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  • 99
    Thermo Fisher drosophila s2 cells
    Ligand-dependency of Torso dimerization, autophosphorylation, and downstream ERK phosphorylation. ( A ) Dimerization of the FLAG-tagged full-length silkworm Torso (BmTorsoFL) was examined using the cross-linking reagent, BS 3 . The Torso dimerization was observed by immunoblotting with an anti-FLAG antibody, either with or without stimulation by the silkworm ligand PTTH (BmPTTH). ( B ) Autophosphorylation of BmTorsoFL in the membrane fractions prepared from the BmTorsoFL-expressing S2 cells was examined by immunoprecipitation with an anti-FLAG antibody and immunoblotting with an anti-phosphotyrosine antibody. The Torso autophosphorylation (shown by the arrow pTorso) was promoted by the BmPTTH stimulation. ( C ) Phosphorylation of the ERK enzyme downstream of Torso was examined in the BmTorsoFL-expressing S2 cells, by immunoblotting with an anti-phospho-ERK antibody. ERK phosphorylation (the arrow pERK) was robustly increased by the BmPTTH stimulation. Since an antibody specifically recognizing the fruit-fly ERK (DmERK) was not available, c-Myc-tagged ERK (DmERK-c-Myc) was co-expressed in the cells, as a protein loading control.
    Drosophila S2 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 764 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore rabbit anti drosophila unc 45 antibody
    Requirement for <t>UNC-45</t> during cardiac remodeling. ( A–F ) 1 week old adult hearts were analyzed after unc-45 KD before or after metamorphosis. The left two bars show data from control and unc-45 KD hearts when KD occurred prior to metamorphosis, i.e., starting at the third instar larval stage. The right two bars in each panel represent data from hearts when KD was performed after metamorphosis, i.e., in the adults. Data show mean values ± SD; statistical significance was determined using an unpaired Student's t test (*** = p
    Rabbit Anti Drosophila Unc 45 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher drosophila s2r
    Integrins and talin modulate the JNK response to mechanical stress. <t>S2R+</t> cells transiently transfected with dJun-FRET biosensors were plated on collagen- (A – treated with Mys dsRNA and C – treated with talin dsRNA) or Con-A-coated (B) silicone membranes on a Stage Flexer set up. Donor mCFP FLs were collected before stretching (blue) and at 3 hours after continuous static stretch (green). In each graph are also represented the data obtained for untreated cells plated on collagen-coated silicone membranes (black– before stretch; red– 3 hours after continuous stretch). In the absence of Mys, talin or by plating cells on Con-A, the activity of JNK increases in relation to WT cells at rest. However, only the talin minus cells were able to increase their dJun-FRET sensor activity upon stretch. Mys minus cells plated on collagen-coated silicone membranes show a pseudopolygonal shape and emit multiple thick short protrusions (D). They rounded up upon stretch emitting very thin filopodia (G). Talin minus cells were more rounded and showed few, long branched filopodia (F). They collapsed in response to stretch showing occasional, very short, thin protrusions (I). Cells plated on Con-A-coated silicone membranes showed a flat shape (E) that was not affected by mechanical stress (H). The data acquired suggests a hierarchical model for the roles of β-integrin and talin regulating the level of Jun sensor activation of S2R+ cells and its response to mechanical stress (J). In the absence of mechanical input both β-integrin and talin restrain the activity of the JNK signaling. In the absence of any of them, the pathway gets moderately activated in response to an independent input. Mechanical stress results in β-integrin activation and the establishment of a, probably talin-independent, positive contribution to JNK signaling leading to a maximum level of dJun-FRET biosensor activity.
    Drosophila S2r, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    ATCC drosophila sl2 cells
    Relish Is Displaced from the Attacin-A Promoter by the Repressosome (A) Left panel: soluble chromatin extracts were prepared from <t>SL2</t> cells with (15 min or 8 h) or without (0 min) LPS/PGN treatment, and immunoprecipitated with antibodies against Relish, Jun, or Stat92E as described in Figure 3 . Right panel: double ChIP assays. The precipitates obtained from the first ChIP of cells with (15 min) or without LPS/PGN treatment were analyzed separately in a second ChIP with the antibodies indicated at the top (second ChIP). The amounts of Attacin-A promoter fragments co-precipitated with the indicated antibody are shown. (B) The transcript levels of each target gene are shown under Luciferase, Relish, Jra, Stat92E, or Dsp1 knock-down conditions with LPS/PGN treatment (10 μg/ml; 1h). The averages and standard deviations of triplicates assays are shown.
    Drosophila Sl2 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 88/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Developmental Studies Hybridoma Bank monoclonal mouse anti drosophila hp1
    STAT92E contributes to H1-dependent heterochromatin formation. Polytene chromosomes of salivary gland cells from L3 larvae were analyzed by indirect immunofluorescence (IF) staining with antibodies against <t>HP1</t> or H1 ( red ) and STAT92E ( green ). DNA was stained with DAPI ( blue ). Scale bar represents 10 μm. (A) Polytene chromosome structure in wild type larvae ( top ), H1-depleted larvae ( middle ), and H1-depleted larvae that overexpress nonphosphorylatable STAT, STAT92E(Y704F) ( bottom ). HP1 signal is strongly enriched in a single chromocenter region in the wild type. The chromocenter is not discernable (DAPI), and HP1 staining is dispersed in multiple foci upon H1 depletion (see also Figure 1 A). The phenotype is partially rescued by STAT92E(Y704F) expression. (B) STAT92E(Y704F) overexpressed in H1-depleted larvae, co-localizes with residual H1 in polytene chromosomes ( top ). Ectopically overexpressed transgenic wild type ( WT ) STAT92E fails to restore the single chromocenter and does not co-localize with residual H1 ( bottom ).
    Monoclonal Mouse Anti Drosophila Hp1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 83/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    TaKaRa embryonic drosophila melanogaster poly a rna
    Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) <t>RNA.</t> B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.
    Embryonic Drosophila Melanogaster Poly A Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 82/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Sturtevant young drosophila athabasca species complex
    Phylogenetic and principal component clustering analyses of populations from the three species: Drosophila <t>athabasca</t> ( WN , blue, squares), D. mahican ( EA , red, circles), and D. lenape ( EB , orange, triangles/diamonds). D. affinis was the outgroup. See text and Figure 1 for locations of abbreviations of specific locations. (a) Phylogeny inferred using the neighbor‐joining ( NJ ) method based on average pairwise Fst values across all 52 gene fragments (see Materials and Methods ). Sum of branch length = 1.7 ( MEGA 7). Minimum number of sequences allowed per population was six chromosomes. Branch lengths are shown. Phylogeny based on Dxy distances showed qualitatively similar results (data not shown). (b) A maximum likelihood consensus phylogeny with bootstrap values based on 22,261‐bp consensus sequences across populations ( D. mahican QB , MVS / MKSP , and SAIJ populations were not included due to small sample sizes). Bootstrap supports (500 replicates) are shown next to the branches. (c) Principal component analysis ( PCA ) based on covariances across a total of 1,820‐bp SNP sites across populations of each species (same populations as in panel b, except D. lenape LK population was not included due to small sample size). PC 1 explains 36%, and PC 2 explains 20% of total genetic variation across populations. Results were qualitatively similar when different nonoverlapping sets (500 bp per set) were used to generate PCA . Some sympatric locations are labeled
    Young Drosophila Athabasca Species Complex, supplied by Sturtevant, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Developmental Studies Hybridoma Bank drosophila hsp70 monoclonal antibody
    DEVDase (caspase 3-like) activity in the brain of PQ-exposed Drosophila . Graphical representation of DEVDase activity in the brain homogenate of Drosophila after their exposure to PQ for 12 and 24 h; A significant decrease in enzyme activity was observed in the brain of PQ-exposed <t>hsp70</t> over-expressing strain. Data are mean ± SD (n = 3). Significance ascribed as ** p
    Drosophila Hsp70 Monoclonal Antibody, supplied by Developmental Studies Hybridoma Bank, 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
    GE Healthcare full length drosophila e2f1
    <t>E2f1</t> Stable acts acutely to trigger apoptosis. A–E) Detection of S phase by EdU labeling (red) and apoptosis by CC3 staining (green) in GMR-Gal4 third instar larval eye imaginal discs expressing GFP or the indicated GFP-E2f1 fusion proteins. Arrowheads indicate the position of the MF, with anterior to the left and posterior to the right. Bars = 5 µM. F) Quantification of the number of CC3 positive cells posterior to the MF. * p
    Full Length Drosophila E2f1, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore drosophila vps13a
    Endogenous <t>VPS13A</t> is enriched at fractions containing LDs upon OA induction. Ratio of VPS13A in the LD fraction in starved (Stv) ( Figure 7A ) and OA fed cells ( Figure 7B ). The amount of VPS13A Stv is set to 1.
    Drosophila Vps13a, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Diagenode cdk9 drosophila embryos
    P-TEFb and the Super Elongation Complex (SEC) display similar phenotypes. ( A ) RNA in situ hybridization using a digoxigenin-labeled Serendipity-α (Sry-α) probe demonstrates decreased levels of Sry-α mRNA in embryos depleted of maternal <t>Cdk9</t> (ii) or Cyclin T (iii) compared to wild-type (i). ( B ) DIC micrographs of wild-type (wt, i), or embryos derived from females with germline clones of the SEC component dAFF4/Lilliputian ( lilli -/- , ii) or females expressing TubGal4 and a shmiRNA targeting the SEC subunit dEll (dELLi, iii) show the same posterior phenotype as in P-TEFb embryos. A close up of the same embryos is shown below (iv-vi). ( C ) Expression of the cellularization genes bottleneck ( bnk , i, ii), slow-as-molasses ( slam , iii, iv), and nullo (v, vi) is comparable between wild-type and embryos depleted of maternal Cdk9. ( D ) Quantification of cellularization gene expression by RT-qPCR. Columns show average values in 2–4h embryos with S.E.M. (n = 5–6) and control values were set to 100%. Relative expression was normalized to a mean of four reference genes ( beta-tubulin , GAPDH , RpL32 , and 28SrRNA ). *indicates P
    Cdk9 Drosophila Embryos, supplied by Diagenode, 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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    78
    Developmental Studies Hybridoma Bank rabbit anti drosophila p53
    Dissemination phenotypes induced by four- and five-hit models. ( a – d ) Mmp1 staining of control and ras G12V <t>p53</t> Ri pten Ri apc Ri hindguts. ( b , d ) Mmp1 channel only. ( e – l ) Cross-section views of control and ras G12V p53 Ri pten Ri apc Ri hindguts. ( f , h , j , l ) Laminin channel only; arrows indicate reduced/absent laminin staining. ( m – s ) Examples of dissemination phenotype. Arrows indicate GFP-positive foci inside the abdominal cavity ( m ), underneath the abdomen epidermis ( n ), ovaries ( o ), head ( p ) and legs ( q , r ) (f, fat body, n, nephrocyte; t, trachea). ( m ) Inset: close-up view showing close association of GFP foci with tracheal branches. ( s ) Live confocal image of a multicellular GFP cluster inside the abdominal cavity. Nuclei are visualized by a nuclear dsRed transgene (nls-dsRed). ( t ) Quantification of dissemination into the abdominal cavity. Each animal is dissected and assigned into one of the following categories based on the number of disseminated foci inside the abdominal cavity: none, no dissemination; weak, 1–3 GFP-positive foci inside the abdominal cavity; moderate, 4–10 GFP-positive foci; strong, > 10 GFP-positive foci ( n =2 replicates, 20–30 flies per replicate; error bars: s.e.m.). Scale bars, 25 mm.
    Rabbit Anti Drosophila P53, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Wohlwend GmbH stage 17 drosophila embryos
    Notopleural  is required for embryonic tracheal gas filling and encodes a serine protease related to human matriptase. (A-D) Bright field light microscopic images of stage 17 wild-type (A),  btl- Gal4; UAS-RNAi- GD13443  (B),  Np P6 /Np C2  mutant (C) and  Np P6 , btl -Gal4/ Np C2 ,UAS- Np  mutant (D) embryos. Wild-type embryos show gas filled tracheal tubes at the end of embryogenesis (arrow in A). RNAi-mediated tracheal knock-down of  CG34350  ( Np ) leads to lack of tracheal gas filling (arrow in B).  Np P6 /Np C2  mutant embryos lack gas filling (arrow in C) while  Np  mutant embryos with tracheal expression of  Np  show normal gas filling of the tracheal system (arrow in D). (E) Schema showing the protein domain organisations of  Drosophila  Np and human matriptase. The transmembrane domains (yellow), the SEA ( s ea urchin sperm protein/ e nteropeptidase/ a grin), CUB ( C ls/Clr,  u rchin embryonic growth factor,  b one morphogenetic protein-1), LDLa ( l ow- d ensity  l ipoprotein receptor class  A ) and the catalytic protease domains are shown. Conserved disulphide bridges (-S-S-) and zymogen activation cleavage sites (V) are indicated. (F-J”) Confocal LSM images of whole-mount antibody stainings of  Np ::GFP embryos at stage 16 (F-F”, H-J”) and stage 17 (G-G”) stained with anti-Spectrin (magenta) and anti-GFP (green, Np::GFP) antibodies. Np::GFP is expressed in the tracheal system (F-G”), the hindgut (H-H”), the epidermis (I-I”) and the salivary glands (J-J”). Np::GFP is localized in the tracheal lumen during stage 16 (arrow in F’) and 17 (arrow in G’) and localizes to the apical membrane of tracheal cells during stage 17 (arrowheads in G’). In the hindgut and epidermis, Np::GFP is localized exclusively at the apical cell membranes (arrowheads in H’ and I’). In the salivary glands, Np::GFP is localized exclusively in the lumen (arrow in J’). Scale bars correspond to 10 μm.
    Stage 17 Drosophila Embryos, supplied by Wohlwend GmbH, 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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    77
    TaKaRa drosophila cdk8
    Dynamics of human <t>CDK8</t> A-loop during the 50-ns molecular dynamic simulation
    Drosophila Cdk8, supplied by TaKaRa, 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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    76
    Custom Monoclonals International drosophila dfz2
    Endosomal acidification is necessary for <t>Wg–DFz2</t> interaction and signaling. ( A–C ) Confocal images of wing discs, surface labeled on ice with A568 anti-Wg and A647 anti-DFz2 ( A ), or surface labeled and pulsed for 3–5 min followed
    Drosophila Dfz2, supplied by Custom Monoclonals International, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Ligand-dependency of Torso dimerization, autophosphorylation, and downstream ERK phosphorylation. ( A ) Dimerization of the FLAG-tagged full-length silkworm Torso (BmTorsoFL) was examined using the cross-linking reagent, BS 3 . The Torso dimerization was observed by immunoblotting with an anti-FLAG antibody, either with or without stimulation by the silkworm ligand PTTH (BmPTTH). ( B ) Autophosphorylation of BmTorsoFL in the membrane fractions prepared from the BmTorsoFL-expressing S2 cells was examined by immunoprecipitation with an anti-FLAG antibody and immunoblotting with an anti-phosphotyrosine antibody. The Torso autophosphorylation (shown by the arrow pTorso) was promoted by the BmPTTH stimulation. ( C ) Phosphorylation of the ERK enzyme downstream of Torso was examined in the BmTorsoFL-expressing S2 cells, by immunoblotting with an anti-phospho-ERK antibody. ERK phosphorylation (the arrow pERK) was robustly increased by the BmPTTH stimulation. Since an antibody specifically recognizing the fruit-fly ERK (DmERK) was not available, c-Myc-tagged ERK (DmERK-c-Myc) was co-expressed in the cells, as a protein loading control.

