B cells of EHD2 by siRNA treatment for 24 hours and then transfecting them with GFP alone, GPF-EHD2, or GFP-EHD2-T72A (ATPase-dead) for an additional 24 hours in the presence or absence of CQ. LD breakdown is represented as the average ORO-stained area per cell from n = 3 experiments in 25 transfected cells per condition. Cells were preloaded with 150 μM oleic acid overnight. ( E ) Subcellular fractionation of oleate-loaded Hep3B cells starved for 2 hours in HBSS through a 0 to 30% discontinuous iodixanol (OptiPrep) gradient. Fractions were collected from the top of the gradient and blotted for EHD2, the endosomal marker Rab5, or the lysosomal marker LAMP1. ( F ) Representative fluorescence images of basal or serum-starved Hep3B cells treated with siNT, Rab10 siRNA, or EHD2 siRNA and expressing a dual-fluorescent red fluorescent protien (RFP)–GFP–PLIN2 construct that had been serum-starved for 24 hours to measure the appearance of “RFP-only” PLIN2-positive puncta, indicative of interactions between the LD and the acidic lysosomal compartment. Cells were preloaded with 150 μM oleic acid overnight. ( G ) Quantification of the number of “RFP-only” PLIN2-positive puncta per Hep3B cell, reflective of active lipophagy, from n = 3 independent experiments, measuring 22 transfected cells per condition. The data are represented as mean ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Scale bars, 10 μm.

    Journal: Science Advances

    Article Title: A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    doi: 10.1126/sciadv.1601470

    Figure Lengend Snippet: EHD2 and EHBP1 are involved in Rab10-mediated LD breakdown. ( A and B ) Representative immunoblots showing the efficiency of a 48-hour EHD2 (A) or EHBP1 (B) siRNA–mediated knockdown in the Hep3B hepatoma cell line. ( C ) Quantification of the effect of EHD2 or EHBP1 knockdown on LD breakdown in Hep3B cells following 48-hour knockdown and 48-hour serum starvation. Average ORO-stained LD area (in pixels) per cell was calculated from n = 3 independent experiments in 100 cells per condition. Cells were preloaded with 150 μM oleic acid overnight. ( D ) The dependence of LD catabolism on EHD2 activity was examined by depleting Hep3B cells of EHD2 by siRNA treatment for 24 hours and then transfecting them with GFP alone, GPF-EHD2, or GFP-EHD2-T72A (ATPase-dead) for an additional 24 hours in the presence or absence of CQ. LD breakdown is represented as the average ORO-stained area per cell from n = 3 experiments in 25 transfected cells per condition. Cells were preloaded with 150 μM oleic acid overnight. ( E ) Subcellular fractionation of oleate-loaded Hep3B cells starved for 2 hours in HBSS through a 0 to 30% discontinuous iodixanol (OptiPrep) gradient. Fractions were collected from the top of the gradient and blotted for EHD2, the endosomal marker Rab5, or the lysosomal marker LAMP1. ( F ) Representative fluorescence images of basal or serum-starved Hep3B cells treated with siNT, Rab10 siRNA, or EHD2 siRNA and expressing a dual-fluorescent red fluorescent protien (RFP)–GFP–PLIN2 construct that had been serum-starved for 24 hours to measure the appearance of “RFP-only” PLIN2-positive puncta, indicative of interactions between the LD and the acidic lysosomal compartment. Cells were preloaded with 150 μM oleic acid overnight. ( G ) Quantification of the number of “RFP-only” PLIN2-positive puncta per Hep3B cell, reflective of active lipophagy, from n = 3 independent experiments, measuring 22 transfected cells per condition. The data are represented as mean ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Scale bars, 10 μm.

    Article Snippet: The siRNA oligos targeting Rab10, EHBP1, EHD2, Rab7, and Atg7 were purchased from Thermo Fisher Scientific, and cells were transfected with the RNAiMAX reagent (Invitrogen).

    Techniques: Western Blot, Staining, Activity Assay, Transfection, Fractionation, Marker, Fluorescence, Expressing, Construct

    Malat1 interacts and recruits Suv39h1 to MyoD-binding loci. ( a ) The expression levels of Suv39h1, HDAC1 and Hp1β proteins in Vector or sh Malat1 C2C12 cells were determined by western blotting in proliferating myoblasts. α-Tubulin was used as a normalization control. ( b ) Nuclear extracts from the above cells were immunoprecipitated by IgG or an antibody against Suv39h1. The retrieved RNAs were detected by qRT-PCR. ( c ) In vitro -transcribed biotinylated Malat1 FL transcripts were used to pull down its binding proteins. The indicated proteins were detected by western blotting. Beads only or antisense Malat1 transcripts were used as negative controls. ( d ) Upper: schematic illustration of the Malat1 FL or fragments (F1, F2, F3 or F4) that were used in the RNA pull-down assays for mapping the functional domain. Beads only or GFP transcripts were used as negative controls. Middle: the retrieved Suv39h1 proteins were detected by western blotting. The numbers below indicate the quantification of the intensity of each band. Lower: the in vitro generated transcripts were visualized on an agarose gel. ( e ) EMSA assays were performed to detect the association between Malat1 FL or various fragments with purified GST-Suv39h1 protein (for 200 fmol Malat1 FL RNA, 100, 400, 800 or 1 600 n M GST-Suv39h1 were used. For 400 fmol F1–F4 fragments, 800, 1 600, 2 400 or 3 200 n M GST-Suv39h1 protein were used). Antisense Malat1 transcript was used as an unrelated negative control for RNA. GST only protein (3 200 n M ) was used as a negative control for GST-Suv39h1 protein. A shifted band (lanes 5 and 6) ran higher than the control bands (lanes 1 and 2) and also displayed lower intensity. ( f ) Malat1 ChIRP with both even and odd antisense oligos retrieved a significant amount of genomic DNAs corresponding to the promoter regions of Myogenin , MyHC , Tnni2 and Ccnd3 genes but not GAPDH locus. LacZ ChIRP retrieved no signal. ( g – k ) ChIP-PCR analysis of Suv39h1, HP1β, HDAC1, H3K9me3 and MyoD enrichment on the promoter or enhancer of Myogenin , MCK , MyHC , Troponin , Ccnd3 and p21 loci in Vector or sh Malat1 expressing C2C12 cells. The enrichment fold was calculated as a fraction of DNA present in the input samples. ( l ) 10T1/2 cells were transfected with the indicated combination of plasmids and si Malat1 oligos. The expression of the myogenic marker genes was analyzed by qRT-PCR. ( m ) 10T1/2 cells were co-transfected with a Myogenin or 4RE luciferase reporter, the indicated expression plasmids and si Malat1 oligos. Luciferase activities were measured after differentiating the cells for 48 h. All PCR data were normalized to GAPDH mRNA and represent the average of three independent experiments±s.d. All luciferase data were normalized to Renilla luciferase activities and represent the average of three independent experiments±s.d. * P

    Journal: Cell Discovery

    Article Title: Malat1 regulates myogenic differentiation and muscle regeneration through modulating MyoD transcriptional activity

    doi: 10.1038/celldisc.2017.2

    Figure Lengend Snippet: Malat1 interacts and recruits Suv39h1 to MyoD-binding loci. ( a ) The expression levels of Suv39h1, HDAC1 and Hp1β proteins in Vector or sh Malat1 C2C12 cells were determined by western blotting in proliferating myoblasts. α-Tubulin was used as a normalization control. ( b ) Nuclear extracts from the above cells were immunoprecipitated by IgG or an antibody against Suv39h1. The retrieved RNAs were detected by qRT-PCR. ( c ) In vitro -transcribed biotinylated Malat1 FL transcripts were used to pull down its binding proteins. The indicated proteins were detected by western blotting. Beads only or antisense Malat1 transcripts were used as negative controls. ( d ) Upper: schematic illustration of the Malat1 FL or fragments (F1, F2, F3 or F4) that were used in the RNA pull-down assays for mapping the functional domain. Beads only or GFP transcripts were used as negative controls. Middle: the retrieved Suv39h1 proteins were detected by western blotting. The numbers below indicate the quantification of the intensity of each band. Lower: the in vitro generated transcripts were visualized on an agarose gel. ( e ) EMSA assays were performed to detect the association between Malat1 FL or various fragments with purified GST-Suv39h1 protein (for 200 fmol Malat1 FL RNA, 100, 400, 800 or 1 600 n M GST-Suv39h1 were used. For 400 fmol F1–F4 fragments, 800, 1 600, 2 400 or 3 200 n M GST-Suv39h1 protein were used). Antisense Malat1 transcript was used as an unrelated negative control for RNA. GST only protein (3 200 n M ) was used as a negative control for GST-Suv39h1 protein. A shifted band (lanes 5 and 6) ran higher than the control bands (lanes 1 and 2) and also displayed lower intensity. ( f ) Malat1 ChIRP with both even and odd antisense oligos retrieved a significant amount of genomic DNAs corresponding to the promoter regions of Myogenin , MyHC , Tnni2 and Ccnd3 genes but not GAPDH locus. LacZ ChIRP retrieved no signal. ( g – k ) ChIP-PCR analysis of Suv39h1, HP1β, HDAC1, H3K9me3 and MyoD enrichment on the promoter or enhancer of Myogenin , MCK , MyHC , Troponin , Ccnd3 and p21 loci in Vector or sh Malat1 expressing C2C12 cells. The enrichment fold was calculated as a fraction of DNA present in the input samples. ( l ) 10T1/2 cells were transfected with the indicated combination of plasmids and si Malat1 oligos. The expression of the myogenic marker genes was analyzed by qRT-PCR. ( m ) 10T1/2 cells were co-transfected with a Myogenin or 4RE luciferase reporter, the indicated expression plasmids and si Malat1 oligos. Luciferase activities were measured after differentiating the cells for 48 h. All PCR data were normalized to GAPDH mRNA and represent the average of three independent experiments±s.d. All luciferase data were normalized to Renilla luciferase activities and represent the average of three independent experiments±s.d. * P

    Article Snippet: Precursor miRNA oligos were obtained from Life Technologies. siRNA oligos against mouse Malat1 and Ago2 were obtained from Ribobio Technologies (Guangzhou, China).

    Techniques: Binding Assay, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation, Quantitative RT-PCR, In Vitro, Functional Assay, Generated, Agarose Gel Electrophoresis, Purification, Negative Control, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Transfection, Marker, Luciferase

    Malat1 functions to repress myoblast differentiation. ( a , b ) The knockdown of Malat1 in C2C12 cells by a stably expressed shRNA oligo was confirmed by qRT-PCR ( a ) and northern blotting in proliferating myoblasts ( b ). ( c ) The knockdown of Malat1 increased the levels of the indicated myogenic genes, Myogenin, MyHC, α-Actin and Troponin at protein level during a 5-day differentiation course. ( d ) The knockdown of Malat1 increased the above myogenic genes at mRNA levels. ( e ) The above cells were visualized on day 2 in DM (phase images). IF staining for Myogenin or MyHC was performed on day 1 and day 3 in DM, respectively. ( f ) The number of positively stained cells was counted from at least 10 fields. ( g ) The knockdown of Malat1 by a siRNA oligo was confirmed by qRT-PCR on day 2 post transfection. ( h ) The above knockdown increased the levels of the indicated myogenic genes α-Actin and Troponin at the protein level during a 4-day differentiation course. ( i ) The above si Malat1 transfection also increased the number of MyHC-positive cells by IF staining on day 2 in DM. ( j ) The knockdown of Malat1 in C2C12 cells increased the luciferase activities of the indicated myogenic reporters on day 2 in DM. RLU, relative luciferase unit. All PCR data were normalized to GAPDH mRNA and represent the average of three independent experiments±s.d. All luciferase data were normalized to Renilla luciferase activities and represent the average of three independent experiments±s.d. * P

    Journal: Cell Discovery

    Article Title: Malat1 regulates myogenic differentiation and muscle regeneration through modulating MyoD transcriptional activity

    doi: 10.1038/celldisc.2017.2

    Figure Lengend Snippet: Malat1 functions to repress myoblast differentiation. ( a , b ) The knockdown of Malat1 in C2C12 cells by a stably expressed shRNA oligo was confirmed by qRT-PCR ( a ) and northern blotting in proliferating myoblasts ( b ). ( c ) The knockdown of Malat1 increased the levels of the indicated myogenic genes, Myogenin, MyHC, α-Actin and Troponin at protein level during a 5-day differentiation course. ( d ) The knockdown of Malat1 increased the above myogenic genes at mRNA levels. ( e ) The above cells were visualized on day 2 in DM (phase images). IF staining for Myogenin or MyHC was performed on day 1 and day 3 in DM, respectively. ( f ) The number of positively stained cells was counted from at least 10 fields. ( g ) The knockdown of Malat1 by a siRNA oligo was confirmed by qRT-PCR on day 2 post transfection. ( h ) The above knockdown increased the levels of the indicated myogenic genes α-Actin and Troponin at the protein level during a 4-day differentiation course. ( i ) The above si Malat1 transfection also increased the number of MyHC-positive cells by IF staining on day 2 in DM. ( j ) The knockdown of Malat1 in C2C12 cells increased the luciferase activities of the indicated myogenic reporters on day 2 in DM. RLU, relative luciferase unit. All PCR data were normalized to GAPDH mRNA and represent the average of three independent experiments±s.d. All luciferase data were normalized to Renilla luciferase activities and represent the average of three independent experiments±s.d. * P

    Article Snippet: Precursor miRNA oligos were obtained from Life Technologies. siRNA oligos against mouse Malat1 and Ago2 were obtained from Ribobio Technologies (Guangzhou, China).