    Journal: Scientific Reports

    Article Title: Ligand-dependent responses of the silkworm prothoracicotropic hormone receptor, Torso, are maintained by unusual intermolecular disulfide bridges in the transmembrane region

    doi: 10.1038/srep22437

    Figure Lengend Snippet: Ligand-dependency of Torso dimerization, autophosphorylation, and downstream ERK phosphorylation. ( A ) Dimerization of the FLAG-tagged full-length silkworm Torso (BmTorsoFL) was examined using the cross-linking reagent, BS 3 . The Torso dimerization was observed by immunoblotting with an anti-FLAG antibody, either with or without stimulation by the silkworm ligand PTTH (BmPTTH). ( B ) Autophosphorylation of BmTorsoFL in the membrane fractions prepared from the BmTorsoFL-expressing S2 cells was examined by immunoprecipitation with an anti-FLAG antibody and immunoblotting with an anti-phosphotyrosine antibody. The Torso autophosphorylation (shown by the arrow pTorso) was promoted by the BmPTTH stimulation. ( C ) Phosphorylation of the ERK enzyme downstream of Torso was examined in the BmTorsoFL-expressing S2 cells, by immunoblotting with an anti-phospho-ERK antibody. ERK phosphorylation (the arrow pERK) was robustly increased by the BmPTTH stimulation. Since an antibody specifically recognizing the fruit-fly ERK (DmERK) was not available, c-Myc-tagged ERK (DmERK-c-Myc) was co-expressed in the cells, as a protein loading control.

    Article Snippet: For the transient expression of the Torso proteins, we used cultured Drosophila S2 cells, which were a component of the DES® Inducible Kit (Life Technologies, Inc., Carlsbad, CA, USA).

    Techniques: Expressing, Immunoprecipitation

    Oligomerization of silkworm Torso on the surface of cultured S2 cells. ( A ) FLAG-tagged full-length silkworm Torso (BmTorsoFL) was transiently expressed in cultured  Drosophila  S2 cells. After successive treatments with 10 nM silkworm PTTH (BmPTTH) and 0.3 mM water-soluble cross-linking reagent, the stimulated receptor was analyzed by reducing Bis-Tris SDS-PAGE. Only a cross-linker with an 11.4-Å spacer, BS 3  (Sulfo-DSS), successfully generated an oligomer band of BmTorsoFL (shown by the arrow D), while the others, Sulfo-DST, Sulfo-BSOCOES, and Sulfo-EGS, showed only unlinked-monomer bands (the arrow M). ( B ) To estimate the precise molecular mass of the receptor complex, the BmTorsoFL-expressing cells were treated with a much lower concentration (0.03 mM) of BS 3  and fractionated by reducing Tris-Acetate SDS-PAGE. The single logarithmic plot of the protein bands shows that the BS 3 -linked oligomer and unlinked monomer of Torso have molecular masses of 236 and 105 kDa, respectively, indicating that the BS 3 -linked complex is probably a Torso dimer.

    Journal: Scientific Reports

    Article Title: Ligand-dependent responses of the silkworm prothoracicotropic hormone receptor, Torso, are maintained by unusual intermolecular disulfide bridges in the transmembrane region

    doi: 10.1038/srep22437

    Figure Lengend Snippet: Oligomerization of silkworm Torso on the surface of cultured S2 cells. ( A ) FLAG-tagged full-length silkworm Torso (BmTorsoFL) was transiently expressed in cultured Drosophila S2 cells. After successive treatments with 10 nM silkworm PTTH (BmPTTH) and 0.3 mM water-soluble cross-linking reagent, the stimulated receptor was analyzed by reducing Bis-Tris SDS-PAGE. Only a cross-linker with an 11.4-Å spacer, BS 3 (Sulfo-DSS), successfully generated an oligomer band of BmTorsoFL (shown by the arrow D), while the others, Sulfo-DST, Sulfo-BSOCOES, and Sulfo-EGS, showed only unlinked-monomer bands (the arrow M). ( B ) To estimate the precise molecular mass of the receptor complex, the BmTorsoFL-expressing cells were treated with a much lower concentration (0.03 mM) of BS 3 and fractionated by reducing Tris-Acetate SDS-PAGE. The single logarithmic plot of the protein bands shows that the BS 3 -linked oligomer and unlinked monomer of Torso have molecular masses of 236 and 105 kDa, respectively, indicating that the BS 3 -linked complex is probably a Torso dimer.

    Article Snippet: For the transient expression of the Torso proteins, we used cultured Drosophila S2 cells, which were a component of the DES® Inducible Kit (Life Technologies, Inc., Carlsbad, CA, USA).

    Techniques: Cell Culture, SDS Page, Generated, Expressing, Concentration Assay

    Expression and oligomerization states of the silkworm Torso extracellular region. ( A ) FLAG-tagged full-length silkworm Torso (BmTorsoFL) and its extracellular region (BmTorsoEC), expressed in cultured S2 cells, were analyzed by reducing Bis-Tris SDS-PAGE. While BmTorsoFL was found in the cell lysate, BmTorsoEC was not detected in the lysate, but was present in the culture medium of the cells. ( B ) The BmTorsoEC secreted in the culture medium was not detected as the dimer band on the non-reducing Bis-Tris gel.

    Journal: Scientific Reports

    Article Title: Ligand-dependent responses of the silkworm prothoracicotropic hormone receptor, Torso, are maintained by unusual intermolecular disulfide bridges in the transmembrane region

    doi: 10.1038/srep22437

    Figure Lengend Snippet: Expression and oligomerization states of the silkworm Torso extracellular region. ( A ) FLAG-tagged full-length silkworm Torso (BmTorsoFL) and its extracellular region (BmTorsoEC), expressed in cultured S2 cells, were analyzed by reducing Bis-Tris SDS-PAGE. While BmTorsoFL was found in the cell lysate, BmTorsoEC was not detected in the lysate, but was present in the culture medium of the cells. ( B ) The BmTorsoEC secreted in the culture medium was not detected as the dimer band on the non-reducing Bis-Tris gel.

    Article Snippet: For the transient expression of the Torso proteins, we used cultured Drosophila S2 cells, which were a component of the DES® Inducible Kit (Life Technologies, Inc., Carlsbad, CA, USA).

    Techniques: Expressing, Cell Culture, SDS Page

    Disulfide-bond-mediated dimerization of silkworm Torso without ligand stimulation. FLAG-tagged full-length silkworm Torso (BmTorsoFL), expressed in cultured S2 cells, was analyzed by non-reducing ( A ) and reducing ( B ) Bis-Tris SDS-PAGE, without the cross-linking treatment. Even without the BmPTTH stimulation, Torso was detected as an apparent dimer band (shown by the arrow D), in addition to a faint monomer band (the arrow M), on the non-reducing gel, while it was only detected as the monomer band on the reducing gel.

    Journal: Scientific Reports

    Article Title: Ligand-dependent responses of the silkworm prothoracicotropic hormone receptor, Torso, are maintained by unusual intermolecular disulfide bridges in the transmembrane region

    doi: 10.1038/srep22437

    Figure Lengend Snippet: Disulfide-bond-mediated dimerization of silkworm Torso without ligand stimulation. FLAG-tagged full-length silkworm Torso (BmTorsoFL), expressed in cultured S2 cells, was analyzed by non-reducing ( A ) and reducing ( B ) Bis-Tris SDS-PAGE, without the cross-linking treatment. Even without the BmPTTH stimulation, Torso was detected as an apparent dimer band (shown by the arrow D), in addition to a faint monomer band (the arrow M), on the non-reducing gel, while it was only detected as the monomer band on the reducing gel.

    Article Snippet: For the transient expression of the Torso proteins, we used cultured Drosophila S2 cells, which were a component of the DES® Inducible Kit (Life Technologies, Inc., Carlsbad, CA, USA).

    Techniques: Cell Culture, SDS Page

    Expression and oligomerization states of silkworm Torso mutants, in which three cysteine residues in the transmembrane region were replaced by alanines (C381/393/394A) and phenylalanines (C381/393/394F). After transfection with a vector for the expression of wild-type Torso or its substitution mutant, the S2 cells without the ligand stimulation were solubilized and directly analyzed by reducing ( A ) and non-reducing ( B ) Bis-Tris SDS-PAGE. The wild-type and phenylalanine mutant proteins were observed as similar monomer bands (shown by the arrow M) on the reducing gel, while that of the alanine mutant was not detected. Wild-type Torso was detected as both the monomer and dimer bands (the arrows M and D, respectively) on the non-reducing gel, while the phenylalanine mutant was only detected as the monomer.

    Journal: Scientific Reports

    Article Title: Ligand-dependent responses of the silkworm prothoracicotropic hormone receptor, Torso, are maintained by unusual intermolecular disulfide bridges in the transmembrane region

    doi: 10.1038/srep22437

    Figure Lengend Snippet: Expression and oligomerization states of silkworm Torso mutants, in which three cysteine residues in the transmembrane region were replaced by alanines (C381/393/394A) and phenylalanines (C381/393/394F). After transfection with a vector for the expression of wild-type Torso or its substitution mutant, the S2 cells without the ligand stimulation were solubilized and directly analyzed by reducing ( A ) and non-reducing ( B ) Bis-Tris SDS-PAGE. The wild-type and phenylalanine mutant proteins were observed as similar monomer bands (shown by the arrow M) on the reducing gel, while that of the alanine mutant was not detected. Wild-type Torso was detected as both the monomer and dimer bands (the arrows M and D, respectively) on the non-reducing gel, while the phenylalanine mutant was only detected as the monomer.

    Article Snippet: For the transient expression of the Torso proteins, we used cultured Drosophila S2 cells, which were a component of the DES® Inducible Kit (Life Technologies, Inc., Carlsbad, CA, USA).

    Techniques: Expressing, Transfection, Plasmid Preparation, Mutagenesis, SDS Page

    Three constructs of silkworm Torso, designed for heterologous expression in cultured  Drosophila  S2 cells. The pIZT vector was used for the expression of full-length silkworm Torso (BmTorsoFL) and the mutant that lacks the latter half of the kinase domain and its following tail (BmTorsoΔK), while the pMT vector was used for the expression of its extracellular region (BmTorsoEC). All of these proteins possessed a common secretory signal sequence at the N-terminus and an additional FLAG-tag sequence (DYKDDDDK) at the C-terminus, to facilitate detection by immunoblotting with an anti-FLAG antibody. The transmembrane regions of BmTorsoFL and BmTorsoΔK have three atypical cysteine residues, at positions 381, 393, and 394.

    Journal: Scientific Reports

    Article Title: Ligand-dependent responses of the silkworm prothoracicotropic hormone receptor, Torso, are maintained by unusual intermolecular disulfide bridges in the transmembrane region

    doi: 10.1038/srep22437

    Figure Lengend Snippet: Three constructs of silkworm Torso, designed for heterologous expression in cultured Drosophila S2 cells. The pIZT vector was used for the expression of full-length silkworm Torso (BmTorsoFL) and the mutant that lacks the latter half of the kinase domain and its following tail (BmTorsoΔK), while the pMT vector was used for the expression of its extracellular region (BmTorsoEC). All of these proteins possessed a common secretory signal sequence at the N-terminus and an additional FLAG-tag sequence (DYKDDDDK) at the C-terminus, to facilitate detection by immunoblotting with an anti-FLAG antibody. The transmembrane regions of BmTorsoFL and BmTorsoΔK have three atypical cysteine residues, at positions 381, 393, and 394.

    Article Snippet: For the transient expression of the Torso proteins, we used cultured Drosophila S2 cells, which were a component of the DES® Inducible Kit (Life Technologies, Inc., Carlsbad, CA, USA).

    Techniques: Construct, Expressing, Cell Culture, Plasmid Preparation, Mutagenesis, Sequencing, FLAG-tag

    Receptor autophosphorylation, dimerization, and downstream ERK phosphorylation of the Torso phenylalanine mutant (C381/393/394F). The receptor autophosphorylation ( A ), dimerization ( B ), and downstream ERK phosphorylation ( C ) by the phenylalanine mutant were assessed in the same manner as described in  Fig. 3 . The mutant receptor was similarly autophosphorylated either with or without the ligand stimulation, and it formed a non-covalent dimer independently of the ligand stimulation. However, ERK phosphorylation was not facilitated by ligand stimulation in the phenylalanine-mutant-expressing S2 cells, while it was promoted by the ligand in those expressing wild-type Torso (positive control).

    Journal: Scientific Reports

    Article Title: Ligand-dependent responses of the silkworm prothoracicotropic hormone receptor, Torso, are maintained by unusual intermolecular disulfide bridges in the transmembrane region

    doi: 10.1038/srep22437

    Figure Lengend Snippet: Receptor autophosphorylation, dimerization, and downstream ERK phosphorylation of the Torso phenylalanine mutant (C381/393/394F). The receptor autophosphorylation ( A ), dimerization ( B ), and downstream ERK phosphorylation ( C ) by the phenylalanine mutant were assessed in the same manner as described in Fig. 3 . The mutant receptor was similarly autophosphorylated either with or without the ligand stimulation, and it formed a non-covalent dimer independently of the ligand stimulation. However, ERK phosphorylation was not facilitated by ligand stimulation in the phenylalanine-mutant-expressing S2 cells, while it was promoted by the ligand in those expressing wild-type Torso (positive control).

    Article Snippet: For the transient expression of the Torso proteins, we used cultured Drosophila S2 cells, which were a component of the DES® Inducible Kit (Life Technologies, Inc., Carlsbad, CA, USA).

    Techniques: Mutagenesis, Expressing, Positive Control

    The PX-BAR domain is required for tubule and protrusion formation.  (A) Schematic of the domain organization of Sh3px1. The amino acid residues that comprise the domains are listed. Also shown is a comparison of the percent identity and similarity in the BAR domains between Sh3px1 and Snx9, Snx18 and Snx33. (B) S2 cells were transfected with a GFP-BAR construct. Two days after transfection, the cells were spotted onto con A coverslips, allowed to adhere, fixed and counterstained to reveal F-actin (red). (C,D) S2 cells were co-transfected with a GFP-PX-BAR construct and a control shRNA (C) or an shRNA targeting endogenous  sh3px1  (D). Three days after transfection, the cells were treated as in panel B. 64.7% (±8) cells expressing the control shRNA formed protrusions in comparison to 63% (±3) for cells expressing shRNA targeting  sh3px1 . This difference is not statistically significant ( P =0.7477). (E,F) S2 cells were transfected with GFP-PX-BAR mutant Y256A (E) or with GFP-PX-BAR mutant K496E, R500E (F). Two days after transfection, the cells were treated as in panel B. Scale bars: 15 μm. (G) Cells from the experiments represented in panels B-F were quantified. As before, cells in which GFP-Sh3px1 displayed no particular localization pattern were counted as ‘Diffuse’. Cells in which membrane protrusions were observed were scored as ‘Protrusions’. Cells containing tubular structures were scored as ‘Tubules’. Cells containing a strong enrichment of GFP signal close to the cytoplasmic face of the nuclear envelope were scored as ‘Perinuclear’. For each experiment, 100 cells were counted for each construct. The experiment was done three times.

    Journal: Biology Open

    Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

    doi: 10.1242/bio.013755

    Figure Lengend Snippet: The PX-BAR domain is required for tubule and protrusion formation. (A) Schematic of the domain organization of Sh3px1. The amino acid residues that comprise the domains are listed. Also shown is a comparison of the percent identity and similarity in the BAR domains between Sh3px1 and Snx9, Snx18 and Snx33. (B) S2 cells were transfected with a GFP-BAR construct. Two days after transfection, the cells were spotted onto con A coverslips, allowed to adhere, fixed and counterstained to reveal F-actin (red). (C,D) S2 cells were co-transfected with a GFP-PX-BAR construct and a control shRNA (C) or an shRNA targeting endogenous sh3px1 (D). Three days after transfection, the cells were treated as in panel B. 64.7% (±8) cells expressing the control shRNA formed protrusions in comparison to 63% (±3) for cells expressing shRNA targeting sh3px1 . This difference is not statistically significant ( P =0.7477). (E,F) S2 cells were transfected with GFP-PX-BAR mutant Y256A (E) or with GFP-PX-BAR mutant K496E, R500E (F). Two days after transfection, the cells were treated as in panel B. Scale bars: 15 μm. (G) Cells from the experiments represented in panels B-F were quantified. As before, cells in which GFP-Sh3px1 displayed no particular localization pattern were counted as ‘Diffuse’. Cells in which membrane protrusions were observed were scored as ‘Protrusions’. Cells containing tubular structures were scored as ‘Tubules’. Cells containing a strong enrichment of GFP signal close to the cytoplasmic face of the nuclear envelope were scored as ‘Perinuclear’. For each experiment, 100 cells were counted for each construct. The experiment was done three times.

    Article Snippet: Drosophila S2 cells were obtained from Life Technologies and were grown in Schneider's media containing 10% Fetal Bovine Serum.

    Techniques: Transfection, Construct, shRNA, Expressing, Mutagenesis

    Localization of endogenous Sh3px1.  (A,B)  Drosophila  S2 cells were treated with dsRNAs against  gfp  (A) or  sh3px1  (B). Four days after dsRNA treatment, the cells were spotted onto concanavalin A (con A) coated coverslips and allowed to adhere for 2 h. The cells were then fixed and analyzed using an antibody against Sh3px1 (green). The cells were also counterstained with DAPI to reveal nuclei (red). (C) S2 cells were treated with dsRNAs against  gfp  (lane 1) or  sh3px1  (lane 2). Lysates were prepared from these cells and run on an SDS-PAGE gel. The proteins were transferred to nitrocellulose and processed for western blot analysis using the indicated antibodies. (D) S2 cells were spotted onto con A coverslips and allowed to adhere for 2 h. The cells were then fixed and processed for immunofluorescence using an antibody against Sh3px1 (green). The cells were also counterstained with TRITC conjugated Phalloidin to reveal F-actin (red). Sh3px1 localized adjacent to the cortical actin network (arrow) as well as to internal foci. (E) S2 cells were transfected with a plasmid expressing RFP-tagged Wasp. Four days after transfection, the cells were spotted onto Con A coverslips and allowed to adhere for 2 h. The cells were then fixed and processed for immunofluorescence using an antibody against Sh3px1 (green). Sh3px1 co-localized with RFP-Wasp at the cell cortex (arrow). (F) S2 cells were spotted onto con A coverslips and allowed to adhere for 2 h. The cells were then fixed and processed for immunofluorescence using an antibody against Sh3px1 (green) and Scar (red). Sh3px1 co-localized with endogenous Scar at the cell cortex (arrow). In addition, a perinuclear enrichment of Sh3px1 could be detected in approximately 45% of cells (arrowhead). Scale bars: 15 μm.

    Journal: Biology Open

    Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

    doi: 10.1242/bio.013755

    Figure Lengend Snippet: Localization of endogenous Sh3px1. (A,B) Drosophila S2 cells were treated with dsRNAs against gfp (A) or sh3px1 (B). Four days after dsRNA treatment, the cells were spotted onto concanavalin A (con A) coated coverslips and allowed to adhere for 2 h. The cells were then fixed and analyzed using an antibody against Sh3px1 (green). The cells were also counterstained with DAPI to reveal nuclei (red). (C) S2 cells were treated with dsRNAs against gfp (lane 1) or sh3px1 (lane 2). Lysates were prepared from these cells and run on an SDS-PAGE gel. The proteins were transferred to nitrocellulose and processed for western blot analysis using the indicated antibodies. (D) S2 cells were spotted onto con A coverslips and allowed to adhere for 2 h. The cells were then fixed and processed for immunofluorescence using an antibody against Sh3px1 (green). The cells were also counterstained with TRITC conjugated Phalloidin to reveal F-actin (red). Sh3px1 localized adjacent to the cortical actin network (arrow) as well as to internal foci. (E) S2 cells were transfected with a plasmid expressing RFP-tagged Wasp. Four days after transfection, the cells were spotted onto Con A coverslips and allowed to adhere for 2 h. The cells were then fixed and processed for immunofluorescence using an antibody against Sh3px1 (green). Sh3px1 co-localized with RFP-Wasp at the cell cortex (arrow). (F) S2 cells were spotted onto con A coverslips and allowed to adhere for 2 h. The cells were then fixed and processed for immunofluorescence using an antibody against Sh3px1 (green) and Scar (red). Sh3px1 co-localized with endogenous Scar at the cell cortex (arrow). In addition, a perinuclear enrichment of Sh3px1 could be detected in approximately 45% of cells (arrowhead). Scale bars: 15 μm.

    Article Snippet: Drosophila S2 cells were obtained from Life Technologies and were grown in Schneider's media containing 10% Fetal Bovine Serum.

    Techniques: SDS Page, Western Blot, Immunofluorescence, Transfection, Plasmid Preparation, Expressing

    Depletion of Sh3px1 results in defective lamellipodia formation .  (A,B) S2 cells were transfected with a plasmid expressing a control shRNA targeting the yeast  gal80  gene (A) or with a plasmid expressing shRNA targeting  sh3px1  (B). The cells were also transfected with the Act5c-Gal4 plasmid in order to induce expression of the shRNA. Three days after transfection, the cells were spotted onto con A coverslips and allowed to adhere for 2 h. The cells were then fixed and processed for immunofluorescence using an antibody against Sh3px1 (green). The cells were also counterstained to reveal F-actin (red). The percentage of Sh3px1 depleted cells displaying the lamellipodia defect is indicated in panel B. (C,D) S2 cells were transfected with a plasmid expressing the  gal80  control shRNA (C) or with a plasmid expressing an shRNA targeting  scar  (D). Three days after transfection, the cells were fixed, processed using an antibody against Scar (green), and were counterstained to reveal F-actin (red). (E,F) S2 cells were transfected with a plasmid expressing the  gal80  control shRNA (E) or with a plasmid expressing an shRNA targeting  sh3px1  (F). Three days post transfection, the cells were fixed and processed using antibodies against Scar (red) and Sh3px1 (green). (G,H) S2 cells were transfected with a plasmid expressing the  gal80  control shRNA (G) or with a plasmid expressing an shRNA targeting  scar  (H). Three days later, the cells were fixed and processed using an antibody against Sh3px1 (green). The cells were also counterstained to reveal F-actin (red). Scale bars: 15 μm.

    Journal: Biology Open

    Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

    doi: 10.1242/bio.013755

    Figure Lengend Snippet: Depletion of Sh3px1 results in defective lamellipodia formation . (A,B) S2 cells were transfected with a plasmid expressing a control shRNA targeting the yeast gal80 gene (A) or with a plasmid expressing shRNA targeting sh3px1 (B). The cells were also transfected with the Act5c-Gal4 plasmid in order to induce expression of the shRNA. Three days after transfection, the cells were spotted onto con A coverslips and allowed to adhere for 2 h. The cells were then fixed and processed for immunofluorescence using an antibody against Sh3px1 (green). The cells were also counterstained to reveal F-actin (red). The percentage of Sh3px1 depleted cells displaying the lamellipodia defect is indicated in panel B. (C,D) S2 cells were transfected with a plasmid expressing the gal80 control shRNA (C) or with a plasmid expressing an shRNA targeting scar (D). Three days after transfection, the cells were fixed, processed using an antibody against Scar (green), and were counterstained to reveal F-actin (red). (E,F) S2 cells were transfected with a plasmid expressing the gal80 control shRNA (E) or with a plasmid expressing an shRNA targeting sh3px1 (F). Three days post transfection, the cells were fixed and processed using antibodies against Scar (red) and Sh3px1 (green). (G,H) S2 cells were transfected with a plasmid expressing the gal80 control shRNA (G) or with a plasmid expressing an shRNA targeting scar (H). Three days later, the cells were fixed and processed using an antibody against Sh3px1 (green). The cells were also counterstained to reveal F-actin (red). Scale bars: 15 μm.

    Article Snippet: Drosophila S2 cells were obtained from Life Technologies and were grown in Schneider's media containing 10% Fetal Bovine Serum.