    Techniques: Stable Transfection, shRNA, Quantitative RT-PCR, Northern Blot, Staining, Transfection, Luciferase, Polymerase Chain Reaction

    Malat1 modulates the transcriptional activity of MyoD. ( a ) RNAs with PolyA tails (PolyA + RNAs) were isolated from C2C12 cells transfected with siNC or si Malat1 oligos and differentiated for 2 days and subjected to RNA-seq for transcriptomic analysis. The normalized fragment density was calculated by counting the fragments per kilobase of genomic regions of interests (exonic, intronic and intergenic) per million mapped reads (FPKM). ( b ) Differentially expressed genes between siNC ( x axis) and si Malat1 ( y axis) cells were determined from the above RNA-seq data and shown as red dots in the scatter plot. Gray dots represent genes unaffected by si Malat1 treatment. ( c ) Genomic snapshots showing an example of significantly up- ( Myh1 , left) or down- ( Ccnd1 , right) regulated genes by si Malat1 treatment. The y axis is the normalized signal density. ( d ) GO analysis of genes that were up- or downregulated in si Malat1 cells. The y axis shows GO terms and the x axis shows statistical significance (that is, −log (p), multicorrected P -values) for the top eight enriched terms. ( e ) A high percentage (29.3%; in red color) of the above upregulated genes contain a MyoD-binding site at their promoter regions based on the analysis of MyoD ChIP-seq data from C2C12 cells. ( f ) 10T1/2 cells were co-transfected with a Myogenin or MCK luciferase reporter, a MyoD expression plasmid (0.5 μg) and a Malat1 expression plasmid (0, 0.125, 0.25 or 0.5 μg). Luciferase activities were measured after differentiating the cells for 48 h. ( g ) 10T1/2 cells were transfected with the indicated reporter, the MyoD plasmid and the siRNAs against Malat1 or negative control (siNC) oligos. Luciferase activities were measured after differentiating the cells for 48 h. ( h ) Knockdown of Malat1 did not affect the expression of MyoD at protein (upper, western blotting) or RNA (bottom, qRT-PCR) level. ( i ) Schematic illustration of the Gal4-MyoD reporter system. A Gal4-binding motif is fused to the luciferase gene; the expression of a fusion protein with Gal4-binding domain fused with MyoD (Gal4-MyoD) will lead to its binding to the Gal4 motif and activate the luciferase reporter. ( j ) 10T1/2 cells were co-transfected with the above Gal4 luciferase reporter, the Malat1 expression vector (0.5 μg) or a Suv39h1 expression plasmid in the presence of the vector expressing Gal4-MyoD fusion protein. Luciferase activities were measured after differentiating the cells for 48 h. ( k ) 10T1/2 cells were co-transfected with the Gal4 luciferase reporter, si Malat1 or siNC oligos and the Gal4-MyoD vector. Luciferase activities were measured after differentiating the cells for 48 h. All PCR data were normalized to GAPDH mRNA and represent the average of three independent experiments±s.d. All luciferase data were normalized to Renilla luciferase activities and represent the average of three independent experiments±s.d. * P

    Journal: Cell Discovery

    Article Title: Malat1 regulates myogenic differentiation and muscle regeneration through modulating MyoD transcriptional activity

    doi: 10.1038/celldisc.2017.2

    Figure Lengend Snippet: Malat1 modulates the transcriptional activity of MyoD. ( a ) RNAs with PolyA tails (PolyA + RNAs) were isolated from C2C12 cells transfected with siNC or si Malat1 oligos and differentiated for 2 days and subjected to RNA-seq for transcriptomic analysis. The normalized fragment density was calculated by counting the fragments per kilobase of genomic regions of interests (exonic, intronic and intergenic) per million mapped reads (FPKM). ( b ) Differentially expressed genes between siNC ( x axis) and si Malat1 ( y axis) cells were determined from the above RNA-seq data and shown as red dots in the scatter plot. Gray dots represent genes unaffected by si Malat1 treatment. ( c ) Genomic snapshots showing an example of significantly up- ( Myh1 , left) or down- ( Ccnd1 , right) regulated genes by si Malat1 treatment. The y axis is the normalized signal density. ( d ) GO analysis of genes that were up- or downregulated in si Malat1 cells. The y axis shows GO terms and the x axis shows statistical significance (that is, −log (p), multicorrected P -values) for the top eight enriched terms. ( e ) A high percentage (29.3%; in red color) of the above upregulated genes contain a MyoD-binding site at their promoter regions based on the analysis of MyoD ChIP-seq data from C2C12 cells. ( f ) 10T1/2 cells were co-transfected with a Myogenin or MCK luciferase reporter, a MyoD expression plasmid (0.5 μg) and a Malat1 expression plasmid (0, 0.125, 0.25 or 0.5 μg). Luciferase activities were measured after differentiating the cells for 48 h. ( g ) 10T1/2 cells were transfected with the indicated reporter, the MyoD plasmid and the siRNAs against Malat1 or negative control (siNC) oligos. Luciferase activities were measured after differentiating the cells for 48 h. ( h ) Knockdown of Malat1 did not affect the expression of MyoD at protein (upper, western blotting) or RNA (bottom, qRT-PCR) level. ( i ) Schematic illustration of the Gal4-MyoD reporter system. A Gal4-binding motif is fused to the luciferase gene; the expression of a fusion protein with Gal4-binding domain fused with MyoD (Gal4-MyoD) will lead to its binding to the Gal4 motif and activate the luciferase reporter. ( j ) 10T1/2 cells were co-transfected with the above Gal4 luciferase reporter, the Malat1 expression vector (0.5 μg) or a Suv39h1 expression plasmid in the presence of the vector expressing Gal4-MyoD fusion protein. Luciferase activities were measured after differentiating the cells for 48 h. ( k ) 10T1/2 cells were co-transfected with the Gal4 luciferase reporter, si Malat1 or siNC oligos and the Gal4-MyoD vector. Luciferase activities were measured after differentiating the cells for 48 h. All PCR data were normalized to GAPDH mRNA and represent the average of three independent experiments±s.d. All luciferase data were normalized to Renilla luciferase activities and represent the average of three independent experiments±s.d. * P

    Article Snippet: Precursor miRNA oligos were obtained from Life Technologies. siRNA oligos against mouse Malat1 and Ago2 were obtained from Ribobio Technologies (Guangzhou, China).

    Techniques: Activity Assay, Isolation, Transfection, RNA Sequencing Assay, Binding Assay, Chromatin Immunoprecipitation, Luciferase, Expressing, Plasmid Preparation, Negative Control, Western Blot, Quantitative RT-PCR, Polymerase Chain Reaction

    Malat1 is directly targeted by miR-181a in the nucleus of myoblast through a Ago2-dependent nRISC machinery. ( a ) Upper: the predicted binding site between miR-181a and Malat1- WT sequence (5′-3916–3941-3′). A mutant was generated by mutating the seed sequence (UUGAAUGU, underlined) to CCUGCGAC. Lower: the hypothetical model depicting that Malat1 transcript is directly targeted by miR-181a/Ago2 machinery in the myoblast nucleus. ( b ) The above-identified WT or mutated miR-181a-binding site was cloned into a pMIR luciferase reporter vector and transfected into the C2C12 cells with miR-181a (pre-181a) or miR-193a (pre-193a) precursor oligos. Luciferase activities were determined 48 h post transfection. ( c ) Pre-181a or negative control (Pre-NC) precursor oligos were transfected into C2C12 cells. The expression level of Malat1 was examined by qRT-PCR after the cells were switched to DM or maintained in the growth medium (GM) for 48 h. ( d ) An antagomiR of miR-181a (Anti-181a) or a negative control (Anti-NC) oligo was transfected into C2C12 and the expression of Malat1 was determined as above. ( e ) The expression level of miR-181a during C2C12 differentiation at 0, 9 and 24 h was determined by qRT-PCR. ( f ) The distribution of miR-181a in C2C12 cytoplasmic (Cyto) or nuclear (Nuc) fraction was determined by qRT-PCR. Xist , Gapdh and U6 RNAs were used as controls to compare with. ( g ) Upper panel: cytoplasmic or nuclear level of Ago2 proteins was detected by western blotting. MyoD and HDAC1 were used as nuclear protein controls and α-Tubulin was used as cytoplasmic protein control. Lower panel: relative quantification of the band intensity was plotted. ( h ) RNA pull-down assay was performed with biotinylated Malat1 FL transcripts to retrieve Ago2 in C2C12 nuclear lysates. Beads only or FL antisense transcripts were used as negative controls. The in vitro generated transcripts were visualized on the Agarose gel to show equal loading. ( i ) The C2C12 nuclear lysate was immunoprecipitated by an Ago2 antibody. The retrieved Malat1 RNAs were detected by qRT-PCR. Yam1 and 7SK RNAs were also detected as controls. ( j ) Ago2 was knocked down by an siRNA oligo in C2C12 myoblast and the Malat1 expression was examined by qRT-PCR after the cells were switched to DM or maintained in GM for 48 h. ( k ) Nuclear run-on assay was performed in C2C12 nuclei transfected with pre-181a or pre-NC oligos. Primers were designed to detect nascent transcription from five different locations of the Malat1 gene. ( l ) Left: cytoplasmic or nuclear levels of Dicer, Tnrc6a and Trbp proteins were detected by western blotting. GAPDH and α-Tubulin were used as cytoplasmic protein control and HDAC1 was used as nuclear protein control. Right: relative quantification of the band intensity was also plotted. ( m ) The nuclear extract of C2C12 cells was immunoprecipitated by the indicated antibodies. The retrieved Neat1 , Dum , Xist , U1 or Malat1 RNAs were detected by qRT-PCR. ( n ) C2C12 cells were transfected with the indicated oligos. Expression of the indicated myogenic genes w as analyzed by qRT-PCR. All PCR data were normalized to GAPDH mRNA and represent the average of three independent experiments±s.d. All luciferase data were normalized to Renilla luciferase activities and represent the average of three independent experiments±s.d. * P

    Journal: Cell Discovery

    Article Title: Malat1 regulates myogenic differentiation and muscle regeneration through modulating MyoD transcriptional activity

    doi: 10.1038/celldisc.2017.2

    Figure Lengend Snippet: Malat1 is directly targeted by miR-181a in the nucleus of myoblast through a Ago2-dependent nRISC machinery. ( a ) Upper: the predicted binding site between miR-181a and Malat1- WT sequence (5′-3916–3941-3′). A mutant was generated by mutating the seed sequence (UUGAAUGU, underlined) to CCUGCGAC. Lower: the hypothetical model depicting that Malat1 transcript is directly targeted by miR-181a/Ago2 machinery in the myoblast nucleus. ( b ) The above-identified WT or mutated miR-181a-binding site was cloned into a pMIR luciferase reporter vector and transfected into the C2C12 cells with miR-181a (pre-181a) or miR-193a (pre-193a) precursor oligos. Luciferase activities were determined 48 h post transfection. ( c ) Pre-181a or negative control (Pre-NC) precursor oligos were transfected into C2C12 cells. The expression level of Malat1 was examined by qRT-PCR after the cells were switched to DM or maintained in the growth medium (GM) for 48 h. ( d ) An antagomiR of miR-181a (Anti-181a) or a negative control (Anti-NC) oligo was transfected into C2C12 and the expression of Malat1 was determined as above. ( e ) The expression level of miR-181a during C2C12 differentiation at 0, 9 and 24 h was determined by qRT-PCR. ( f ) The distribution of miR-181a in C2C12 cytoplasmic (Cyto) or nuclear (Nuc) fraction was determined by qRT-PCR. Xist , Gapdh and U6 RNAs were used as controls to compare with. ( g ) Upper panel: cytoplasmic or nuclear level of Ago2 proteins was detected by western blotting. MyoD and HDAC1 were used as nuclear protein controls and α-Tubulin was used as cytoplasmic protein control. Lower panel: relative quantification of the band intensity was plotted. ( h ) RNA pull-down assay was performed with biotinylated Malat1 FL transcripts to retrieve Ago2 in C2C12 nuclear lysates. Beads only or FL antisense transcripts were used as negative controls. The in vitro generated transcripts were visualized on the Agarose gel to show equal loading. ( i ) The C2C12 nuclear lysate was immunoprecipitated by an Ago2 antibody. The retrieved Malat1 RNAs were detected by qRT-PCR. Yam1 and 7SK RNAs were also detected as controls. ( j ) Ago2 was knocked down by an siRNA oligo in C2C12 myoblast and the Malat1 expression was examined by qRT-PCR after the cells were switched to DM or maintained in GM for 48 h. ( k ) Nuclear run-on assay was performed in C2C12 nuclei transfected with pre-181a or pre-NC oligos. Primers were designed to detect nascent transcription from five different locations of the Malat1 gene. ( l ) Left: cytoplasmic or nuclear levels of Dicer, Tnrc6a and Trbp proteins were detected by western blotting. GAPDH and α-Tubulin were used as cytoplasmic protein control and HDAC1 was used as nuclear protein control. Right: relative quantification of the band intensity was also plotted. ( m ) The nuclear extract of C2C12 cells was immunoprecipitated by the indicated antibodies. The retrieved Neat1 , Dum , Xist , U1 or Malat1 RNAs were detected by qRT-PCR. ( n ) C2C12 cells were transfected with the indicated oligos. Expression of the indicated myogenic genes w as analyzed by qRT-PCR. All PCR data were normalized to GAPDH mRNA and represent the average of three independent experiments±s.d. All luciferase data were normalized to Renilla luciferase activities and represent the average of three independent experiments±s.d. * P

    Article Snippet: Precursor miRNA oligos were obtained from Life Technologies. siRNA oligos against mouse Malat1 and Ago2 were obtained from Ribobio Technologies (Guangzhou, China).

    Techniques: Binding Assay, Sequencing, Mutagenesis, Generated, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Negative Control, Expressing, Quantitative RT-PCR, Western Blot, Pull Down Assay, In Vitro, Agarose Gel Electrophoresis, Immunoprecipitation, Nuclear Run-on Assay, Polymerase Chain Reaction

    Spo11-oligo complexes in msh5 and zip1 Representative time courses are shown.

    Journal: Nature

    Article Title: Homologue engagement controls meiotic DNA break number and distribution

    doi: 10.1038/nature13120

    Figure Lengend Snippet: Spo11-oligo complexes in msh5 and zip1 Representative time courses are shown.

    Article Snippet: Precipitated Spo11-oligo complexes were end-labeled in NEBuffer 4 (New England Biolabs) containing 3–10 μCi of [α-32 P]dCTP and terminal deoxynucleotidyl transferase (TdT) .

    Techniques:

    Separable effects of ndt80 and zmm mutations a , b , Spo11-oligo complex labeling from representative time courses is in a and quantification from ≥ 3 cultures (mean ± SD) is in b . c , Meiotic progression (percent of cells completing the first division).

    Journal: Nature

    Article Title: Homologue engagement controls meiotic DNA break number and distribution

    doi: 10.1038/nature13120

    Figure Lengend Snippet: Separable effects of ndt80 and zmm mutations a , b , Spo11-oligo complex labeling from representative time courses is in a and quantification from ≥ 3 cultures (mean ± SD) is in b . c , Meiotic progression (percent of cells completing the first division).

    Article Snippet: Precipitated Spo11-oligo complexes were end-labeled in NEBuffer 4 (New England Biolabs) containing 3–10 μCi of [α-32 P]dCTP and terminal deoxynucleotidyl transferase (TdT) .

    Techniques: Labeling

    Spo11-oligo mapping in wild type and zip3 a , b , Quantitative reproducibility of Spo11-oligo maps. In a , comparisons are shown for individual wild type (WT) or zip3 datasets from the present study, or the previously published spo11-HA data (from ref 11 ). Uniquely mapped Spo11 oligos were summed in non-overlapping 5-kb bins and expressed as RPM per kb (plotted on a log scale). In b , pairwise correlation coefficients for the datasets from the current study are shown (Pearson's r; box colors scaled from blue to red proportional to strength of correlation). For the comparison of this study's wild-type average with data from Pan et al., r = 0.949. Note that Pan et al. used a different strain background with different auxotrophies, which may alter DSB distributions 61,62 , and a hypomorphic spo11 allele ( spo11-HA ), which may affect DSBs to different extents at different locations 56 . Note that biological replicates (WT-1 vs. WT-2 or zip3-1 vs. zip3-2 ) agreed better than comparisons between cultures of different genotype. c , DSBs form at the same hotspots and with similar distribution within and between hotspots in wild type and zip3 . Unsmoothed Spo11 oligo maps are shown in the vicinity of the well-characterized ARE1 ( YCR048w) hotspot.

    Journal: Nature

    Article Title: Homologue engagement controls meiotic DNA break number and distribution

    doi: 10.1038/nature13120

    Figure Lengend Snippet: Spo11-oligo mapping in wild type and zip3 a , b , Quantitative reproducibility of Spo11-oligo maps. In a , comparisons are shown for individual wild type (WT) or zip3 datasets from the present study, or the previously published spo11-HA data (from ref 11 ). Uniquely mapped Spo11 oligos were summed in non-overlapping 5-kb bins and expressed as RPM per kb (plotted on a log scale). In b , pairwise correlation coefficients for the datasets from the current study are shown (Pearson's r; box colors scaled from blue to red proportional to strength of correlation). For the comparison of this study's wild-type average with data from Pan et al., r = 0.949. Note that Pan et al. used a different strain background with different auxotrophies, which may alter DSB distributions 61,62 , and a hypomorphic spo11 allele ( spo11-HA ), which may affect DSBs to different extents at different locations 56 . Note that biological replicates (WT-1 vs. WT-2 or zip3-1 vs. zip3-2 ) agreed better than comparisons between cultures of different genotype. c , DSBs form at the same hotspots and with similar distribution within and between hotspots in wild type and zip3 . Unsmoothed Spo11 oligo maps are shown in the vicinity of the well-characterized ARE1 ( YCR048w) hotspot.

    Article Snippet: Precipitated Spo11-oligo complexes were end-labeled in NEBuffer 4 (New England Biolabs) containing 3–10 μCi of [α-32 P]dCTP and terminal deoxynucleotidyl transferase (TdT) .

    Techniques: Hemagglutination Assay

    Hyper-rec phenotype of zmm mutants a , Schematic of arg4 heteroalleles, showing ORFs and mutated restriction sites. Below, Spo11-oligo profile shows DSB distribution (RPM, reads per million mapped; smoothed with 201-bp Hann window). b , Heteroallele recombination frequencies (mean ± SD). *, significantly different from wild type (p

    Journal: Nature

    Article Title: Homologue engagement controls meiotic DNA break number and distribution

    doi: 10.1038/nature13120

    Figure Lengend Snippet: Hyper-rec phenotype of zmm mutants a , Schematic of arg4 heteroalleles, showing ORFs and mutated restriction sites. Below, Spo11-oligo profile shows DSB distribution (RPM, reads per million mapped; smoothed with 201-bp Hann window). b , Heteroallele recombination frequencies (mean ± SD). *, significantly different from wild type (p

    Article Snippet: Precipitated Spo11-oligo complexes were end-labeled in NEBuffer 4 (New England Biolabs) containing 3–10 μCi of [α-32 P]dCTP and terminal deoxynucleotidyl transferase (TdT) .

    Techniques:

    Changes in the DSB landscape in zip3 a, Change in Spo11-oligo counts in hotspots grouped by chromosomal context. Tel, within 20 kb of telomeres; Cen, within ±10 kb of centromeres; rDNA, from 60 kb leftward to 30 kb rightward of rDNA; Interstitial, all others. Dashed lines mark values assumed as no change and average change (1.8-fold). Boxes indicate median and interquartile range; whiskers indicate the most extreme data points which are ≤1.5 times the interquartile range from the box; individual points are outliers. Sub-telomeric and pericentric zones show less increase in zip3 on average, thus, ZMM-dependent feedback contributes less than other, unknown factors to suppressing DSBs in these regions. The zone near the rDNA showed no increase or was even decreased; thus, zip3 mutants are competent for this region's DSB suppression, which is dependent on the ATPase Pch2 and the replication factor Orc1 (ref 63). Note that the remaining interstitial hotspots showed highly variable response to zip3 mutation ( > 20 fold). b, Correlation between log-fold change in Spo11-oligo counts in zip3 and the binding of the indicated proteins, binned in non-overlapping windows of varying size. Closed symbols, p

    Journal: Nature

    Article Title: Homologue engagement controls meiotic DNA break number and distribution

    doi: 10.1038/nature13120

    Figure Lengend Snippet: Changes in the DSB landscape in zip3 a, Change in Spo11-oligo counts in hotspots grouped by chromosomal context. Tel, within 20 kb of telomeres; Cen, within ±10 kb of centromeres; rDNA, from 60 kb leftward to 30 kb rightward of rDNA; Interstitial, all others. Dashed lines mark values assumed as no change and average change (1.8-fold). Boxes indicate median and interquartile range; whiskers indicate the most extreme data points which are ≤1.5 times the interquartile range from the box; individual points are outliers. Sub-telomeric and pericentric zones show less increase in zip3 on average, thus, ZMM-dependent feedback contributes less than other, unknown factors to suppressing DSBs in these regions. The zone near the rDNA showed no increase or was even decreased; thus, zip3 mutants are competent for this region's DSB suppression, which is dependent on the ATPase Pch2 and the replication factor Orc1 (ref 63). Note that the remaining interstitial hotspots showed highly variable response to zip3 mutation ( > 20 fold). b, Correlation between log-fold change in Spo11-oligo counts in zip3 and the binding of the indicated proteins, binned in non-overlapping windows of varying size. Closed symbols, p

    Article Snippet: Precipitated Spo11-oligo complexes were end-labeled in NEBuffer 4 (New England Biolabs) containing 3–10 μCi of [α-32 P]dCTP and terminal deoxynucleotidyl transferase (TdT) .