    Techniques: Transfection, Plasmid Preparation, Expressing, shRNA, Immunofluorescence

    Formation of tubules and protrusions by GFP-Sh3px1.  (A-E) S2 cells were transfected with a plasmid encoding GFP (A) or GFP-Sh3px1 (B-E) and the Act5c-Gal4 plasmid that was required for driving expression of the fusion proteins. Two days after transfection, the cells were spotted onto con A coverslips and allowed to adhere for 2 h. The cells were then fixed, counterstained with Phalloidin-TRITC, and imaged. (F) S2 cells were transfected with a plasmid encoding GFP-Sh3px1 and spotted onto con A coverslips as noted above. The cells were then fixed in methanol at −20°C and stained with an antibody against Alpha-tubulin to reveal the microtubule network (red). Panel F′ represents a magnified view of the region outlined by the rectangle in F. (G) S2 cells were transfected with a plasmid encoding GFP-Sh3px1. Two days after transfection, the cells were spotted onto con A coated glass bottom dishes and allowed to adhere. The cells were then imaged live. Select time points (0 s, 168 s, and 300 s) from one such imaging experiment are shown. The panel on the far right represents a temporal color coded image. Images at the start of the experiment are depicted in purple and those at the end of the experiment are shown in white. The arrows indicate accumulation of GFP-Sh3px1 in small membrane protrusions that continue to elongate during the course of the experiment. (H) Similar treatment as panel G. Select time points (0 s, 72 s, and 150 s) from a live imaging experiment are shown here. The panel on the far right represents a temporal color-coded image. Arrows indicate membrane protrusions that continue to elongate during the time course of the experiment. Scale bars: 15 μm. (I) Quantification of the phenotypes observed in panels A-E. Cells in which no particular localization pattern was detected were counted as ‘Diffuse’. Cells in which membrane protrusions were observed were scored as ‘Protrusions’. Cells containing tubular structures were scored as ‘Tubules’. The graph represents data from three independent experiments. For each experiment, 100 cells were counted for each construct. The error bars represent standard deviation. ** P =0.0007, *** P ≤0.0001, Unpaired  t -test. (J-M) S2 cells were transfected with the following expression constructs, Cip4-GFP (J), Syndapin-mCherry (K), Mim-GFP (L), and Nwk-GFP (M). Two days after transfection, the cells were spotted onto con A coated coverslips and allowed to adhere. The cells were then fixed, counterstained to reveal F-actin, and imaged.

    Journal: Biology Open

    Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

    doi: 10.1242/bio.013755

    Figure Lengend Snippet: Formation of tubules and protrusions by GFP-Sh3px1. (A-E) S2 cells were transfected with a plasmid encoding GFP (A) or GFP-Sh3px1 (B-E) and the Act5c-Gal4 plasmid that was required for driving expression of the fusion proteins. Two days after transfection, the cells were spotted onto con A coverslips and allowed to adhere for 2 h. The cells were then fixed, counterstained with Phalloidin-TRITC, and imaged. (F) S2 cells were transfected with a plasmid encoding GFP-Sh3px1 and spotted onto con A coverslips as noted above. The cells were then fixed in methanol at −20°C and stained with an antibody against Alpha-tubulin to reveal the microtubule network (red). Panel F′ represents a magnified view of the region outlined by the rectangle in F. (G) S2 cells were transfected with a plasmid encoding GFP-Sh3px1. Two days after transfection, the cells were spotted onto con A coated glass bottom dishes and allowed to adhere. The cells were then imaged live. Select time points (0 s, 168 s, and 300 s) from one such imaging experiment are shown. The panel on the far right represents a temporal color coded image. Images at the start of the experiment are depicted in purple and those at the end of the experiment are shown in white. The arrows indicate accumulation of GFP-Sh3px1 in small membrane protrusions that continue to elongate during the course of the experiment. (H) Similar treatment as panel G. Select time points (0 s, 72 s, and 150 s) from a live imaging experiment are shown here. The panel on the far right represents a temporal color-coded image. Arrows indicate membrane protrusions that continue to elongate during the time course of the experiment. Scale bars: 15 μm. (I) Quantification of the phenotypes observed in panels A-E. Cells in which no particular localization pattern was detected were counted as ‘Diffuse’. Cells in which membrane protrusions were observed were scored as ‘Protrusions’. Cells containing tubular structures were scored as ‘Tubules’. The graph represents data from three independent experiments. For each experiment, 100 cells were counted for each construct. The error bars represent standard deviation. ** P =0.0007, *** P ≤0.0001, Unpaired t -test. (J-M) S2 cells were transfected with the following expression constructs, Cip4-GFP (J), Syndapin-mCherry (K), Mim-GFP (L), and Nwk-GFP (M). Two days after transfection, the cells were spotted onto con A coated coverslips and allowed to adhere. The cells were then fixed, counterstained to reveal F-actin, and imaged.

    Article Snippet: Drosophila S2 cells were obtained from Life Technologies and were grown in Schneider's media containing 10% Fetal Bovine Serum.

    Techniques: Transfection, Plasmid Preparation, Expressing, Staining, Imaging, Construct, Standard Deviation

    Localization of Snx9, Snx18 and Snx33 in S2 cells.  (A,B) S2 cells were transfected with a plasmid encoding GFP-Snx9. Two days after transfection, the cells were spotted onto con A coverslips and allowed to adhere. The cells were then fixed and counterstained with Phalloidin to visualize F-actin (red). Panel A represents an example of a tubule containing cell and panel B represent a cell that has formed protrusions. (C,D) S2 cells were transfected with a plasmid encoding GFP-Snx18. The cells were treated as in the above panels. C is an example of a GFP-Snx18 cell that has formed tubules and panel D is an example of a cell with long protrusions. (E,F) S2 cells were transfected with a plasmid encoding GFP-Snx33 and treated as above. E is an example of a tubule containing cell and panel F is an example of a cell that has formed protrusions. Scale bars: 15 μm. (G) Quantification of phenotypes observed upon over-expressing GFP-Snx9, Sxn18 and Snx33. The quantification criteria for determining Diffuse, Tubule or Protrusion was the same as previously described. The graph represents data from three independent experiments. For each experiment, 100 cells were counted per construct. The error bars represent standard deviation.

    Journal: Biology Open

    Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

    doi: 10.1242/bio.013755

    Figure Lengend Snippet: Localization of Snx9, Snx18 and Snx33 in S2 cells. (A,B) S2 cells were transfected with a plasmid encoding GFP-Snx9. Two days after transfection, the cells were spotted onto con A coverslips and allowed to adhere. The cells were then fixed and counterstained with Phalloidin to visualize F-actin (red). Panel A represents an example of a tubule containing cell and panel B represent a cell that has formed protrusions. (C,D) S2 cells were transfected with a plasmid encoding GFP-Snx18. The cells were treated as in the above panels. C is an example of a GFP-Snx18 cell that has formed tubules and panel D is an example of a cell with long protrusions. (E,F) S2 cells were transfected with a plasmid encoding GFP-Snx33 and treated as above. E is an example of a tubule containing cell and panel F is an example of a cell that has formed protrusions. Scale bars: 15 μm. (G) Quantification of phenotypes observed upon over-expressing GFP-Snx9, Sxn18 and Snx33. The quantification criteria for determining Diffuse, Tubule or Protrusion was the same as previously described. The graph represents data from three independent experiments. For each experiment, 100 cells were counted per construct. The error bars represent standard deviation.

    Article Snippet: Drosophila S2 cells were obtained from Life Technologies and were grown in Schneider's media containing 10% Fetal Bovine Serum.

    Techniques: Transfection, Plasmid Preparation, Expressing, Construct, Standard Deviation

    Co-localization between endogenous Sh3px1 and BAR domain constructs.  (A-G) S2 cells were transfected with GFP-Snx9 (A,B), GFP-Snx18 (C,D), GFP-Snx33 (E), GFP-Mim (F) or GFP-Nwk (G). Two days after the transfection, the cells were fixed and processed for immunofluorescence using an antibody against endogenous Sh3px1 (red). Scale bars: 15 μm. (H) The Pearson's co-localization coefficient was quantified for endogenous Sh3px1 and the indicated GFP-tagged constructs. For each construct, 25 cells were imaged and quantified. The co-localization was quantified for the entire cell rather than using a specific region within the cell. The error bars represent standard deviation. The  P  values for comparing the level of co-localization observed between the indicated GFP-tagged constructs and endogenous Sh3px1 are shown in  Fig. S4 .

    Journal: Biology Open

    Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

    doi: 10.1242/bio.013755

    Figure Lengend Snippet: Co-localization between endogenous Sh3px1 and BAR domain constructs. (A-G) S2 cells were transfected with GFP-Snx9 (A,B), GFP-Snx18 (C,D), GFP-Snx33 (E), GFP-Mim (F) or GFP-Nwk (G). Two days after the transfection, the cells were fixed and processed for immunofluorescence using an antibody against endogenous Sh3px1 (red). Scale bars: 15 μm. (H) The Pearson's co-localization coefficient was quantified for endogenous Sh3px1 and the indicated GFP-tagged constructs. For each construct, 25 cells were imaged and quantified. The co-localization was quantified for the entire cell rather than using a specific region within the cell. The error bars represent standard deviation. The P values for comparing the level of co-localization observed between the indicated GFP-tagged constructs and endogenous Sh3px1 are shown in Fig. S4 .

    Article Snippet: Drosophila S2 cells were obtained from Life Technologies and were grown in Schneider's media containing 10% Fetal Bovine Serum.

    Techniques: Construct, Transfection, Immunofluorescence, Standard Deviation

    The potential involvement of F-actin in protrusion formation.  (A,B) S2 cells were transfected with a plasmid encoding GFP-Sh3px1. Two days after transfection, the cells were spotted onto con A coverslips, allowed to adhere, and then fixed. Next, the cells were counterstained with Phalloidin-TRITC (red) to reveal F-actin. Panel A depicts a cell with long protrusions and panel B depicts a cell that is just starting to form protrusions. A′ and B′ represent magnified views of the regions outlined by rectangles in A and B, respectively. The arrows in A′ indicate that signal for Sh3px1 could be detected on the membrane of the long protrusions, whereas F-actin was observed more internally. The arrow in B′ indicates a short protrusion that contains more signal for Sh3px1 than F-actin. (C,D) Panels C and D represent select time frames from two independent live imaging experiments. S2 cells were co-transfected with plasmids encoding GFP-Sh3px1 (green) and mRuby2-Lifeact (red). Individual and merged images are shown. The arrows indicate protrusions that form and elongate during the course of the experiment. (E,F) S2 cells were co-transfected with the following combination of plasmids; GFP-Sh3px1 and the  gal80  control shRNA (E) and GFP-Sh3px1 and  wasp  shRNA-2 (F). Three days after transfection, the cells were spotted onto con A coverslips, allowed to adhere, and then fixed. Scale bars: 15 μm. (G) Quantification of phenotypes from the experiment depicted in panels E and F. The quantification criteria for determining Diffuse, Tubule, and Protrusion were as described previously. The graph represents data from three independent experiments. For each experiment, 100 cells were counted per construct. The error bars represent standard deviation. *** P =0.0018, unpaired  t -test.

    Journal: Biology Open

    Article Title: Depletion or over-expression of Sh3px1 results in dramatic changes in cell morphology

    doi: 10.1242/bio.013755

    Figure Lengend Snippet: The potential involvement of F-actin in protrusion formation. (A,B) S2 cells were transfected with a plasmid encoding GFP-Sh3px1. Two days after transfection, the cells were spotted onto con A coverslips, allowed to adhere, and then fixed. Next, the cells were counterstained with Phalloidin-TRITC (red) to reveal F-actin. Panel A depicts a cell with long protrusions and panel B depicts a cell that is just starting to form protrusions. A′ and B′ represent magnified views of the regions outlined by rectangles in A and B, respectively. The arrows in A′ indicate that signal for Sh3px1 could be detected on the membrane of the long protrusions, whereas F-actin was observed more internally. The arrow in B′ indicates a short protrusion that contains more signal for Sh3px1 than F-actin. (C,D) Panels C and D represent select time frames from two independent live imaging experiments. S2 cells were co-transfected with plasmids encoding GFP-Sh3px1 (green) and mRuby2-Lifeact (red). Individual and merged images are shown. The arrows indicate protrusions that form and elongate during the course of the experiment. (E,F) S2 cells were co-transfected with the following combination of plasmids; GFP-Sh3px1 and the gal80 control shRNA (E) and GFP-Sh3px1 and wasp shRNA-2 (F). Three days after transfection, the cells were spotted onto con A coverslips, allowed to adhere, and then fixed. Scale bars: 15 μm. (G) Quantification of phenotypes from the experiment depicted in panels E and F. The quantification criteria for determining Diffuse, Tubule, and Protrusion were as described previously. The graph represents data from three independent experiments. For each experiment, 100 cells were counted per construct. The error bars represent standard deviation. *** P =0.0018, unpaired t -test.

    Article Snippet: Drosophila S2 cells were obtained from Life Technologies and were grown in Schneider's media containing 10% Fetal Bovine Serum.