    Techniques: Mutagenesis, Binding Assay

    Altered DSB distribution in zip3 mutants a , Upper, reproducibility of Spo11-oligo maps. Lower, DSBs form at the same hotspots in zip3 as wild type. Smoothed with 201-bp Hann window. b , Zip3 is required for chromosome size-dependent variation in Spo11-oligo density. Lines, least squares fits (dashed = not significant). c , Larger chromosomes experience greater increase in Spo11 oligos. Fold change is the per-chromosome Spo11-oligo density in zip3 over wild type (WT). Open circles, Chr XII (“12”, omitting rDNA length) and the portions of Chr XII left or right of the rDNA (“12L”, “12R”). Regression line treats 12L and 12R as separate chromosomes. d , Regional variation in response to zip3 mutation. Each point is the change at a hotspot (plotted on log scale). Red lines, local regression (loess); green circles, centromeres. e , Local domains of correlated behavior. Each point compares hotspots to their neighbors in 5-kb-wide windows the indicated distance away. Nearby hotspots show correlated behavior for fold change in zip3 (red), but not heat (Spo11-oligo frequency) in wild type (black). Shaded areas denote 95% CI estimates for hotspots randomized within-chromosome (randomized r > 0 for zip3- fold-change because of the chromosome size effect). f , Correlation between log-fold-change in zip3 and binding of indicated proteins, binned in non-overlapping windows of varying size. For clarity, other proteins are in Extended Data Fig. 9b . Pericentric, sub-telomeric, and rDNA-proximal regions were censored. Closed symbols, p

    Journal: Nature

    Article Title: Homologue engagement controls meiotic DNA break number and distribution

    doi: 10.1038/nature13120

    Figure Lengend Snippet: Altered DSB distribution in zip3 mutants a , Upper, reproducibility of Spo11-oligo maps. Lower, DSBs form at the same hotspots in zip3 as wild type. Smoothed with 201-bp Hann window. b , Zip3 is required for chromosome size-dependent variation in Spo11-oligo density. Lines, least squares fits (dashed = not significant). c , Larger chromosomes experience greater increase in Spo11 oligos. Fold change is the per-chromosome Spo11-oligo density in zip3 over wild type (WT). Open circles, Chr XII (“12”, omitting rDNA length) and the portions of Chr XII left or right of the rDNA (“12L”, “12R”). Regression line treats 12L and 12R as separate chromosomes. d , Regional variation in response to zip3 mutation. Each point is the change at a hotspot (plotted on log scale). Red lines, local regression (loess); green circles, centromeres. e , Local domains of correlated behavior. Each point compares hotspots to their neighbors in 5-kb-wide windows the indicated distance away. Nearby hotspots show correlated behavior for fold change in zip3 (red), but not heat (Spo11-oligo frequency) in wild type (black). Shaded areas denote 95% CI estimates for hotspots randomized within-chromosome (randomized r > 0 for zip3- fold-change because of the chromosome size effect). f , Correlation between log-fold-change in zip3 and binding of indicated proteins, binned in non-overlapping windows of varying size. For clarity, other proteins are in Extended Data Fig. 9b . Pericentric, sub-telomeric, and rDNA-proximal regions were censored. Closed symbols, p

    Article Snippet: Precipitated Spo11-oligo complexes were end-labeled in NEBuffer 4 (New England Biolabs) containing 3–10 μCi of [α-32 P]dCTP and terminal deoxynucleotidyl transferase (TdT) .

    Techniques: Mutagenesis, Binding Assay

    More DSBs form in zmm mutants a , Spo11 generates a covalent protein-linked DSB; endonucleolytic cleavage releases Spo11 bound to a short oligo (detection method at left). Resection is followed by strand invasion and ZMM-dependent stabilization of intermediates fated to become crossovers. b , c , Representative pulsed-field gel Southern blots probed for Chr IX are in b and Poisson-corrected DSB quantification in c (mean ± SD, 3 cultures). P, parental; W, wells. d , e , Representative Spo11-oligo complex time courses are in d and quantification in e (mean ± SD for 3 cultures, except at 10 hr for msh5 and zip3 analyses (1 culture)). Radiolabeled Spo11-oligo complexes were detected by autoradiography (top panels) and total Spo11 was detected by anti-flag western blot (WB, middle). The main labeled species differ in oligo size 10 . Nearly all of the WB signal is Spo11 that has not made a DSB 10 . Asterisk, species co-migrating with upper Spo11-oligo complexes; arrowhead, proteolytic product. Extract samples run separately and stained with Coomassie control for input to the IPs (bottom). In panels c and e , mutants are plotted with wild-type data collected in parallel.

    Journal: Nature

    Article Title: Homologue engagement controls meiotic DNA break number and distribution

    doi: 10.1038/nature13120

    Figure Lengend Snippet: More DSBs form in zmm mutants a , Spo11 generates a covalent protein-linked DSB; endonucleolytic cleavage releases Spo11 bound to a short oligo (detection method at left). Resection is followed by strand invasion and ZMM-dependent stabilization of intermediates fated to become crossovers. b , c , Representative pulsed-field gel Southern blots probed for Chr IX are in b and Poisson-corrected DSB quantification in c (mean ± SD, 3 cultures). P, parental; W, wells. d , e , Representative Spo11-oligo complex time courses are in d and quantification in e (mean ± SD for 3 cultures, except at 10 hr for msh5 and zip3 analyses (1 culture)). Radiolabeled Spo11-oligo complexes were detected by autoradiography (top panels) and total Spo11 was detected by anti-flag western blot (WB, middle). The main labeled species differ in oligo size 10 . Nearly all of the WB signal is Spo11 that has not made a DSB 10 . Asterisk, species co-migrating with upper Spo11-oligo complexes; arrowhead, proteolytic product. Extract samples run separately and stained with Coomassie control for input to the IPs (bottom). In panels c and e , mutants are plotted with wild-type data collected in parallel.

    Article Snippet: Precipitated Spo11-oligo complexes were end-labeled in NEBuffer 4 (New England Biolabs) containing 3–10 μCi of [α-32 P]dCTP and terminal deoxynucleotidyl transferase (TdT) .

    Techniques: Pulsed-Field Gel, Autoradiography, Western Blot, Labeling, Staining

    Spo11-oligo complexes in msh5 ndt80 Representative time courses are shown.

    Journal: Nature

    Article Title: Homologue engagement controls meiotic DNA break number and distribution

    doi: 10.1038/nature13120

    Figure Lengend Snippet: Spo11-oligo complexes in msh5 ndt80 Representative time courses are shown.

    Article Snippet: Precipitated Spo11-oligo complexes were end-labeled in NEBuffer 4 (New England Biolabs) containing 3–10 μCi of [α-32 P]dCTP and terminal deoxynucleotidyl transferase (TdT) .

    Techniques:

    Analysis of recombination at three natural DSB hotspots a , b , Recombination reporters at the ERG1 ( a ) and GAT1 ( b ) hotspots. c , d , e , Representative Southern blots of parental and recombinant DNA molecules at CCT6 ( c ), ERG1 ( d ), and GAT1 ( e ). The arrowhead in e indicates a non-reproducible radiolabeled species. f , Local distribution of DSBs around recombination reporter locations is not altered in zip3 mutants. Spo11-oligo profiles (averages for wild type and zip3 ) are smoothed with 201-bp Hann window; zip3 values are offset to separate profiles.

    Journal: Nature

    Article Title: Homologue engagement controls meiotic DNA break number and distribution

    doi: 10.1038/nature13120

    Figure Lengend Snippet: Analysis of recombination at three natural DSB hotspots a , b , Recombination reporters at the ERG1 ( a ) and GAT1 ( b ) hotspots. c , d , e , Representative Southern blots of parental and recombinant DNA molecules at CCT6 ( c ), ERG1 ( d ), and GAT1 ( e ). The arrowhead in e indicates a non-reproducible radiolabeled species. f , Local distribution of DSBs around recombination reporter locations is not altered in zip3 mutants. Spo11-oligo profiles (averages for wild type and zip3 ) are smoothed with 201-bp Hann window; zip3 values are offset to separate profiles.

    Article Snippet: Precipitated Spo11-oligo complexes were end-labeled in NEBuffer 4 (New England Biolabs) containing 3–10 μCi of [α-32 P]dCTP and terminal deoxynucleotidyl transferase (TdT) .

    Techniques: Recombinant

    Model for a role of Dyn2 in autophagic breakdown of lipid droplets in hepatocytes. After initiation of starvation, lipid droplets are enclosed by the double-membrane isolation membrane (phagophore), which engulfs the droplet, forming an autophagosome. Subsequent fusion with lysosomes results in the release of hydrolytic enzymes and lysosomal lipases into the autolysosomal compartment, which degrade the lipid droplets within. During the process of autophagic lysosomal reformation (top), nascent lysosomes are generated from membrane tubule extensions of this autolysosomal compartment. Dyn2 mediates the scission of these tubular structures. Depletion of Dyn2 by siRNA knockdown or inhibition of Dyn2 activity by pharmacological reagents (bottom) prevents tubular scission, eventually resulting in depleted lysosomal pools within the cell. Continued fusion of remaining lysosomes causes enlargement of the autolysosomal structures. A complete decrease in the recycled lysosomal population will prevent autophagic-mediated breakdown of hepatic lipid droplets.

    Journal: The Journal of Cell Biology

    Article Title: Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

    doi: 10.1083/jcb.201306140

    Figure Lengend Snippet: Model for a role of Dyn2 in autophagic breakdown of lipid droplets in hepatocytes. After initiation of starvation, lipid droplets are enclosed by the double-membrane isolation membrane (phagophore), which engulfs the droplet, forming an autophagosome. Subsequent fusion with lysosomes results in the release of hydrolytic enzymes and lysosomal lipases into the autolysosomal compartment, which degrade the lipid droplets within. During the process of autophagic lysosomal reformation (top), nascent lysosomes are generated from membrane tubule extensions of this autolysosomal compartment. Dyn2 mediates the scission of these tubular structures. Depletion of Dyn2 by siRNA knockdown or inhibition of Dyn2 activity by pharmacological reagents (bottom) prevents tubular scission, eventually resulting in depleted lysosomal pools within the cell. Continued fusion of remaining lysosomes causes enlargement of the autolysosomal structures. A complete decrease in the recycled lysosomal population will prevent autophagic-mediated breakdown of hepatic lipid droplets.

    Article Snippet: The siRNA oligos targeting Dyn2 and clathrin heavy chain were purchased from Thermo Fisher Scientific and cells were transfected with the RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Isolation, Generated, Inhibition, Activity Assay

    Dyn2 knockdown disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and stained with an antibody specific for LAMP1 after a 24-h starvation period in media containing 0.1% FBS. Control cells displayed normal lysosomal compartments, with LAMP1-stained structures dispersed throughout the cytosol, some containing short reformation tubules (B′). After Dyn2 knockdown, many cells contained enlarged autolysosomes with a noticeable increase in persistent and lengthy reformation tubules emanating from LAMP1-positive compartments (C′ and D′). Bars: (A–D) 20 µM; (A′ and B′) 5 µM; (C′ and D′) 10 µM. (E and F) Measurements of the length (E) and distribution (F) of LAMP1-stained ALR tubules in control (siNT) and siDyn2-treated cells. Data represent the mean derived from measurements of tubules in a minimum of 40 cells for each experimental group over 6 independent experiments. Error bars represent SE; *, P ≤ 0.05; **, P ≤ 0.01. (G) TEM of siDyn2-treated Hep3B hepatocyte exhibiting enlarged autolysosomal structures with extensive tubulation (arrows). Bar, 2 µm. (G′) Inset showing enlargement of autolysosomal tubule (arrows). Bar, 1 µm. (H) Tubulating autolysosome with engulfed lipid droplet. Bar, 0.5 µm.

    Journal: The Journal of Cell Biology

    Article Title: Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

    doi: 10.1083/jcb.201306140

    Figure Lengend Snippet: Dyn2 knockdown disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and stained with an antibody specific for LAMP1 after a 24-h starvation period in media containing 0.1% FBS. Control cells displayed normal lysosomal compartments, with LAMP1-stained structures dispersed throughout the cytosol, some containing short reformation tubules (B′). After Dyn2 knockdown, many cells contained enlarged autolysosomes with a noticeable increase in persistent and lengthy reformation tubules emanating from LAMP1-positive compartments (C′ and D′). Bars: (A–D) 20 µM; (A′ and B′) 5 µM; (C′ and D′) 10 µM. (E and F) Measurements of the length (E) and distribution (F) of LAMP1-stained ALR tubules in control (siNT) and siDyn2-treated cells. Data represent the mean derived from measurements of tubules in a minimum of 40 cells for each experimental group over 6 independent experiments. Error bars represent SE; *, P ≤ 0.05; **, P ≤ 0.01. (G) TEM of siDyn2-treated Hep3B hepatocyte exhibiting enlarged autolysosomal structures with extensive tubulation (arrows). Bar, 2 µm. (G′) Inset showing enlargement of autolysosomal tubule (arrows). Bar, 1 µm. (H) Tubulating autolysosome with engulfed lipid droplet. Bar, 0.5 µm.