    Techniques: Transfection, Plasmid Preparation, Imaging, shRNA, Construct, Standard Deviation

    Drosophila  Trabid significantly decreases K63-linked polyubiquitination in dTAK1 through its catalytic C518 and NZF domains. (A) Schematic representation of dTrabid and sequence alignment of human and  Drosophila  Trabid in the OTU domain. The putative catalytic residue is in red    is indicated by an arrow. (B) Point mutations in C518 and the 3 NZF domains significantly affect dTrabid function in Imd signalling. Expression vectors containing dTrabidC518S, dTrabidC518S+3xNZFDel, dTAK1 and dTAB2 were co-transfected in S2 cells in the combinations shown and  diptericin  and  puckered  expression was assayed by qPCR 48 hrs post-transfection.   Results  represent mean values of 3 independent experiments and the error bars SEM. Asterisk (*) indicates significance value of the results when compared to dTAK1+dTAB2 as determined by Student's t-Test (t = 9.8004,  p

    Journal: PLoS Genetics

    Article Title: Loss of Trabid, a New Negative Regulator of the Drosophila Immune-Deficiency Pathway at the Level of TAK1, Reduces Life Span

    doi: 10.1371/journal.pgen.1004117

    Figure Lengend Snippet: Drosophila Trabid significantly decreases K63-linked polyubiquitination in dTAK1 through its catalytic C518 and NZF domains. (A) Schematic representation of dTrabid and sequence alignment of human and Drosophila Trabid in the OTU domain. The putative catalytic residue is in red is indicated by an arrow. (B) Point mutations in C518 and the 3 NZF domains significantly affect dTrabid function in Imd signalling. Expression vectors containing dTrabidC518S, dTrabidC518S+3xNZFDel, dTAK1 and dTAB2 were co-transfected in S2 cells in the combinations shown and diptericin and puckered expression was assayed by qPCR 48 hrs post-transfection. Results represent mean values of 3 independent experiments and the error bars SEM. Asterisk (*) indicates significance value of the results when compared to dTAK1+dTAB2 as determined by Student's t-Test (t = 9.8004, p

    Article Snippet: Drosophila S2 cells (Invitrogen) were maintained at 25°C in Schneider's Drosophila Medium (BioWhittaker/Lonza), supplemented with 10% heat-inactivated FBS and antibiotics 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate – all Invitrogen).

    Techniques: Sequencing, Radial Immuno Diffusion, Expressing, Transfection, Real-time Polymerase Chain Reaction

    Drosophila  TAB2 ZnF domain regulates IMD signalling. (A) Sequence alignment of the C-terminal ZnF domain of hTAB2, hTAB3 and dTAB2. Conserved Cys residues are in red and indicated by arrows. (B) IMD signalling is significantly increased in dTAB2 ZnFDel  mutant. S2 cells were co-transfected with expression vectors in combinations shown and  diptericin  expression was assayed 48 hrs post-transfection by qPCR.   Results  are mean values from 3 independent experiments and the error bars represent the Standard Error. Single, and triple asterisk indicates significance value of the result when compared to EGFP as determined by Student's t-Test (t = 17.8435, p

    Journal: PLoS Genetics

    Article Title: Loss of Trabid, a New Negative Regulator of the Drosophila Immune-Deficiency Pathway at the Level of TAK1, Reduces Life Span

    doi: 10.1371/journal.pgen.1004117

    Figure Lengend Snippet: Drosophila TAB2 ZnF domain regulates IMD signalling. (A) Sequence alignment of the C-terminal ZnF domain of hTAB2, hTAB3 and dTAB2. Conserved Cys residues are in red and indicated by arrows. (B) IMD signalling is significantly increased in dTAB2 ZnFDel mutant. S2 cells were co-transfected with expression vectors in combinations shown and diptericin expression was assayed 48 hrs post-transfection by qPCR. Results are mean values from 3 independent experiments and the error bars represent the Standard Error. Single, and triple asterisk indicates significance value of the result when compared to EGFP as determined by Student's t-Test (t = 17.8435, p

    Article Snippet: Drosophila S2 cells (Invitrogen) were maintained at 25°C in Schneider's Drosophila Medium (BioWhittaker/Lonza), supplemented with 10% heat-inactivated FBS and antibiotics 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate – all Invitrogen).

    Techniques: Radial Immuno Diffusion, Sequencing, Mutagenesis, Transfection, Expressing, Real-time Polymerase Chain Reaction

    Drosophila  Trabid binds to dTAK1 and negatively regulates IMD signalling. (A)  Drosophila  Trabid interacted with dTAK1.  Drosophila  TAK1-HA was co-transfected with or without dTrabid-V5 in S2 cells, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE and immunoblotted with anti-HA antibody. (B) AMP gene expression was highly elevated in  trabid  mutants in the absence of infection. Expression levels of various AMPs ( attacinA; drosocin; diptericin; cecropinA1 ) were checked in whole flies (3–6 days old) using qPCR compared with  yw  as control for the genetic background (wild type) of  pirk  and  trbd   [30] ,   [31] . We also used the  w 118  strain as an additional control (to check the range between two independent controls). Error bars represent the Standard Error of 3 separate experiments. *p

    Journal: PLoS Genetics

    Article Title: Loss of Trabid, a New Negative Regulator of the Drosophila Immune-Deficiency Pathway at the Level of TAK1, Reduces Life Span

    doi: 10.1371/journal.pgen.1004117

    Figure Lengend Snippet: Drosophila Trabid binds to dTAK1 and negatively regulates IMD signalling. (A) Drosophila Trabid interacted with dTAK1. Drosophila TAK1-HA was co-transfected with or without dTrabid-V5 in S2 cells, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE and immunoblotted with anti-HA antibody. (B) AMP gene expression was highly elevated in trabid mutants in the absence of infection. Expression levels of various AMPs ( attacinA; drosocin; diptericin; cecropinA1 ) were checked in whole flies (3–6 days old) using qPCR compared with yw as control for the genetic background (wild type) of pirk and trbd [30] , [31] . We also used the w 118 strain as an additional control (to check the range between two independent controls). Error bars represent the Standard Error of 3 separate experiments. *p

    Article Snippet: Drosophila S2 cells (Invitrogen) were maintained at 25°C in Schneider's Drosophila Medium (BioWhittaker/Lonza), supplemented with 10% heat-inactivated FBS and antibiotics 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate – all Invitrogen).

    Techniques: Radial Immuno Diffusion, Hemagglutination Assay, Transfection, Immunoprecipitation, SDS Page, Expressing, Infection, Real-time Polymerase Chain Reaction

    Ubiquitination profile is altered in the dTAK1 K142R  mutant. (A) Mutant dTAK1 K142R  showed enhanced ubiquitination. Expression vectors encoding dTAK1-V5 or dTAK1 K142R -V5 were transfected in S2 cells along with dTAB2-HA and cMyc-Ub in combinations as shown. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc. Observe the enhanced degradation of dTAK1 K142R -V5 in the cell lysate (right lane). (B) Mutant dTAK1 K142R  showed enhanced K48-linked ubiquitination and very little K63-linked ubiquitination. Expression vectors encoding dTAK1-V5 or dTAK1 K142R -V5 were transfected in S2 cells along with dTAB2-HA and cMyc-UbK63 (left panel) or cMyc-UbK48 (right panel) in combinations as shown. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc antibody. Protein size markers (NEB) are depicted adjacent to the top panels with values given in kDa.

    Journal: PLoS Genetics

    Article Title: Loss of Trabid, a New Negative Regulator of the Drosophila Immune-Deficiency Pathway at the Level of TAK1, Reduces Life Span

    doi: 10.1371/journal.pgen.1004117

    Figure Lengend Snippet: Ubiquitination profile is altered in the dTAK1 K142R mutant. (A) Mutant dTAK1 K142R showed enhanced ubiquitination. Expression vectors encoding dTAK1-V5 or dTAK1 K142R -V5 were transfected in S2 cells along with dTAB2-HA and cMyc-Ub in combinations as shown. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc. Observe the enhanced degradation of dTAK1 K142R -V5 in the cell lysate (right lane). (B) Mutant dTAK1 K142R showed enhanced K48-linked ubiquitination and very little K63-linked ubiquitination. Expression vectors encoding dTAK1-V5 or dTAK1 K142R -V5 were transfected in S2 cells along with dTAB2-HA and cMyc-UbK63 (left panel) or cMyc-UbK48 (right panel) in combinations as shown. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc antibody. Protein size markers (NEB) are depicted adjacent to the top panels with values given in kDa.

    Article Snippet: Drosophila S2 cells (Invitrogen) were maintained at 25°C in Schneider's Drosophila Medium (BioWhittaker/Lonza), supplemented with 10% heat-inactivated FBS and antibiotics 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate – all Invitrogen).

    Techniques: Mutagenesis, Expressing, Transfection, Hemagglutination Assay, Immunoprecipitation, SDS Page

    Decreased ubiquitination in the dTAK1 K156R  mutant. (A) Mutant dTAK1 K156R  showed decreased polyubiquitination when compared with dTAK1. Expression vectors encoding dTAK1-V5 or dTAK1 K156R  -V5 were transfected along with dTAB2-HA and cMyc-Ub in combinations shown in S2 cells. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc antibody. (B) K48-linked polyubiquitination was decreased in the dTAK1 K156R  mutant while K63-linked polyubiquitination was somewhat reduced. Expression vectors encoding dTAK1-V5 or dTAK1 K156R -V5 were transfected along with dTAB2-HA and cMyc-Ub, cMyc-UbK63 (left panel) or cMyc-UbK48 (right panel) in combinations shown in S2 cells. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc antibody. Protein size marker is depicted adjacent to the top panels with values in kDa.

    Journal: PLoS Genetics

    Article Title: Loss of Trabid, a New Negative Regulator of the Drosophila Immune-Deficiency Pathway at the Level of TAK1, Reduces Life Span

    doi: 10.1371/journal.pgen.1004117

    Figure Lengend Snippet: Decreased ubiquitination in the dTAK1 K156R mutant. (A) Mutant dTAK1 K156R showed decreased polyubiquitination when compared with dTAK1. Expression vectors encoding dTAK1-V5 or dTAK1 K156R -V5 were transfected along with dTAB2-HA and cMyc-Ub in combinations shown in S2 cells. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc antibody. (B) K48-linked polyubiquitination was decreased in the dTAK1 K156R mutant while K63-linked polyubiquitination was somewhat reduced. Expression vectors encoding dTAK1-V5 or dTAK1 K156R -V5 were transfected along with dTAB2-HA and cMyc-Ub, cMyc-UbK63 (left panel) or cMyc-UbK48 (right panel) in combinations shown in S2 cells. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc antibody. Protein size marker is depicted adjacent to the top panels with values in kDa.

    Article Snippet: Drosophila S2 cells (Invitrogen) were maintained at 25°C in Schneider's Drosophila Medium (BioWhittaker/Lonza), supplemented with 10% heat-inactivated FBS and antibiotics 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate – all Invitrogen).

    Techniques: Mutagenesis, Expressing, Transfection, Hemagglutination Assay, Immunoprecipitation, SDS Page, Marker

    Lys 142 and Lys 156 of dTAK1 are essential for immune signalling. (A) Sequence alignment of 1 to 230 amino acids of human TAK1 (hTAK1) and  Drosophila  TAK1 (dTAK1). Lys 142 (dark grey) and 156 (light grey) are indicated (arrows) and the kinase activation loop is underlined. (B)  Drosophila  TAK1 K142R  and TAK1 K156R  mutants failed to activate  diptericin .  Drosophila  TAK1 or dTAK1 K142R  or dTAK1 K156R  mutant was expressed along with or without dTAB2, in S2 cells in combinations shown.  Diptericin  expression was assayed 48 hrs post-transfection. Error bars represent Standard Error of 3 separate experiments. *p

    Journal: PLoS Genetics

    Article Title: Loss of Trabid, a New Negative Regulator of the Drosophila Immune-Deficiency Pathway at the Level of TAK1, Reduces Life Span

    doi: 10.1371/journal.pgen.1004117

    Figure Lengend Snippet: Lys 142 and Lys 156 of dTAK1 are essential for immune signalling. (A) Sequence alignment of 1 to 230 amino acids of human TAK1 (hTAK1) and Drosophila TAK1 (dTAK1). Lys 142 (dark grey) and 156 (light grey) are indicated (arrows) and the kinase activation loop is underlined. (B) Drosophila TAK1 K142R and TAK1 K156R mutants failed to activate diptericin . Drosophila TAK1 or dTAK1 K142R or dTAK1 K156R mutant was expressed along with or without dTAB2, in S2 cells in combinations shown. Diptericin expression was assayed 48 hrs post-transfection. Error bars represent Standard Error of 3 separate experiments. *p

    Article Snippet: Drosophila S2 cells (Invitrogen) were maintained at 25°C in Schneider's Drosophila Medium (BioWhittaker/Lonza), supplemented with 10% heat-inactivated FBS and antibiotics 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate – all Invitrogen).

    Techniques: Sequencing, Activation Assay, Mutagenesis, Expressing, Transfection

    Slh is not required for mitophagy or endocytosis. (A) Live-cell imaging of control S2 cells and cells knocked down for Slh, treated for 2 h with 5 μM FCCP and stained with Mitotracker Green and Lysotracker Red. The two probes overlap almost completely in both cases, indicating that Slh knockdown does not interfere with global mitophagy. (B) Visualization of acidified late endosomes and endolysosomes in untreated S2 cells and in cells knocked down for Bet1 or Slh or treated with an inert dsRNA targeted on GFP. Red fluorescence due to the pH-sensitive nonpermeant dextran-conjugated dye pHrodo Red Dextran is visible in all cells.

    Journal: Molecular Biology of the Cell

    Article Title: Intracellular vesicle trafficking plays an essential role in mitochondrial quality control

    doi: 10.1091/mbc.E17-10-0619

    Figure Lengend Snippet: Slh is not required for mitophagy or endocytosis. (A) Live-cell imaging of control S2 cells and cells knocked down for Slh, treated for 2 h with 5 μM FCCP and stained with Mitotracker Green and Lysotracker Red. The two probes overlap almost completely in both cases, indicating that Slh knockdown does not interfere with global mitophagy. (B) Visualization of acidified late endosomes and endolysosomes in untreated S2 cells and in cells knocked down for Bet1 or Slh or treated with an inert dsRNA targeted on GFP. Red fluorescence due to the pH-sensitive nonpermeant dextran-conjugated dye pHrodo Red Dextran is visible in all cells.