    Article Snippet: The siRNA oligos targeting Dyn2 and clathrin heavy chain were purchased from Thermo Fisher Scientific and cells were transfected with the RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Staining, Derivative Assay, Transmission Electron Microscopy

    Dyn2 inhibition leads to enlarged autolysosomal structures and prevents the autophagy of lipid droplets. Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and co-stained with antibodies specific for LAMP1 (red) and LC3 (green). After Dyn2 knockdown, a juxtanuclear aggregation and enlargement of the LAMP1-positive compartment is observed (C and D, arrows). Increased labeling of LC3 is also detectable after knockdown of Dyn2. (E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure. (F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.

    Journal: The Journal of Cell Biology

    Article Title: Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

    doi: 10.1083/jcb.201306140

    Figure Lengend Snippet: Dyn2 inhibition leads to enlarged autolysosomal structures and prevents the autophagy of lipid droplets. Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and co-stained with antibodies specific for LAMP1 (red) and LC3 (green). After Dyn2 knockdown, a juxtanuclear aggregation and enlargement of the LAMP1-positive compartment is observed (C and D, arrows). Increased labeling of LC3 is also detectable after knockdown of Dyn2. (E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure. (F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.

    Article Snippet: The siRNA oligos targeting Dyn2 and clathrin heavy chain were purchased from Thermo Fisher Scientific and cells were transfected with the RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Inhibition, Staining, Labeling, Western Blot, Quantitation Assay

    Dyn2 knockdown results in the formation of enlarged autophagic structures. (A–D) Transmission electron micrographs (TEMs) of oleate-loaded Hep3B hepatocytes treated with nontargeting control (siNT) or Dyn2 (siDyn2) siRNA for 72 h. Bars: (A and B) 2 µm; (A′ and B′) 1 µm; (C and D) 0.5 µm. Insets in A and B show fluorescent micrographs of LAMP1-stained cells (bars, 10 µM). Control cells (A) contain an abundance of small (

    Journal: The Journal of Cell Biology

    Article Title: Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

    doi: 10.1083/jcb.201306140

    Figure Lengend Snippet: Dyn2 knockdown results in the formation of enlarged autophagic structures. (A–D) Transmission electron micrographs (TEMs) of oleate-loaded Hep3B hepatocytes treated with nontargeting control (siNT) or Dyn2 (siDyn2) siRNA for 72 h. Bars: (A and B) 2 µm; (A′ and B′) 1 µm; (C and D) 0.5 µm. Insets in A and B show fluorescent micrographs of LAMP1-stained cells (bars, 10 µM). Control cells (A) contain an abundance of small (

    Article Snippet: The siRNA oligos targeting Dyn2 and clathrin heavy chain were purchased from Thermo Fisher Scientific and cells were transfected with the RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Transmission Assay, Staining

    Dyn2 knockdown interferes with starvation-induced LD breakdown in Hep3B hepatocytes. Hep3B hepatocytes were treated with nontargeting control siRNA (siNT) or siRNA targeting Dyn2 (siDyn2), followed by re-expression of either vector alone, or GFP-tagged versions of Dyn2, loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS. LDs were visualized by immunofluorescence using Oil Red O staining. Cell boundaries are outlined and those cells re-expressing GFP, GFP-wtDyn2, or GFP--K44A are denoted with asterisks. Bars, 20 µM. (B) Representative blot showing the efficiency of the Dyn2 knockdown in these cells. (C and D) Quantitation of the average LD number and area (in pixels 2 ) per cell from three independent experiments. The data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. NS, not significant.

    Journal: The Journal of Cell Biology

    Article Title: Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

    doi: 10.1083/jcb.201306140

    Figure Lengend Snippet: Dyn2 knockdown interferes with starvation-induced LD breakdown in Hep3B hepatocytes. Hep3B hepatocytes were treated with nontargeting control siRNA (siNT) or siRNA targeting Dyn2 (siDyn2), followed by re-expression of either vector alone, or GFP-tagged versions of Dyn2, loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS. LDs were visualized by immunofluorescence using Oil Red O staining. Cell boundaries are outlined and those cells re-expressing GFP, GFP-wtDyn2, or GFP--K44A are denoted with asterisks. Bars, 20 µM. (B) Representative blot showing the efficiency of the Dyn2 knockdown in these cells. (C and D) Quantitation of the average LD number and area (in pixels 2 ) per cell from three independent experiments. The data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. NS, not significant.

    Article Snippet: The siRNA oligos targeting Dyn2 and clathrin heavy chain were purchased from Thermo Fisher Scientific and cells were transfected with the RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Expressing, Plasmid Preparation, Immunofluorescence, Staining, Quantitation Assay

    The effect of GnRH-E1 RNA knockdown on Gnrh1 gene expression. (A-D) GT1-7 neurons were transfected with either negative siRNA control (Neg; black bars) or siRNA targeting both strands of the mouse GnRH-E1 RNA (siRNA; white bars). Total RNA was harvested at 36 hours, 48 hours, and 72 hours after siRNA transfection. RT-qPCR analysis was performed to quantify endogenous mouse GnRH-E1 RNA (A), transgene-derived rat (rTg) GnRH-E1 RNA (B), Gnrh1 pre-mRNA (C), and Gnrh1 mRNA (D) expression. Relative RNA expression is normalized to control histone 2A.Z ( H2afz ) mRNA expression at each time point. Data are displayed as the mean ± SD, where statistical significance was determined by Student’s t-test compared between negative control and siRNA treatment at each time point. Asterisk indicates statistical significance, where p

    Journal: PLoS ONE

    Article Title: A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models

    doi: 10.1371/journal.pone.0158597

    Figure Lengend Snippet: The effect of GnRH-E1 RNA knockdown on Gnrh1 gene expression. (A-D) GT1-7 neurons were transfected with either negative siRNA control (Neg; black bars) or siRNA targeting both strands of the mouse GnRH-E1 RNA (siRNA; white bars). Total RNA was harvested at 36 hours, 48 hours, and 72 hours after siRNA transfection. RT-qPCR analysis was performed to quantify endogenous mouse GnRH-E1 RNA (A), transgene-derived rat (rTg) GnRH-E1 RNA (B), Gnrh1 pre-mRNA (C), and Gnrh1 mRNA (D) expression. Relative RNA expression is normalized to control histone 2A.Z ( H2afz ) mRNA expression at each time point. Data are displayed as the mean ± SD, where statistical significance was determined by Student’s t-test compared between negative control and siRNA treatment at each time point. Asterisk indicates statistical significance, where p

    Article Snippet: Negative siRNA control targeting luciferase mRNA and custom-designed siRNA duplex oligos targeting GnRH-E1 RNA (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA) and Lipofectamine RNAiMax transfection reagent (Thermo Fisher Scientific, Carlsbad, CA) were prepared according to the manufacturer’s recommendations, in Gibco OptiMEM (Thermo Fisher Scientific, Carlsbad, CA) at a concentration of 10 μM. siRNA was transfected into GT1-7 cells at a concentration of 900 pmol per well.

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Derivative Assay, RNA Expression, Negative Control

    GnRH-E1 RNA is localized in the GT1-7 neuron nucleus. (A) Schematic diagram of the conserved regulatory elements upstream of the mouse Gnrh1 TSS, which contains enhancers 1, 2, and 3 (E3, E2, E1, respectively), the promoter (P), and the Gnrh1 gene with four exons (white boxes). Coordinates above the regulatory elements indicate positions with respect to the Gnrh1 TSS. RT-PCR primers used in B-D are indicated by arrows, and expected PCR products are represented by a connecting line. Positions of PCR primers are aligned to the mouse conserved regulatory region diagrammed above. Nuclear and cytoplasmic extracts from GT1-7 neurons were analyzed for GnRH-E1 RNA (B), Gnrh1 pre-mRNA (C), Gnrh1 mRNA (D), and H2afz mRNA control (E) by RT-PCR. RT-PCR analysis was performed on random hexamer-primed cDNA, where cDNA synthesized with (+) and without (-) reverse transcriptase were analyzed in parallel. PCR loading controls are plasmid containing the -3568/-1128 bp segment upstream of the Gnrh1 TSS and no-template control (NTC). The sizes of the PCR amplicons were marked by a 100 bp DNA ladder or a 1 kbp DNA ladder where indicated, that were resolved on the agarose gel in parallel.

    Journal: PLoS ONE

    Article Title: A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models

    doi: 10.1371/journal.pone.0158597

    Figure Lengend Snippet: GnRH-E1 RNA is localized in the GT1-7 neuron nucleus. (A) Schematic diagram of the conserved regulatory elements upstream of the mouse Gnrh1 TSS, which contains enhancers 1, 2, and 3 (E3, E2, E1, respectively), the promoter (P), and the Gnrh1 gene with four exons (white boxes). Coordinates above the regulatory elements indicate positions with respect to the Gnrh1 TSS. RT-PCR primers used in B-D are indicated by arrows, and expected PCR products are represented by a connecting line. Positions of PCR primers are aligned to the mouse conserved regulatory region diagrammed above. Nuclear and cytoplasmic extracts from GT1-7 neurons were analyzed for GnRH-E1 RNA (B), Gnrh1 pre-mRNA (C), Gnrh1 mRNA (D), and H2afz mRNA control (E) by RT-PCR. RT-PCR analysis was performed on random hexamer-primed cDNA, where cDNA synthesized with (+) and without (-) reverse transcriptase were analyzed in parallel. PCR loading controls are plasmid containing the -3568/-1128 bp segment upstream of the Gnrh1 TSS and no-template control (NTC). The sizes of the PCR amplicons were marked by a 100 bp DNA ladder or a 1 kbp DNA ladder where indicated, that were resolved on the agarose gel in parallel.

    Article Snippet: Negative siRNA control targeting luciferase mRNA and custom-designed siRNA duplex oligos targeting GnRH-E1 RNA (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA) and Lipofectamine RNAiMax transfection reagent (Thermo Fisher Scientific, Carlsbad, CA) were prepared according to the manufacturer’s recommendations, in Gibco OptiMEM (Thermo Fisher Scientific, Carlsbad, CA) at a concentration of 10 μM. siRNA was transfected into GT1-7 cells at a concentration of 900 pmol per well.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Random Hexamer Labeling, Synthesized, Plasmid Preparation, Agarose Gel Electrophoresis

    Strand-specific cDNA and RT-PCR analysis of the mouse GnRH-E1 RNA variants. (A) Schematic diagram of the sense GnRH-E1 RNA variant, with a 3’ polyA site located downstream of GnRH-E1 as predicted by RACE. PCR primers in reverse direction at -1128 bp or -1271 bp (reverse arrows) were used for strand-specific cDNA synthesis to capture the sense GnRH-E1 RNA. PCR analysis was performed using -1271 bp or -1128 bp reverse primer paired with the forward primers at -3560 bp, -3606 bp, -3779 bp, and -3746 bp (forward arrows) from the mouse Gnrh1 TSS. Primer positions are aligned to the mouse conserved regulatory elements and coordinates diagrammed above. (B) Strand-specific cDNA synthesized using the -1271 bp reverse primer was subject to PCR analysis using the following primer pairs: -3560F/-1271R, -3606F/-1271R, -3779F/-1271R, -3746F/-1271R. Strand-specific cDNA synthesized using -1128 bp reverse primer was subject to PCR analysis using the primer pair -3560F/-1128R. (C) Schematic diagram of the mouse antisense GnRH-E1 RNA variant, with a 3’ polyA site predicted upstream of GnRH-E2 by RACE. PCR primers in the forward direction at -3560 bp or -3371 bp (forward arrows) was used for strand-specific cDNA synthesis to capture the antisense GnRH-E1 RNA variant. PCR analysis was performed using -3371 bp or -3560 bp forward primer paired with the reverse primers at -1443 bp, -1271 bp, -1128 bp from the Gnrh1 TSS. D) Strand-specific cDNA synthesized using the -3371 bp forward primer was subject to PCR analysis using the following PCR primer pairs -3371F/-1443R, -3371F/-1271R, and -3371F/-1128R. Strand-specific cDNA synthesized using the -3560 bp forward primer was subject to PCR analysis using the primer pair at -3560F/-1443R. All reverse transcription reactions were performed on total RNA samples with (+) and without (-) reverse transcriptase and were amplified by PCR in parallel with GT1-7 genomic DNA control and no-template water control (NTC). The size of PCR amplicons was marked by 1 kbp DNA ladder.

    Journal: PLoS ONE

    Article Title: A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models

    doi: 10.1371/journal.pone.0158597

    Figure Lengend Snippet: Strand-specific cDNA and RT-PCR analysis of the mouse GnRH-E1 RNA variants. (A) Schematic diagram of the sense GnRH-E1 RNA variant, with a 3’ polyA site located downstream of GnRH-E1 as predicted by RACE. PCR primers in reverse direction at -1128 bp or -1271 bp (reverse arrows) were used for strand-specific cDNA synthesis to capture the sense GnRH-E1 RNA. PCR analysis was performed using -1271 bp or -1128 bp reverse primer paired with the forward primers at -3560 bp, -3606 bp, -3779 bp, and -3746 bp (forward arrows) from the mouse Gnrh1 TSS. Primer positions are aligned to the mouse conserved regulatory elements and coordinates diagrammed above. (B) Strand-specific cDNA synthesized using the -1271 bp reverse primer was subject to PCR analysis using the following primer pairs: -3560F/-1271R, -3606F/-1271R, -3779F/-1271R, -3746F/-1271R. Strand-specific cDNA synthesized using -1128 bp reverse primer was subject to PCR analysis using the primer pair -3560F/-1128R. (C) Schematic diagram of the mouse antisense GnRH-E1 RNA variant, with a 3’ polyA site predicted upstream of GnRH-E2 by RACE. PCR primers in the forward direction at -3560 bp or -3371 bp (forward arrows) was used for strand-specific cDNA synthesis to capture the antisense GnRH-E1 RNA variant. PCR analysis was performed using -3371 bp or -3560 bp forward primer paired with the reverse primers at -1443 bp, -1271 bp, -1128 bp from the Gnrh1 TSS. D) Strand-specific cDNA synthesized using the -3371 bp forward primer was subject to PCR analysis using the following PCR primer pairs -3371F/-1443R, -3371F/-1271R, and -3371F/-1128R. Strand-specific cDNA synthesized using the -3560 bp forward primer was subject to PCR analysis using the primer pair at -3560F/-1443R. All reverse transcription reactions were performed on total RNA samples with (+) and without (-) reverse transcriptase and were amplified by PCR in parallel with GT1-7 genomic DNA control and no-template water control (NTC). The size of PCR amplicons was marked by 1 kbp DNA ladder.

    Article Snippet: Negative siRNA control targeting luciferase mRNA and custom-designed siRNA duplex oligos targeting GnRH-E1 RNA (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA) and Lipofectamine RNAiMax transfection reagent (Thermo Fisher Scientific, Carlsbad, CA) were prepared according to the manufacturer’s recommendations, in Gibco OptiMEM (Thermo Fisher Scientific, Carlsbad, CA) at a concentration of 10 μM. siRNA was transfected into GT1-7 cells at a concentration of 900 pmol per well.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Variant Assay, Polymerase Chain Reaction, Synthesized, Amplification

    RT-qPCR analysis of rat and mouse GnRH-E1 RNA expression in cell lines. (A) A schematic diagram of conserved regulatory elements upstream of the mouse Gnrh1 transcription start site (TSS, curved arrow) and Gnrh1 gene coding region. The Gnrh1 gene regulatory region contains Gnrh1 enhancers 1, 2, and 3 (E3, E2, E1, respectively), the promoter (P), and the Gnrh1 gene consists of four exons (white boxes). PCR primers used in Fig 2C are indicated by arrows, and the predicted PCR product mouse GnRH-E1 RNA is represented by a connecting line. (B) Schematic diagram of transgene embedded in GT1-7 neurons carrying the 3’ portion of the rat GnRH-E2, GnRH-E1, GnRH-P, and the Gnrh1 TSS, driving the SV40 T-antigen oncogene. PCR primers used in Fig 2C are indicated by arrows, and the predicted PCR product rat GnRH-E1 RNA from the transgene is represented by a connecting line. (C) RT-qPCR analysis of endogenous mouse GnRH-E1 RNA (black) and GnRH-E1 RNA expressed from the rat Gnrh1 promoter transgene (white) in GT1-7, GN11, and NIH3T3 cells. Relative GnRH-E1 RNA expression is normalized to peptidylprolyl isomerase A ( Ppia ) mRNA control. Data are displayed as means ± SD. Asterisk indicates statistical significance by Student’s T-test on the comparison between mouse and rat GnRH-E1 RNA, where p

    Journal: PLoS ONE

    Article Title: A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models

    doi: 10.1371/journal.pone.0158597

    Figure Lengend Snippet: RT-qPCR analysis of rat and mouse GnRH-E1 RNA expression in cell lines. (A) A schematic diagram of conserved regulatory elements upstream of the mouse Gnrh1 transcription start site (TSS, curved arrow) and Gnrh1 gene coding region. The Gnrh1 gene regulatory region contains Gnrh1 enhancers 1, 2, and 3 (E3, E2, E1, respectively), the promoter (P), and the Gnrh1 gene consists of four exons (white boxes). PCR primers used in Fig 2C are indicated by arrows, and the predicted PCR product mouse GnRH-E1 RNA is represented by a connecting line. (B) Schematic diagram of transgene embedded in GT1-7 neurons carrying the 3’ portion of the rat GnRH-E2, GnRH-E1, GnRH-P, and the Gnrh1 TSS, driving the SV40 T-antigen oncogene. PCR primers used in Fig 2C are indicated by arrows, and the predicted PCR product rat GnRH-E1 RNA from the transgene is represented by a connecting line. (C) RT-qPCR analysis of endogenous mouse GnRH-E1 RNA (black) and GnRH-E1 RNA expressed from the rat Gnrh1 promoter transgene (white) in GT1-7, GN11, and NIH3T3 cells. Relative GnRH-E1 RNA expression is normalized to peptidylprolyl isomerase A ( Ppia ) mRNA control. Data are displayed as means ± SD. Asterisk indicates statistical significance by Student’s T-test on the comparison between mouse and rat GnRH-E1 RNA, where p

    Article Snippet: Negative siRNA control targeting luciferase mRNA and custom-designed siRNA duplex oligos targeting GnRH-E1 RNA (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA) and Lipofectamine RNAiMax transfection reagent (Thermo Fisher Scientific, Carlsbad, CA) were prepared according to the manufacturer’s recommendations, in Gibco OptiMEM (Thermo Fisher Scientific, Carlsbad, CA) at a concentration of 10 μM. siRNA was transfected into GT1-7 cells at a concentration of 900 pmol per well.

    Techniques: Quantitative RT-PCR, RNA Expression, Polymerase Chain Reaction

    Increased reduction of MM-AN melanoma xenograft tumors by TOP complex in nu/nu mice. First, 2.5×10 6 MM-AN cells were injected into the flank of nu/nu mice (n=10). After 24 hours, the mice were treated daily with diluent, T-oligo (52 nmol), TOP complex (52 nmol T-oligo and 0.025 mg/mL PVBLG) intravenously for 3 weeks. Mice treated with T-oligo demonstrated reduced tumors, while most TOP complex treated mice showed no visible tumors. The graph shows a 3-fold and 9-fold reduction in tumor volume after treatment with T-oligo or the TOP complex, respectively, as compared with diluent ( P ,0.001). Abbreviations: TOP complex, T-oligo and PVBLG nanocomplex.

    Journal: International Journal of Nanomedicine

    Article Title: Novel delivery system for T-oligo using a nanocomplex formed with an alpha helical peptide for melanoma therapy

    doi: 10.2147/IJN.S55133

    Figure Lengend Snippet: Increased reduction of MM-AN melanoma xenograft tumors by TOP complex in nu/nu mice. First, 2.5×10 6 MM-AN cells were injected into the flank of nu/nu mice (n=10). After 24 hours, the mice were treated daily with diluent, T-oligo (52 nmol), TOP complex (52 nmol T-oligo and 0.025 mg/mL PVBLG) intravenously for 3 weeks. Mice treated with T-oligo demonstrated reduced tumors, while most TOP complex treated mice showed no visible tumors. The graph shows a 3-fold and 9-fold reduction in tumor volume after treatment with T-oligo or the TOP complex, respectively, as compared with diluent ( P ,0.001). Abbreviations: TOP complex, T-oligo and PVBLG nanocomplex.

    Article Snippet: T-oligo and PVBLG solutions were prepared separately in DNAse/RNAse-free distilled water (Invitrogen, Grand Island, NY, USA) and complexed at various weight ratios.

    Techniques: Mouse Assay, Injection

    Particle size analysis of the TOP complex. T-oligo and PVBLG solutions were prepared at a DNA to polymer weight ratio of 15:1 in DNAse/RNAse-free distilled water. T-oligo and PVBLG solutions were then mixed and incubated at room temperature for 20 minutes. The particle size was analyzed by dynamic light scattering. The results obtained are shown in NICOMP (dynamic light scattering) distribution, indicating that the TOP complex had a mean diameter of 147.3 nm. PVBLG or T-oligo alone was used as the control. Abbreviation: TOP complex, T-oligo-PVBLG nanocomplex.

    Journal: International Journal of Nanomedicine

    Article Title: Novel delivery system for T-oligo using a nanocomplex formed with an alpha helical peptide for melanoma therapy

    doi: 10.2147/IJN.S55133

    Figure Lengend Snippet: Particle size analysis of the TOP complex. T-oligo and PVBLG solutions were prepared at a DNA to polymer weight ratio of 15:1 in DNAse/RNAse-free distilled water. T-oligo and PVBLG solutions were then mixed and incubated at room temperature for 20 minutes. The particle size was analyzed by dynamic light scattering. The results obtained are shown in NICOMP (dynamic light scattering) distribution, indicating that the TOP complex had a mean diameter of 147.3 nm. PVBLG or T-oligo alone was used as the control. Abbreviation: TOP complex, T-oligo-PVBLG nanocomplex.

    Article Snippet: T-oligo and PVBLG solutions were prepared separately in DNAse/RNAse-free distilled water (Invitrogen, Grand Island, NY, USA) and complexed at various weight ratios.

    Techniques: Particle Size Analysis, Incubation

    E nhanced antitumor effects of the TOP complex in xenograft tumors of MM-AN melanoma cells. First, 2.5×10 6 MM-AN cells were injected into the flank of each immunodeficient mouse (n=10). After 24 hours, the mice were treated daily with diluent, T-oligo (52 nmol), or TOP complex (52 nmol T-oligo and 0.025 mg/mL PVBLG) intravenously for 3 weeks. Xenograft tumors were resected on day 21 and subjected to immunohistochemistry for VEGF and TSP-1. Apoptosis was evaluated by TUNEL assay, which was increased in tumors treated with the TOP complex compared with T-oligo. Immunohistochemical staining showed enhanced expression of TSP-1 and decreased expression of VEGF in mice treated with the TOP complex as compared with T-oligo alone. Abbreviations: C, complement ary oligonucleotide (C-oligo); T, T-oligo; T + P, TOP complex; TOP complex, T-oligo and PVBLG nanocomplex; TSP-1, thrombospondin 1; VEGF, vascular endothelial growth factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

    Journal: International Journal of Nanomedicine

    Article Title: Novel delivery system for T-oligo using a nanocomplex formed with an alpha helical peptide for melanoma therapy

    doi: 10.2147/IJN.S55133

    Figure Lengend Snippet: E nhanced antitumor effects of the TOP complex in xenograft tumors of MM-AN melanoma cells. First, 2.5×10 6 MM-AN cells were injected into the flank of each immunodeficient mouse (n=10). After 24 hours, the mice were treated daily with diluent, T-oligo (52 nmol), or TOP complex (52 nmol T-oligo and 0.025 mg/mL PVBLG) intravenously for 3 weeks. Xenograft tumors were resected on day 21 and subjected to immunohistochemistry for VEGF and TSP-1. Apoptosis was evaluated by TUNEL assay, which was increased in tumors treated with the TOP complex compared with T-oligo. Immunohistochemical staining showed enhanced expression of TSP-1 and decreased expression of VEGF in mice treated with the TOP complex as compared with T-oligo alone. Abbreviations: C, complement ary oligonucleotide (C-oligo); T, T-oligo; T + P, TOP complex; TOP complex, T-oligo and PVBLG nanocomplex; TSP-1, thrombospondin 1; VEGF, vascular endothelial growth factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

    Article Snippet: T-oligo and PVBLG solutions were prepared separately in DNAse/RNAse-free distilled water (Invitrogen, Grand Island, NY, USA) and complexed at various weight ratios.