    Article Snippet: Drosophila S2 cells (Invitrogen) were cultured under standard conditions in Schneider’s medium (Sigma).

    Techniques: Live Cell Imaging, Staining, Fluorescence

    Slh, Bet1, or CG10144 knockdown does not affect mtDNA copy number, topology, or integrity. (A) Relative RNA levels of the indicated genes, normalized against RpL32 RNA and then renormalized against the values for untreated S2 cells, and (B) relative mtDNA copy number (based on qPCR), also normalized against the values for untreated S2 cells. Means ± SD, significant differences from control cells (Student’s  t  test with Bonferroni correction,  n  ≥ 4 biological replicates) denoted by * ( p

    Journal: Molecular Biology of the Cell

    Article Title: Intracellular vesicle trafficking plays an essential role in mitochondrial quality control

    doi: 10.1091/mbc.E17-10-0619

    Figure Lengend Snippet: Slh, Bet1, or CG10144 knockdown does not affect mtDNA copy number, topology, or integrity. (A) Relative RNA levels of the indicated genes, normalized against RpL32 RNA and then renormalized against the values for untreated S2 cells, and (B) relative mtDNA copy number (based on qPCR), also normalized against the values for untreated S2 cells. Means ± SD, significant differences from control cells (Student’s t test with Bonferroni correction, n ≥ 4 biological replicates) denoted by * ( p

    Article Snippet: Drosophila S2 cells (Invitrogen) were cultured under standard conditions in Schneider’s medium (Sigma).

    Techniques: Real-time Polymerase Chain Reaction

    Slh is required for Bet1 coassociation with lysosomal and mitochondrial components. (A) Representative live-cell images of cells transfected with Bet1-BFP, either alone (control S2 cells, con, panels a–d) or with concomitant knockdown of Slh (panels e–h), costained with Lysotracker Red and Mitotracker Green, together with (inset) zoomed images at higher magnification. White arrows indicate vesicles positive for Mitotracker Green and Lysotracker Red, which, in control cells, but not Slh knockdown cells, were mostly positive for Bet1-GFP. Images were optimized on the microscope for brightness and contrast, but have not been manipulated in any other way, apart from the addition of panel labels and arrows as referred to here and in the text. (B) Quantitation (%) of structures costained with Mitotracker Green and Lysotracker Red, which were also positive for Bet1-BFP, based on inspection of all visibly fluorescent structures in eight cells of each class analyzed. con, control S2 cells transfected with Bet1-BFP; Slh KD, cells also knocked down for Slh. * denotes a statistically significant difference,  p

    Journal: Molecular Biology of the Cell

    Article Title: Intracellular vesicle trafficking plays an essential role in mitochondrial quality control

    doi: 10.1091/mbc.E17-10-0619

    Figure Lengend Snippet: Slh is required for Bet1 coassociation with lysosomal and mitochondrial components. (A) Representative live-cell images of cells transfected with Bet1-BFP, either alone (control S2 cells, con, panels a–d) or with concomitant knockdown of Slh (panels e–h), costained with Lysotracker Red and Mitotracker Green, together with (inset) zoomed images at higher magnification. White arrows indicate vesicles positive for Mitotracker Green and Lysotracker Red, which, in control cells, but not Slh knockdown cells, were mostly positive for Bet1-GFP. Images were optimized on the microscope for brightness and contrast, but have not been manipulated in any other way, apart from the addition of panel labels and arrows as referred to here and in the text. (B) Quantitation (%) of structures costained with Mitotracker Green and Lysotracker Red, which were also positive for Bet1-BFP, based on inspection of all visibly fluorescent structures in eight cells of each class analyzed. con, control S2 cells transfected with Bet1-BFP; Slh KD, cells also knocked down for Slh. * denotes a statistically significant difference, p

    Article Snippet: Drosophila S2 cells (Invitrogen) were cultured under standard conditions in Schneider’s medium (Sigma).

    Techniques: Transfection, Microscopy, Quantitation Assay

    Post-Golgi components of the secretory pathway are not needed to maintain mitochondrial quality and cell growth. (A) Relative cell number, compared with control cells, after 5 d of dsRNA treatment against the indicated genes. See also Supplemental Figure S3. (B) Relative level of Hsp22 RNA, normalized against RpL32 RNA and renormalized against the values for untreated S2 cells, after dsRNA treatment against the indicated genes. Because there were so few remaining viable cells after 5 d of βCOP knockdown, RNA levels were assessed after 2 d of knockdown. Means ± SD; significant differences from control cells (Student’s  t  test with Bonferroni correction,  n  ≥ 4 biological replicates) denoted by * ( p

    Journal: Molecular Biology of the Cell

    Article Title: Intracellular vesicle trafficking plays an essential role in mitochondrial quality control

    doi: 10.1091/mbc.E17-10-0619

    Figure Lengend Snippet: Post-Golgi components of the secretory pathway are not needed to maintain mitochondrial quality and cell growth. (A) Relative cell number, compared with control cells, after 5 d of dsRNA treatment against the indicated genes. See also Supplemental Figure S3. (B) Relative level of Hsp22 RNA, normalized against RpL32 RNA and renormalized against the values for untreated S2 cells, after dsRNA treatment against the indicated genes. Because there were so few remaining viable cells after 5 d of βCOP knockdown, RNA levels were assessed after 2 d of knockdown. Means ± SD; significant differences from control cells (Student’s t test with Bonferroni correction, n ≥ 4 biological replicates) denoted by * ( p

    Article Snippet: Drosophila S2 cells (Invitrogen) were cultured under standard conditions in Schneider’s medium (Sigma).

    Techniques:

    Slh knockdown impairs mitochondrial functions. (A) Mitochondrial O 2  consumption, normalized against cell number, (B) TMRM fluorescence per cell, (C) Mitotracker Green fluorescence per cell, (D, E) time course of changes in (D) TMRM fluorescence and (E) Mitotracker Green fluorescence per cell, and (F) relative RNA levels of the indicated genes, normalized against RpL32 RNA; all renormalized against the values for untreated S2 cells, in cells treated with dsRNAs against the indicated genes for the number of days shown. (A–C, F) Means ± SD, significant differences from control cells (Student’s  t  test with Bonferroni correction,  n  ≥ 4 biological replicates) denoted by * ( p

    Journal: Molecular Biology of the Cell

    Article Title: Intracellular vesicle trafficking plays an essential role in mitochondrial quality control

    doi: 10.1091/mbc.E17-10-0619

    Figure Lengend Snippet: Slh knockdown impairs mitochondrial functions. (A) Mitochondrial O 2 consumption, normalized against cell number, (B) TMRM fluorescence per cell, (C) Mitotracker Green fluorescence per cell, (D, E) time course of changes in (D) TMRM fluorescence and (E) Mitotracker Green fluorescence per cell, and (F) relative RNA levels of the indicated genes, normalized against RpL32 RNA; all renormalized against the values for untreated S2 cells, in cells treated with dsRNAs against the indicated genes for the number of days shown. (A–C, F) Means ± SD, significant differences from control cells (Student’s t test with Bonferroni correction, n ≥ 4 biological replicates) denoted by * ( p

    Article Snippet: Drosophila S2 cells (Invitrogen) were cultured under standard conditions in Schneider’s medium (Sigma).

    Techniques: Fluorescence

    Slh and Bet1 knockdown entrains cell growth defects. (A) Growth curves of control S2 cells and cells treated continuously for 10 d, with dsRNAs targeted against the indicated genes, commencing 5 d after transfection. For source data, including SD and statistical analysis, following normalization to cell number on day 5, see Supplemental Table S1. (B) Cell-cycle analysis of control S2 cells and cells knocked down for 10 d by dsRNAs targeted on the indicated genes. (C) Micrographs of control S2 cells and cells after 5 d of treatment with dsRNAs targeted against the indicated genes. Scale bars: 20 μm. (D) Cell diameter of control S2 cells and cells knocked down for the indicated genes (means ± SD of 52 cells of each class).  # , * denote significant differences from the corresponding data class of control cells (Student’s  t  test with Bonferroni correction,  p

    Journal: Molecular Biology of the Cell

    Article Title: Intracellular vesicle trafficking plays an essential role in mitochondrial quality control

    doi: 10.1091/mbc.E17-10-0619

    Figure Lengend Snippet: Slh and Bet1 knockdown entrains cell growth defects. (A) Growth curves of control S2 cells and cells treated continuously for 10 d, with dsRNAs targeted against the indicated genes, commencing 5 d after transfection. For source data, including SD and statistical analysis, following normalization to cell number on day 5, see Supplemental Table S1. (B) Cell-cycle analysis of control S2 cells and cells knocked down for 10 d by dsRNAs targeted on the indicated genes. (C) Micrographs of control S2 cells and cells after 5 d of treatment with dsRNAs targeted against the indicated genes. Scale bars: 20 μm. (D) Cell diameter of control S2 cells and cells knocked down for the indicated genes (means ± SD of 52 cells of each class). # , * denote significant differences from the corresponding data class of control cells (Student’s t test with Bonferroni correction, p

    Article Snippet: Drosophila S2 cells (Invitrogen) were cultured under standard conditions in Schneider’s medium (Sigma).

    Techniques: Transfection, Cell Cycle Assay

    CrPV-3(R146A) infection is attenuated in Drosophila S2 cells. Autoradiography of [ 35 S]Met/Cys pulse-labeled protein lysates from S2 cells infected with CrPV-3 (A) or CrPV-3(R146A) (B) at an MOI of 10 resolved on a 12% SDS-PAGE gel. Cells were metabolically

    Journal:

    Article Title: Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein

    doi: 10.1128/JVI.01779-16

    Figure Lengend Snippet: CrPV-3(R146A) infection is attenuated in Drosophila S2 cells. Autoradiography of [ 35 S]Met/Cys pulse-labeled protein lysates from S2 cells infected with CrPV-3 (A) or CrPV-3(R146A) (B) at an MOI of 10 resolved on a 12% SDS-PAGE gel. Cells were metabolically

    Article Snippet: Drosophila S2 cells (Invitrogen) were maintained as described in reference .

    Techniques: Infection, Autoradiography, Labeling, SDS Page, Metabolic Labelling

    Addition of actinomycin D during CrPV-3(R146A) infection inhibits SG assembly. (A) S2 cells stably expressing RinGFP (green fluorescence) pretreated with 5 μg/μl ActD or DMSO for 1 h were incubated with 50 nM PatA for 1 h to induce SGs.

    Journal:

    Article Title: Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein

    doi: 10.1128/JVI.01779-16

    Figure Lengend Snippet: Addition of actinomycin D during CrPV-3(R146A) infection inhibits SG assembly. (A) S2 cells stably expressing RinGFP (green fluorescence) pretreated with 5 μg/μl ActD or DMSO for 1 h were incubated with 50 nM PatA for 1 h to induce SGs.

    Article Snippet: Drosophila S2 cells (Invitrogen) were maintained as described in reference .

    Techniques: Infection, Stable Transfection, Expressing, Fluorescence, Incubation

    CrPV-1A modulates SG assembly during virus infection. (A) Stable RinGFP S2 cells were mock infected or infected for 2, 4, 6, or 8 h with CrPV-3 or CrPV-3(R146A) virus at an MOI of 50. Cells were fixed and stained with Hoechst (blue fluorescence). Percentages

    Journal:

    Article Title: Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein

    doi: 10.1128/JVI.01779-16

    Figure Lengend Snippet: CrPV-1A modulates SG assembly during virus infection. (A) Stable RinGFP S2 cells were mock infected or infected for 2, 4, 6, or 8 h with CrPV-3 or CrPV-3(R146A) virus at an MOI of 50. Cells were fixed and stained with Hoechst (blue fluorescence). Percentages

    Article Snippet: Drosophila S2 cells (Invitrogen) were maintained as described in reference .

    Techniques: Infection, Staining, Fluorescence

    R146 residue is important for transcriptional repression during CrPV infection in S2 cells. (A) Scheme of metabolic mRNA labeling in mock-, CrPV-3-, or CrPV-3(R146A)-infected S2 cells. Mock-, CrPV-3-, or CrPV-3(R146A)-infected S2 cells (MOI of 50) (adsorbed

    Journal:

    Article Title: Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein

    doi: 10.1128/JVI.01779-16

    Figure Lengend Snippet: R146 residue is important for transcriptional repression during CrPV infection in S2 cells. (A) Scheme of metabolic mRNA labeling in mock-, CrPV-3-, or CrPV-3(R146A)-infected S2 cells. Mock-, CrPV-3-, or CrPV-3(R146A)-infected S2 cells (MOI of 50) (adsorbed

    Article Snippet: Drosophila S2 cells (Invitrogen) were maintained as described in reference .

    Techniques: Infection, Labeling

    Pateamine A treatment induces stress granules in CrPV-3(R146A)-infected S2 cells. (A) Infected cells were treated with 50 nM PatA to induce SGs for 1 h before the indicated time postinfection. (B) Box plot of the number of RinGFP foci in individual cells,

    Journal:

    Article Title: Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein

    doi: 10.1128/JVI.01779-16

    Figure Lengend Snippet: Pateamine A treatment induces stress granules in CrPV-3(R146A)-infected S2 cells. (A) Infected cells were treated with 50 nM PatA to induce SGs for 1 h before the indicated time postinfection. (B) Box plot of the number of RinGFP foci in individual cells,

    Article Snippet: Drosophila S2 cells (Invitrogen) were maintained as described in reference .