    Techniques: Injection, Mouse Assay, Immunohistochemistry, TUNEL Assay, Staining, Expressing

    Uptake studies of T-oligo and TOP complex in melanoma cells using fluorescence-activated cell sorting analysis and fluorescence microscopy. ( A ) 1.5×10 5 MM-AN cells in Minimum Essential Medium were plated in 35 mm 2 cell culture dishes. After 24 hours, the cells were treated with 40 μM FITC-T-oligo or the TOP complex (40 μM FITC-T-oligo and 0.025 mg/mL PVBLG) in Minimum Essential Medium with 2% calf serum for 4 hours. Cells were then washed twice with Hank’s Balanced Salt Solution, trypsinized, pelleted, counted, and fixed with 4% paraformaldehyde and analyzed on a Becton Dickinson fluorescent-activated cell sorter. Data analysis was performed using Cell Quest software. ( B ) 10,000 MM-AN cells were plated in chamber slides, treated with T-oligo or the TOP complex as described above, and observed under a fluorescent microscope (Olympus IX71) at 200× magnification. Abbreviations: C-oligo, complementary oligonucleotide; TOP complex, T-oligo and PVBLG nanocomplex; FITC, fluorescein isothiocyanate; FL1-H, fluorescence 1 signal height.

    Journal: International Journal of Nanomedicine

    Article Title: Novel delivery system for T-oligo using a nanocomplex formed with an alpha helical peptide for melanoma therapy

    doi: 10.2147/IJN.S55133

    Figure Lengend Snippet: Uptake studies of T-oligo and TOP complex in melanoma cells using fluorescence-activated cell sorting analysis and fluorescence microscopy. ( A ) 1.5×10 5 MM-AN cells in Minimum Essential Medium were plated in 35 mm 2 cell culture dishes. After 24 hours, the cells were treated with 40 μM FITC-T-oligo or the TOP complex (40 μM FITC-T-oligo and 0.025 mg/mL PVBLG) in Minimum Essential Medium with 2% calf serum for 4 hours. Cells were then washed twice with Hank’s Balanced Salt Solution, trypsinized, pelleted, counted, and fixed with 4% paraformaldehyde and analyzed on a Becton Dickinson fluorescent-activated cell sorter. Data analysis was performed using Cell Quest software. ( B ) 10,000 MM-AN cells were plated in chamber slides, treated with T-oligo or the TOP complex as described above, and observed under a fluorescent microscope (Olympus IX71) at 200× magnification. Abbreviations: C-oligo, complementary oligonucleotide; TOP complex, T-oligo and PVBLG nanocomplex; FITC, fluorescein isothiocyanate; FL1-H, fluorescence 1 signal height.

    Article Snippet: T-oligo and PVBLG solutions were prepared separately in DNAse/RNAse-free distilled water (Invitrogen, Grand Island, NY, USA) and complexed at various weight ratios.

    Techniques: Fluorescence, FACS, Microscopy, Cell Culture, Software

    E ffect of T-oligo with or without PVBLG on growth and viability of MM-AN cells. First, 5,000 MM-AN cells/well were plated (n=6) in Minimum Essential Medium supplemented with 10% fetal bovine serum. After 24 hours, the cells were treated for 96 hours with 20 μM T-oligo with or without PVBLG at concentrations found to be nontoxic (0.025–0.05 mg/mL). Cell viability was determined using the MTT colorimetric dye reduction assay and percent cell viability was determined relative to the control. T-oligo inhibited growth by 51% while T-oligo with PVBLG (0.025–0.05 mg/mL) inhibited growth by 78%–89%. PVBLG alone had no effect at the tested concentrations. * P ,0.001. Abbreviations: D, diluent; P, PVBLG; T, T-oligo; TOP complex, T-oligo and PVBLG nanocomplex.

    Journal: International Journal of Nanomedicine

    Article Title: Novel delivery system for T-oligo using a nanocomplex formed with an alpha helical peptide for melanoma therapy

    doi: 10.2147/IJN.S55133

    Figure Lengend Snippet: E ffect of T-oligo with or without PVBLG on growth and viability of MM-AN cells. First, 5,000 MM-AN cells/well were plated (n=6) in Minimum Essential Medium supplemented with 10% fetal bovine serum. After 24 hours, the cells were treated for 96 hours with 20 μM T-oligo with or without PVBLG at concentrations found to be nontoxic (0.025–0.05 mg/mL). Cell viability was determined using the MTT colorimetric dye reduction assay and percent cell viability was determined relative to the control. T-oligo inhibited growth by 51% while T-oligo with PVBLG (0.025–0.05 mg/mL) inhibited growth by 78%–89%. PVBLG alone had no effect at the tested concentrations. * P ,0.001. Abbreviations: D, diluent; P, PVBLG; T, T-oligo; TOP complex, T-oligo and PVBLG nanocomplex.

    Article Snippet: T-oligo and PVBLG solutions were prepared separately in DNAse/RNAse-free distilled water (Invitrogen, Grand Island, NY, USA) and complexed at various weight ratios.

    Techniques: MTT Assay

    Detection of a homozygous deletion by the oligo array CGH platform. DNA from the cell lines MDA-MB-468 ( A ) and SUM159 ( C ) was hybridized with normal male reference DNA on a human oligo array. Log2ratios were calculated without moving average and are displayed in black as a function of their position on chromosome 13. The smoothed values of the log2ratios are displayed in red and the position of the RB1 oligo is indicated by the green circle in A and the arrow in C. Note the lack of the deletion in SUM159. Validation of the HD by FISH analysis in cell lines: MDA-MB-468 ( B ) and SUM159 ( D ). The green signal from the RB1 probe clearly shows the presence of RB1 in SUM159 (D), but is absent from MDA-MB-468 (B). Chromosome 13 paint (red) shows the presence of three normal copies of chromosome 13 and two marker chromosomes with chromosome 13 material.

    Journal: Nucleic Acids Research

    Article Title: Human and mouse oligonucleotide-based array CGH

    doi: 10.1093/nar/gni191

    Figure Lengend Snippet: Detection of a homozygous deletion by the oligo array CGH platform. DNA from the cell lines MDA-MB-468 ( A ) and SUM159 ( C ) was hybridized with normal male reference DNA on a human oligo array. Log2ratios were calculated without moving average and are displayed in black as a function of their position on chromosome 13. The smoothed values of the log2ratios are displayed in red and the position of the RB1 oligo is indicated by the green circle in A and the arrow in C. Note the lack of the deletion in SUM159. Validation of the HD by FISH analysis in cell lines: MDA-MB-468 ( B ) and SUM159 ( D ). The green signal from the RB1 probe clearly shows the presence of RB1 in SUM159 (D), but is absent from MDA-MB-468 (B). Chromosome 13 paint (red) shows the presence of three normal copies of chromosome 13 and two marker chromosomes with chromosome 13 material.

    Article Snippet: Although tiling resolution BAC arrays have been developed ( ) and now several commercial oligo array CGH platforms are available (Agilent, Nimblegen), we have shown here that in-house spotting of commercially available oligonucleotide libraries can be a cost-effective way to produce high quality, high resolution results.

    Techniques: Multiple Displacement Amplification, Fluorescence In Situ Hybridization, Marker

    Detection of a heterozygous deletion by the oligo array CGH platform. ( A ) DNA from the cell line SKBR7 was hybridized with normal male reference DNA on a human oligo array. Log2ratios were calculated without moving average and are displayed in black as a function of their position on chromosome 12. The smoothed values of the log2ratios are displayed in red. ( B ) Validation of the heterozygous deletion on 12q24 by FISH analysis of cell line SKBR7. Chromosome 12 paint (dark blue) shows two copies of seemingly normal chromosome 12. FISH using 3 BACs, RP11-340F14 (red), RP11-44F24 (green) and RP11-7M8 (aqua blue) confirmed the interstitial deletion on one copy of chromosome 12 (arrow). Overlapping FISH signals from different BACs show up in white.

    Journal: Nucleic Acids Research

    Article Title: Human and mouse oligonucleotide-based array CGH

    doi: 10.1093/nar/gni191

    Figure Lengend Snippet: Detection of a heterozygous deletion by the oligo array CGH platform. ( A ) DNA from the cell line SKBR7 was hybridized with normal male reference DNA on a human oligo array. Log2ratios were calculated without moving average and are displayed in black as a function of their position on chromosome 12. The smoothed values of the log2ratios are displayed in red. ( B ) Validation of the heterozygous deletion on 12q24 by FISH analysis of cell line SKBR7. Chromosome 12 paint (dark blue) shows two copies of seemingly normal chromosome 12. FISH using 3 BACs, RP11-340F14 (red), RP11-44F24 (green) and RP11-7M8 (aqua blue) confirmed the interstitial deletion on one copy of chromosome 12 (arrow). Overlapping FISH signals from different BACs show up in white.

    Article Snippet: Although tiling resolution BAC arrays have been developed ( ) and now several commercial oligo array CGH platforms are available (Agilent, Nimblegen), we have shown here that in-house spotting of commercially available oligonucleotide libraries can be a cost-effective way to produce high quality, high resolution results.

    Techniques: Fluorescence In Situ Hybridization

    Performance of the oligo array CGH platform. ( A ) DNA from the cell line GM01750 was hybridized against normal female reference DNA. Copy number changes were detected using the smoothing algorithm ( 14 ). Blue: median values and SDs (error bars) were calculated for the areas with different copy numbers and are displayed as a function of the theoretical log2ratio. The different areas were the X-chromosome (theoretical ratio = 1/2, log2ratio = −1), chromosomes 1–8, 10–13 and 15–22 (theoretical ratio = 2/2, log2ratio = 0) and the gain in chromosome 9 (theoretical ratio = 3/2, log2ratio = 0.58). The correlation coefficient is 0.98 and the slope is 0.38. Red: same values calculated after applying a moving average of 3 to the data. Note that the red error bars do not overlap. ( B ) Detailed profile of chromosome 9 of the GM01750 hybridization. Log2ratios were calculated without moving average and are displayed in black as a function of their position on chromosome 9. Smoothed values of the log2ratios (red).

    Journal: Nucleic Acids Research

    Article Title: Human and mouse oligonucleotide-based array CGH

    doi: 10.1093/nar/gni191

    Figure Lengend Snippet: Performance of the oligo array CGH platform. ( A ) DNA from the cell line GM01750 was hybridized against normal female reference DNA. Copy number changes were detected using the smoothing algorithm ( 14 ). Blue: median values and SDs (error bars) were calculated for the areas with different copy numbers and are displayed as a function of the theoretical log2ratio. The different areas were the X-chromosome (theoretical ratio = 1/2, log2ratio = −1), chromosomes 1–8, 10–13 and 15–22 (theoretical ratio = 2/2, log2ratio = 0) and the gain in chromosome 9 (theoretical ratio = 3/2, log2ratio = 0.58). The correlation coefficient is 0.98 and the slope is 0.38. Red: same values calculated after applying a moving average of 3 to the data. Note that the red error bars do not overlap. ( B ) Detailed profile of chromosome 9 of the GM01750 hybridization. Log2ratios were calculated without moving average and are displayed in black as a function of their position on chromosome 9. Smoothed values of the log2ratios (red).