    Techniques: Infection

    CrPV-1A inhibits PatA-induced SG assembly. (A) S2 cells stably expressing a fusion RinGFP protein (green fluorescence) were transiently transfected with RNAs encoding CrPV-1A-HA or CrPV-1A(R146A)-HA for 9 h and then treated with DMSO or 50 nM pateamine

    Journal:

    Article Title: Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein

    doi: 10.1128/JVI.01779-16

    Figure Lengend Snippet: CrPV-1A inhibits PatA-induced SG assembly. (A) S2 cells stably expressing a fusion RinGFP protein (green fluorescence) were transiently transfected with RNAs encoding CrPV-1A-HA or CrPV-1A(R146A)-HA for 9 h and then treated with DMSO or 50 nM pateamine

    Article Snippet: Drosophila S2 cells (Invitrogen) were maintained as described in reference .

    Techniques: Stable Transfection, Expressing, Fluorescence, Transfection, Hemagglutination Assay

    Requirement for UNC-45 during cardiac remodeling. ( A–F ) 1 week old adult hearts were analyzed after unc-45 KD before or after metamorphosis. The left two bars show data from control and unc-45 KD hearts when KD occurred prior to metamorphosis, i.e., starting at the third instar larval stage. The right two bars in each panel represent data from hearts when KD was performed after metamorphosis, i.e., in the adults. Data show mean values ± SD; statistical significance was determined using an unpaired Student's t test (*** = p

    Journal: PLoS ONE

    Article Title: The UNC-45 Chaperone Is Critical for Establishing Myosin-Based Myofibrillar Organization and Cardiac Contractility in the Drosophila Heart Model

    doi: 10.1371/journal.pone.0022579

    Figure Lengend Snippet: Requirement for UNC-45 during cardiac remodeling. ( A–F ) 1 week old adult hearts were analyzed after unc-45 KD before or after metamorphosis. The left two bars show data from control and unc-45 KD hearts when KD occurred prior to metamorphosis, i.e., starting at the third instar larval stage. The right two bars in each panel represent data from hearts when KD was performed after metamorphosis, i.e., in the adults. Data show mean values ± SD; statistical significance was determined using an unpaired Student's t test (*** = p

    Article Snippet: Extracted samples from equal numbers of hearts, indirect flight muscles or embryos were separated by 10% SDS-PAGE and immunoblotting was carried out using a rabbit anti-Drosophila UNC-45 antibody as recently described and mouse anti β-actin antibody (Sigma, St. Louis, MO).

    Techniques:

    M-Mode analysis and quantification of heart rate in unc-45 KD and control third instar larvae, young pupae and adult hearts. Cardiac parameters were determined as described in Materials and Methods . ( A, B ) M-modes (4 sec) from control (top) and unc-45 KD (bottom) third instar larvae and young pupae (as described in Figure 1 ). ( C ) Mean heart rate of unc-45 KD in third instar larvae and young pupae were significantly increased (tachycardia) compared to control. In contrast, heart rates were significantly reduced in adult hearts compared to controls. Heart rate data are shown as means ± SD; statistical significance was determined using a multivariate Student's t test (** = p

    Journal: PLoS ONE

    Article Title: The UNC-45 Chaperone Is Critical for Establishing Myosin-Based Myofibrillar Organization and Cardiac Contractility in the Drosophila Heart Model

    doi: 10.1371/journal.pone.0022579

    Figure Lengend Snippet: M-Mode analysis and quantification of heart rate in unc-45 KD and control third instar larvae, young pupae and adult hearts. Cardiac parameters were determined as described in Materials and Methods . ( A, B ) M-modes (4 sec) from control (top) and unc-45 KD (bottom) third instar larvae and young pupae (as described in Figure 1 ). ( C ) Mean heart rate of unc-45 KD in third instar larvae and young pupae were significantly increased (tachycardia) compared to control. In contrast, heart rates were significantly reduced in adult hearts compared to controls. Heart rate data are shown as means ± SD; statistical significance was determined using a multivariate Student's t test (** = p

    Article Snippet: Extracted samples from equal numbers of hearts, indirect flight muscles or embryos were separated by 10% SDS-PAGE and immunoblotting was carried out using a rabbit anti-Drosophila UNC-45 antibody as recently described and mouse anti β-actin antibody (Sigma, St. Louis, MO).

    Techniques: Size-exclusion Chromatography

    Structural dysfunction in unc-45 KD hearts due to impaired myosin accumulation. Immunofluorescence micrographs of 1 week old flies are shown. ( A, C ) Hearts from controls and ( B, D ) unc-45 KD flies were probed with antibody against muscle myosin. Control cardiac tubes show typical spiral myofibrillar arrangements within the cardiomyocytes (arrow). Myofibrillar organization is completely disrupted in the conical chamber and third segment of unc-45 KD cardiac tubes (indicated by *) with loss of most myosin-containing myofibrils and significant dilation. Enlarged image ( D ) of the third abdominal segment of an unc-45 KD heart demonstrates only a few myofibrils and gaps (missing myofibrils, indicated by *). Scale bars: 100 µM in A–B and 75 µM in C–D. ( E ) Myosin expression is significantly reduced in 1 week old unc-45 KD hearts, however, actin expression remain unchanged as shown by SDS-PAGE.

    Journal: PLoS ONE

    Article Title: The UNC-45 Chaperone Is Critical for Establishing Myosin-Based Myofibrillar Organization and Cardiac Contractility in the Drosophila Heart Model

    doi: 10.1371/journal.pone.0022579

    Figure Lengend Snippet: Structural dysfunction in unc-45 KD hearts due to impaired myosin accumulation. Immunofluorescence micrographs of 1 week old flies are shown. ( A, C ) Hearts from controls and ( B, D ) unc-45 KD flies were probed with antibody against muscle myosin. Control cardiac tubes show typical spiral myofibrillar arrangements within the cardiomyocytes (arrow). Myofibrillar organization is completely disrupted in the conical chamber and third segment of unc-45 KD cardiac tubes (indicated by *) with loss of most myosin-containing myofibrils and significant dilation. Enlarged image ( D ) of the third abdominal segment of an unc-45 KD heart demonstrates only a few myofibrils and gaps (missing myofibrils, indicated by *). Scale bars: 100 µM in A–B and 75 µM in C–D. ( E ) Myosin expression is significantly reduced in 1 week old unc-45 KD hearts, however, actin expression remain unchanged as shown by SDS-PAGE.

    Article Snippet: Extracted samples from equal numbers of hearts, indirect flight muscles or embryos were separated by 10% SDS-PAGE and immunoblotting was carried out using a rabbit anti-Drosophila UNC-45 antibody as recently described and mouse anti β-actin antibody (Sigma, St. Louis, MO).

    Techniques: Immunofluorescence, Expressing, SDS Page

    Physiological cardiac defects associated with unc-45 KD. Cardiac parameters from the region between the second and third abdominal segments of the heart were determined as described in Materials and Methods . ( A, B ) Hearts from 1, 2 and 3 week old unc-45 KD flies show prolonged systolic and diastolic intervals compared to control hearts. ( C, D ) Systolic and diastolic diameters of the KD hearts were significantly higher compared to those of age-matched controls. ( E ) Contractility (% FS) of the unc-45 KD hearts was significantly reduced at all ages ( F ) Significant cardiac arrhythmias were observed in unc-45 KD hearts (quantified as arrhythmia index, see Materials and Methods ). Data are shown as means ± SD; statistical significance was determined using a multivariate Student's t test (*** = p

    Journal: PLoS ONE

    Article Title: The UNC-45 Chaperone Is Critical for Establishing Myosin-Based Myofibrillar Organization and Cardiac Contractility in the Drosophila Heart Model

    doi: 10.1371/journal.pone.0022579

    Figure Lengend Snippet: Physiological cardiac defects associated with unc-45 KD. Cardiac parameters from the region between the second and third abdominal segments of the heart were determined as described in Materials and Methods . ( A, B ) Hearts from 1, 2 and 3 week old unc-45 KD flies show prolonged systolic and diastolic intervals compared to control hearts. ( C, D ) Systolic and diastolic diameters of the KD hearts were significantly higher compared to those of age-matched controls. ( E ) Contractility (% FS) of the unc-45 KD hearts was significantly reduced at all ages ( F ) Significant cardiac arrhythmias were observed in unc-45 KD hearts (quantified as arrhythmia index, see Materials and Methods ). Data are shown as means ± SD; statistical significance was determined using a multivariate Student's t test (*** = p

    Article Snippet: Extracted samples from equal numbers of hearts, indirect flight muscles or embryos were separated by 10% SDS-PAGE and immunoblotting was carried out using a rabbit anti-Drosophila UNC-45 antibody as recently described and mouse anti β-actin antibody (Sigma, St. Louis, MO).

    Techniques:

    Mhc KD mimics the unc-45 KD cardiac phenotype. Cardiac physiological parameters in hearts from 1 week old Mhc KD flies. ( A, B ) systolic and diastolic intervals were prolonged, ( C, D ) diastolic and systolic cardiac diameters were increased, ( E ) % FS was decreased (indicating reduced cardiac efficiency) and ( F ) cardiac arrhythmias were increased compared to age-matched controls. Data are shown as means ± SD; statistical significance was determined using a multivariate Student's t test (*** = p

    Journal: PLoS ONE

    Article Title: The UNC-45 Chaperone Is Critical for Establishing Myosin-Based Myofibrillar Organization and Cardiac Contractility in the Drosophila Heart Model

    doi: 10.1371/journal.pone.0022579

    Figure Lengend Snippet: Mhc KD mimics the unc-45 KD cardiac phenotype. Cardiac physiological parameters in hearts from 1 week old Mhc KD flies. ( A, B ) systolic and diastolic intervals were prolonged, ( C, D ) diastolic and systolic cardiac diameters were increased, ( E ) % FS was decreased (indicating reduced cardiac efficiency) and ( F ) cardiac arrhythmias were increased compared to age-matched controls. Data are shown as means ± SD; statistical significance was determined using a multivariate Student's t test (*** = p

    Article Snippet: Extracted samples from equal numbers of hearts, indirect flight muscles or embryos were separated by 10% SDS-PAGE and immunoblotting was carried out using a rabbit anti-Drosophila UNC-45 antibody as recently described and mouse anti β-actin antibody (Sigma, St. Louis, MO).

    Techniques:

    Cardiac defects associated with KD of unc-45 . ( A ) Immunoblot analysis of UNC-45 expression in hearts from control ( Hand /+) and unc-45 KD ( Hand > UAS- unc-45 RNAi-1 (NIG) and Hand > UAS- unc-45 RNAi-2 (Vienna Drosophila RNAi Center) flies. UNC-45 expression was reduced significantly (∼80%) in the KD hearts; however, actin expression remained unchanged. ( B ) Images of semi-intact conical chamber (CC) and cardiac tubes in third abdominal segments during systole and M-mode records from 1 week old control and unc-45 KD flies. KD hearts showed significant dilation in the third abdominal segment of the cardiac tube, which was also apparent in M-mode analysis. M-mode records show heart wall movements over 15 and 5 (inset) sec time periods. Double-headed arrows in the M-mode traces indicate diastolic and systolic distances between heart walls. In addition to dilation, unc-45 KD hearts developed arrhythmias (prolonged beating and episodes of fibrillation) whereas control hearts did not. ( C ) Cardiac-specific KD of unc-45 directly impacted the life span of the flies; graph plots % survival (n = 250 for each group) vs. time post-eclosion.

    Journal: PLoS ONE

    Article Title: The UNC-45 Chaperone Is Critical for Establishing Myosin-Based Myofibrillar Organization and Cardiac Contractility in the Drosophila Heart Model

    doi: 10.1371/journal.pone.0022579

    Figure Lengend Snippet: Cardiac defects associated with KD of unc-45 . ( A ) Immunoblot analysis of UNC-45 expression in hearts from control ( Hand /+) and unc-45 KD ( Hand > UAS- unc-45 RNAi-1 (NIG) and Hand > UAS- unc-45 RNAi-2 (Vienna Drosophila RNAi Center) flies. UNC-45 expression was reduced significantly (∼80%) in the KD hearts; however, actin expression remained unchanged. ( B ) Images of semi-intact conical chamber (CC) and cardiac tubes in third abdominal segments during systole and M-mode records from 1 week old control and unc-45 KD flies. KD hearts showed significant dilation in the third abdominal segment of the cardiac tube, which was also apparent in M-mode analysis. M-mode records show heart wall movements over 15 and 5 (inset) sec time periods. Double-headed arrows in the M-mode traces indicate diastolic and systolic distances between heart walls. In addition to dilation, unc-45 KD hearts developed arrhythmias (prolonged beating and episodes of fibrillation) whereas control hearts did not. ( C ) Cardiac-specific KD of unc-45 directly impacted the life span of the flies; graph plots % survival (n = 250 for each group) vs. time post-eclosion.

    Article Snippet: Extracted samples from equal numbers of hearts, indirect flight muscles or embryos were separated by 10% SDS-PAGE and immunoblotting was carried out using a rabbit anti-Drosophila UNC-45 antibody as recently described and mouse anti β-actin antibody (Sigma, St. Louis, MO).