    Article Snippet: Although tiling resolution BAC arrays have been developed ( ) and now several commercial oligo array CGH platforms are available (Agilent, Nimblegen), we have shown here that in-house spotting of commercially available oligonucleotide libraries can be a cost-effective way to produce high quality, high resolution results.

    Techniques: Hybridization

    Validation of the mouse oligo array CGH platform. DNA from a mouse tumour was hybridized with normal mouse reference DNA on a 21 K mouse oligo array ( A ). Log2ratios were calculated with a weighted moving average as described ( 11 ) using a window of 250 kb and are displayed as a function of their position in the genome. The same mouse tumour and reference DNA was hybridized in a paired fluor-reversed experiment (dye-swap) to a 1 Mb mouse BAC array ( B ). Log2ratios were calculated as described ( 15 ) and displayed as a function of their position in the genome. Log2ratios of the odd and even chromosomes are shown in aqua blue and black, respectively. Smoothed values of the log2ratios (red).

    Journal: Nucleic Acids Research

    Article Title: Human and mouse oligonucleotide-based array CGH

    doi: 10.1093/nar/gni191

    Figure Lengend Snippet: Validation of the mouse oligo array CGH platform. DNA from a mouse tumour was hybridized with normal mouse reference DNA on a 21 K mouse oligo array ( A ). Log2ratios were calculated with a weighted moving average as described ( 11 ) using a window of 250 kb and are displayed as a function of their position in the genome. The same mouse tumour and reference DNA was hybridized in a paired fluor-reversed experiment (dye-swap) to a 1 Mb mouse BAC array ( B ). Log2ratios were calculated as described ( 15 ) and displayed as a function of their position in the genome. Log2ratios of the odd and even chromosomes are shown in aqua blue and black, respectively. Smoothed values of the log2ratios (red).

    Article Snippet: Although tiling resolution BAC arrays have been developed ( ) and now several commercial oligo array CGH platforms are available (Agilent, Nimblegen), we have shown here that in-house spotting of commercially available oligonucleotide libraries can be a cost-effective way to produce high quality, high resolution results.

    Techniques: BAC Assay

    Reproducibility of the oligo array CGH platform. BT474 DNA was hybridized four times against normal human male reference DNA on four different days and on three different batches of 29 K human oligo arrays. Log2ratios were calculated without moving average and are displayed in different colours as indicated for each experiment as a function of their position on chromosome 2.

    Journal: Nucleic Acids Research

    Article Title: Human and mouse oligonucleotide-based array CGH

    doi: 10.1093/nar/gni191

    Figure Lengend Snippet: Reproducibility of the oligo array CGH platform. BT474 DNA was hybridized four times against normal human male reference DNA on four different days and on three different batches of 29 K human oligo arrays. Log2ratios were calculated without moving average and are displayed in different colours as indicated for each experiment as a function of their position on chromosome 2.

    Article Snippet: Although tiling resolution BAC arrays have been developed ( ) and now several commercial oligo array CGH platforms are available (Agilent, Nimblegen), we have shown here that in-house spotting of commercially available oligonucleotide libraries can be a cost-effective way to produce high quality, high resolution results.

    Techniques:

    Capability of the oligo array CGH platform to detect and map chromosomal aberrations. Genome-wide profiles are shown that were obtained from hybridization of BT474 DNA ( A ) with human male reference DNA or ( B ) normal male with normal female DNA on a 29 K human oligonucleotide array. Log2ratios were calculated with a weighted moving average as described ( 11 ) using a window of 250 kb and are displayed as a function of their position in the genome. Log2ratios of the odd and even chromosomes are shown in aqua blue and black, respectively. Chromosome numbers are indicated. Smoothed values of the log2ratios were calculated using a dedicated smoothing algorithm ( 14 ) (red). Note the many breakpoints, gains, losses and amplifications in the BT474 profile and the lack of those in the male–female profile. Detailed profiles of chromosome 17 for the BT474 ( C ) and male–female ( D ) hybridizations. Log2ratios were calculated without moving average and are displayed in black as a function of their position on chromosome 17. Smoothed values of the log2ratios (red). The arrow in C indicates a fifth amplification in the BT474 profile on chromosome 17 that was not observed in a 1 Mb BAC array ( 3 ).

    Journal: Nucleic Acids Research

    Article Title: Human and mouse oligonucleotide-based array CGH

    doi: 10.1093/nar/gni191

    Figure Lengend Snippet: Capability of the oligo array CGH platform to detect and map chromosomal aberrations. Genome-wide profiles are shown that were obtained from hybridization of BT474 DNA ( A ) with human male reference DNA or ( B ) normal male with normal female DNA on a 29 K human oligonucleotide array. Log2ratios were calculated with a weighted moving average as described ( 11 ) using a window of 250 kb and are displayed as a function of their position in the genome. Log2ratios of the odd and even chromosomes are shown in aqua blue and black, respectively. Chromosome numbers are indicated. Smoothed values of the log2ratios were calculated using a dedicated smoothing algorithm ( 14 ) (red). Note the many breakpoints, gains, losses and amplifications in the BT474 profile and the lack of those in the male–female profile. Detailed profiles of chromosome 17 for the BT474 ( C ) and male–female ( D ) hybridizations. Log2ratios were calculated without moving average and are displayed in black as a function of their position on chromosome 17. Smoothed values of the log2ratios (red). The arrow in C indicates a fifth amplification in the BT474 profile on chromosome 17 that was not observed in a 1 Mb BAC array ( 3 ).

    Article Snippet: Although tiling resolution BAC arrays have been developed ( ) and now several commercial oligo array CGH platforms are available (Agilent, Nimblegen), we have shown here that in-house spotting of commercially available oligonucleotide libraries can be a cost-effective way to produce high quality, high resolution results.

    Techniques: Genome Wide, Hybridization, Amplification, BAC Assay

    Validation of the oligo array CGH platform with DNA obtained from FFPE tissue. DNA from an FFPE gastric tumour was hybridized with normal human reference DNA on a human oligo array ( A ) and a 1 Mb human BAC array ( B ). Log2ratios were calculated without moving average and are displayed for chromosomes 19–21 as a function of their position on the genome. Log2ratios of the odd and even chromosomes are shown in aqua blue and black, respectively. Chromosome numbers are indicated. Smoothed values of the log2ratios (red).

    Journal: Nucleic Acids Research

    Article Title: Human and mouse oligonucleotide-based array CGH

    doi: 10.1093/nar/gni191

    Figure Lengend Snippet: Validation of the oligo array CGH platform with DNA obtained from FFPE tissue. DNA from an FFPE gastric tumour was hybridized with normal human reference DNA on a human oligo array ( A ) and a 1 Mb human BAC array ( B ). Log2ratios were calculated without moving average and are displayed for chromosomes 19–21 as a function of their position on the genome. Log2ratios of the odd and even chromosomes are shown in aqua blue and black, respectively. Chromosome numbers are indicated. Smoothed values of the log2ratios (red).

    Article Snippet: Although tiling resolution BAC arrays have been developed ( ) and now several commercial oligo array CGH platforms are available (Agilent, Nimblegen), we have shown here that in-house spotting of commercially available oligonucleotide libraries can be a cost-effective way to produce high quality, high resolution results.

    Techniques: Formalin-fixed Paraffin-Embedded, BAC Assay

    Cofilin is required for the translocation of CIN at the cell leading edge. ( A , Left ) Depletion of cofilin in MTLn3 cells. MTLn3 cells were transfected with shRNA constructs, stimulated with EGF, and stained for endogenous cofilin (green in merge) and

    Journal:

    Article Title: Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

    doi: 10.1073/pnas.1510945112

    Figure Lengend Snippet: Cofilin is required for the translocation of CIN at the cell leading edge. ( A , Left ) Depletion of cofilin in MTLn3 cells. MTLn3 cells were transfected with shRNA constructs, stimulated with EGF, and stained for endogenous cofilin (green in merge) and

    Article Snippet: Two CIN-directed siRNA oligos duplexes were purchased from Dharmacon RNAtechnologies.

    Techniques: Translocation Assay, Transfection, shRNA, Construct, Staining

    Depletion of CIN increases P-cofilin/cofilin levels in total cell lysates. ( A ) Depletion of CIN in MTLn3 cells using rat siRNA oligo1 and oligo4. MTLn3 cells were treated with control CIN-, human CIN-, or rCIN-targeting oligos (oligo1 and oligo4 sequences

    Journal:

    Article Title: Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

    doi: 10.1073/pnas.1510945112

    Figure Lengend Snippet: Depletion of CIN increases P-cofilin/cofilin levels in total cell lysates. ( A ) Depletion of CIN in MTLn3 cells using rat siRNA oligo1 and oligo4. MTLn3 cells were treated with control CIN-, human CIN-, or rCIN-targeting oligos (oligo1 and oligo4 sequences

    Article Snippet: Two CIN-directed siRNA oligos duplexes were purchased from Dharmacon RNAtechnologies.

    Techniques:

    β-Arrestins are not required for the translocation of CIN at the cell leading edge during EGF stimulation. ( A ) Immunofluorescence of actin and paxillin in MTLn3 cells depleted in ARB1 siRNA, ARB2 siRNA, or both (ARB1 + ARB2 siRNAs). MTLn3 cells

    Journal:

    Article Title: Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

    doi: 10.1073/pnas.1510945112

    Figure Lengend Snippet: β-Arrestins are not required for the translocation of CIN at the cell leading edge during EGF stimulation. ( A ) Immunofluorescence of actin and paxillin in MTLn3 cells depleted in ARB1 siRNA, ARB2 siRNA, or both (ARB1 + ARB2 siRNAs). MTLn3 cells

    Article Snippet: Two CIN-directed siRNA oligos duplexes were purchased from Dharmacon RNAtechnologies.

    Techniques: Translocation Assay, Immunofluorescence

    Leading edge dynamics are regulated by CIN. ( A ). ( B ) Average protrusion area (± SEM) measured in control siRNA ( n = 122 cells) and CIN siRNA ( n = 137 cells) cells at

    Journal:

    Article Title: Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

    doi: 10.1073/pnas.1510945112

    Figure Lengend Snippet: Leading edge dynamics are regulated by CIN. ( A ). ( B ) Average protrusion area (± SEM) measured in control siRNA ( n = 122 cells) and CIN siRNA ( n = 137 cells) cells at

    Article Snippet: Two CIN-directed siRNA oligos duplexes were purchased from Dharmacon RNAtechnologies.

    Techniques:

    Increase of the P-cofilin level in CIN-depleted MTLn3 cells at 3 min after EGF stimulation. P-cofilin and cofilin staining in ( A ) control siRNA (siRNA CTRL) cells or ( B ) CIN siRNA-transfected cells at 0, 1, and 3 min after EGF stimulation. Column 3 in

    Journal:

    Article Title: Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

    doi: 10.1073/pnas.1510945112

    Figure Lengend Snippet: Increase of the P-cofilin level in CIN-depleted MTLn3 cells at 3 min after EGF stimulation. P-cofilin and cofilin staining in ( A ) control siRNA (siRNA CTRL) cells or ( B ) CIN siRNA-transfected cells at 0, 1, and 3 min after EGF stimulation. Column 3 in

    Article Snippet: Two CIN-directed siRNA oligos duplexes were purchased from Dharmacon RNAtechnologies.

    Techniques: Staining, Transfection

    CIN regulates F-actin network organization and turnover at the cell leading edge. ( A ) Kymograph analysis of actin retrograde flow in MTLn3 cells transfected with GFP empty vector control alone or combined with Myc-tagged CIN WT , control siRNA, or CIN siRNA.

    Journal:

    Article Title: Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels

    doi: 10.1073/pnas.1510945112

    Figure Lengend Snippet: CIN regulates F-actin network organization and turnover at the cell leading edge. ( A ) Kymograph analysis of actin retrograde flow in MTLn3 cells transfected with GFP empty vector control alone or combined with Myc-tagged CIN WT , control siRNA, or CIN siRNA.

    Article Snippet: Two CIN-directed siRNA oligos duplexes were purchased from Dharmacon RNAtechnologies.

    Techniques: Flow Cytometry, Transfection, Plasmid Preparation

    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: 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: 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: 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: 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: 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: 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