    Techniques: Expressing, Size-exclusion Chromatography

    Transgenic over-expression of UNC-45 rescues defects associated with unc-45 KD. ( A ) Assessment of cardiac diastolic and systolic intervals, ( B ) diastolic and systolic diameters, ( C ) % FS and cardiac arrhythmia, in 1 week control, unc-45 KD and rescued old flies. Statistical difference of unc-45 KD from control and rescued as well as between KD and rescued are represented as mean ± SD; where *** = p

    Journal: PLoS ONE

    Article Title: The UNC-45 Chaperone Is Critical for Establishing Myosin-Based Myofibrillar Organization and Cardiac Contractility in the Drosophila Heart Model

    doi: 10.1371/journal.pone.0022579

    Figure Lengend Snippet: Transgenic over-expression of UNC-45 rescues defects associated with unc-45 KD. ( A ) Assessment of cardiac diastolic and systolic intervals, ( B ) diastolic and systolic diameters, ( C ) % FS and cardiac arrhythmia, in 1 week control, unc-45 KD and rescued old flies. Statistical difference of unc-45 KD from control and rescued as well as between KD and rescued are represented as mean ± SD; where *** = p

    Article Snippet: Extracted samples from equal numbers of hearts, indirect flight muscles or embryos were separated by 10% SDS-PAGE and immunoblotting was carried out using a rabbit anti-Drosophila UNC-45 antibody as recently described and mouse anti β-actin antibody (Sigma, St. Louis, MO).

    Techniques: Transgenic Assay, Over Expression

    Integrins and talin modulate the JNK response to mechanical stress. S2R+ cells transiently transfected with dJun-FRET biosensors were plated on collagen- (A – treated with Mys dsRNA and C – treated with talin dsRNA) or Con-A-coated (B) silicone membranes on a Stage Flexer set up. Donor mCFP FLs were collected before stretching (blue) and at 3 hours after continuous static stretch (green). In each graph are also represented the data obtained for untreated cells plated on collagen-coated silicone membranes (black– before stretch; red– 3 hours after continuous stretch). In the absence of Mys, talin or by plating cells on Con-A, the activity of JNK increases in relation to WT cells at rest. However, only the talin minus cells were able to increase their dJun-FRET sensor activity upon stretch. Mys minus cells plated on collagen-coated silicone membranes show a pseudopolygonal shape and emit multiple thick short protrusions (D). They rounded up upon stretch emitting very thin filopodia (G). Talin minus cells were more rounded and showed few, long branched filopodia (F). They collapsed in response to stretch showing occasional, very short, thin protrusions (I). Cells plated on Con-A-coated silicone membranes showed a flat shape (E) that was not affected by mechanical stress (H). The data acquired suggests a hierarchical model for the roles of β-integrin and talin regulating the level of Jun sensor activation of S2R+ cells and its response to mechanical stress (J). In the absence of mechanical input both β-integrin and talin restrain the activity of the JNK signaling. In the absence of any of them, the pathway gets moderately activated in response to an independent input. Mechanical stress results in β-integrin activation and the establishment of a, probably talin-independent, positive contribution to JNK signaling leading to a maximum level of dJun-FRET biosensor activity.

    Journal: PLoS ONE

    Article Title: Integrin-Dependent Activation of the JNK Signaling Pathway by Mechanical Stress

    doi: 10.1371/journal.pone.0026182

    Figure Lengend Snippet: Integrins and talin modulate the JNK response to mechanical stress. S2R+ cells transiently transfected with dJun-FRET biosensors were plated on collagen- (A – treated with Mys dsRNA and C – treated with talin dsRNA) or Con-A-coated (B) silicone membranes on a Stage Flexer set up. Donor mCFP FLs were collected before stretching (blue) and at 3 hours after continuous static stretch (green). In each graph are also represented the data obtained for untreated cells plated on collagen-coated silicone membranes (black– before stretch; red– 3 hours after continuous stretch). In the absence of Mys, talin or by plating cells on Con-A, the activity of JNK increases in relation to WT cells at rest. However, only the talin minus cells were able to increase their dJun-FRET sensor activity upon stretch. Mys minus cells plated on collagen-coated silicone membranes show a pseudopolygonal shape and emit multiple thick short protrusions (D). They rounded up upon stretch emitting very thin filopodia (G). Talin minus cells were more rounded and showed few, long branched filopodia (F). They collapsed in response to stretch showing occasional, very short, thin protrusions (I). Cells plated on Con-A-coated silicone membranes showed a flat shape (E) that was not affected by mechanical stress (H). The data acquired suggests a hierarchical model for the roles of β-integrin and talin regulating the level of Jun sensor activation of S2R+ cells and its response to mechanical stress (J). In the absence of mechanical input both β-integrin and talin restrain the activity of the JNK signaling. In the absence of any of them, the pathway gets moderately activated in response to an independent input. Mechanical stress results in β-integrin activation and the establishment of a, probably talin-independent, positive contribution to JNK signaling leading to a maximum level of dJun-FRET biosensor activity.

    Article Snippet: Drosophila S2R+ cells were grown routinely in Schneider's Drosophila medium (GIBCO, Invitrogen) supplemented with 10% heat inactivated Fetal Bovine Serum (GIBCO, Invitrogen) at 25°C.

    Techniques: Transfection, Activity Assay, Activation Assay

    FRET-FLIM quantification of dJun-FRET biosensor is a readout of the activity of the JNK pathway in response to chemical agonists and antagonists. S2R+ cells were transiently transfected with dJun-FRET (A) biosensor, and fluorescence lifetimes (FL) of mCFP were collected 48 hours post transfection. Cells were left untreated (black) or subjected to treatment with LPS, a JNK signaling activator (red) or L-JNKI1, a JNK inhibitor (blue) for 2 hours before FLIM measurements. Curves represent FLIM data recorded from ∼75 cells for each condition. The chemical activator and inhibitor modulated the donor FL of dJun-FRET, while no effect was observed on the controls. A direct measurement of sensor activity on S2R+ cells plated on plastic was performed. Untreated cells (B) or those treated with LPS (C) or L-JNKI1 (D) were stained with anti-Phospho-c-Jun antibody and phalloidin-TRITC. P-Jun staining was quantified by calculation of the average integrated density (the product of Area and Mean Gray Value) of ∼100 cells (nucleus and cytoplasm) (E). LPS treatment led to morphological changes in S2R+ cells (from a pseudopolygonal flat shape to a filopodia-rich compacted aspect) and a statistically significant (p

    Journal: PLoS ONE

    Article Title: Integrin-Dependent Activation of the JNK Signaling Pathway by Mechanical Stress

    doi: 10.1371/journal.pone.0026182

    Figure Lengend Snippet: FRET-FLIM quantification of dJun-FRET biosensor is a readout of the activity of the JNK pathway in response to chemical agonists and antagonists. S2R+ cells were transiently transfected with dJun-FRET (A) biosensor, and fluorescence lifetimes (FL) of mCFP were collected 48 hours post transfection. Cells were left untreated (black) or subjected to treatment with LPS, a JNK signaling activator (red) or L-JNKI1, a JNK inhibitor (blue) for 2 hours before FLIM measurements. Curves represent FLIM data recorded from ∼75 cells for each condition. The chemical activator and inhibitor modulated the donor FL of dJun-FRET, while no effect was observed on the controls. A direct measurement of sensor activity on S2R+ cells plated on plastic was performed. Untreated cells (B) or those treated with LPS (C) or L-JNKI1 (D) were stained with anti-Phospho-c-Jun antibody and phalloidin-TRITC. P-Jun staining was quantified by calculation of the average integrated density (the product of Area and Mean Gray Value) of ∼100 cells (nucleus and cytoplasm) (E). LPS treatment led to morphological changes in S2R+ cells (from a pseudopolygonal flat shape to a filopodia-rich compacted aspect) and a statistically significant (p

    Article Snippet: Drosophila S2R+ cells were grown routinely in Schneider's Drosophila medium (GIBCO, Invitrogen) supplemented with 10% heat inactivated Fetal Bovine Serum (GIBCO, Invitrogen) at 25°C.

    Techniques: Activity Assay, Transfection, Fluorescence, Staining

    Attachment dependent activation of the JNK pathway. S2R+ cells transiently transfected with dJun-FRET biosensor (A) or the controls mCFP alone (B) and mCFP-dJun (C) were plated on different surfaces and the mCFP donor FLs were collected 48 hours post transfection. dJun-FRET biosensor activity (A) was highest on cells plated on glass (black), and much less pronounced on collagen-coated glass (red), and on Con-A-coated glass (blue). FL of control biosensors was not affected by the substrate (panels B, C).

    Journal: PLoS ONE

    Article Title: Integrin-Dependent Activation of the JNK Signaling Pathway by Mechanical Stress

    doi: 10.1371/journal.pone.0026182

    Figure Lengend Snippet: Attachment dependent activation of the JNK pathway. S2R+ cells transiently transfected with dJun-FRET biosensor (A) or the controls mCFP alone (B) and mCFP-dJun (C) were plated on different surfaces and the mCFP donor FLs were collected 48 hours post transfection. dJun-FRET biosensor activity (A) was highest on cells plated on glass (black), and much less pronounced on collagen-coated glass (red), and on Con-A-coated glass (blue). FL of control biosensors was not affected by the substrate (panels B, C).

    Article Snippet: Drosophila S2R+ cells were grown routinely in Schneider's Drosophila medium (GIBCO, Invitrogen) supplemented with 10% heat inactivated Fetal Bovine Serum (GIBCO, Invitrogen) at 25°C.

    Techniques: Activation Assay, Transfection, Activity Assay

    S2R+ cells exhibit different morphologies and matrix attachments on different substrates. S2R+ cells were plated on glass (A, D), collagen-coated glass (B, E) and Con-A-coated glass (C, F). They were stained with phalloidin-TRITC (A–C) and anti- β-integrin antibodies (D–F) and visualized by fluorescence confocal microscopy. S2R+ cells plated on glass shrank around their nuclei showing extensive actin-rich lamellipodia and occasional short filopodia and punctate β-integrin expression at the margins (arrowhead) and dispersed all over the cytoplasm and the cell periphery. Cells plated on collagen-coated glass show abundant long and thin protrusions with punctate β-integrin at their tips (arrowheads). Cells plated on Con-A-coated glass show spread out morphologies, dense peripheral actin staining and circular bundles of actin around the nucleus. These cells present diffuse, just above background, β-integrin staining.

    Journal: PLoS ONE

    Article Title: Integrin-Dependent Activation of the JNK Signaling Pathway by Mechanical Stress

    doi: 10.1371/journal.pone.0026182

    Figure Lengend Snippet: S2R+ cells exhibit different morphologies and matrix attachments on different substrates. S2R+ cells were plated on glass (A, D), collagen-coated glass (B, E) and Con-A-coated glass (C, F). They were stained with phalloidin-TRITC (A–C) and anti- β-integrin antibodies (D–F) and visualized by fluorescence confocal microscopy. S2R+ cells plated on glass shrank around their nuclei showing extensive actin-rich lamellipodia and occasional short filopodia and punctate β-integrin expression at the margins (arrowhead) and dispersed all over the cytoplasm and the cell periphery. Cells plated on collagen-coated glass show abundant long and thin protrusions with punctate β-integrin at their tips (arrowheads). Cells plated on Con-A-coated glass show spread out morphologies, dense peripheral actin staining and circular bundles of actin around the nucleus. These cells present diffuse, just above background, β-integrin staining.

    Article Snippet: Drosophila S2R+ cells were grown routinely in Schneider's Drosophila medium (GIBCO, Invitrogen) supplemented with 10% heat inactivated Fetal Bovine Serum (GIBCO, Invitrogen) at 25°C.

    Techniques: Staining, Fluorescence, Confocal Microscopy, Expressing

    S2R+ cells respond to mechanical stress by changing their morphology and altering their cytoskeletal network. S2R+ cells plated on collagen-coated silicone membranes showed a polygonal shape with actin rich stress fibers and lamellipodia at their periphery (A) and distinctly arranged long microtubules spreading out and intermingling in the center (B). Anti- β-integrin antibodies show high expression at the periphery and dispersed cytoplasmic distribution (C). Upon subjecting cells to static stretch for 1 hour, S2R+ cells rounded up showing spotty cytoplasmic polymerized actin (D), short and diffuse microtubules (E) and punctate expression of β-integrin (F). Red - Phalloidin-TRITC; green - anti-Tubulin-FITC and anti- β-integrin antibodies.

    Journal: PLoS ONE

    Article Title: Integrin-Dependent Activation of the JNK Signaling Pathway by Mechanical Stress

    doi: 10.1371/journal.pone.0026182

    Figure Lengend Snippet: S2R+ cells respond to mechanical stress by changing their morphology and altering their cytoskeletal network. S2R+ cells plated on collagen-coated silicone membranes showed a polygonal shape with actin rich stress fibers and lamellipodia at their periphery (A) and distinctly arranged long microtubules spreading out and intermingling in the center (B). Anti- β-integrin antibodies show high expression at the periphery and dispersed cytoplasmic distribution (C). Upon subjecting cells to static stretch for 1 hour, S2R+ cells rounded up showing spotty cytoplasmic polymerized actin (D), short and diffuse microtubules (E) and punctate expression of β-integrin (F). Red - Phalloidin-TRITC; green - anti-Tubulin-FITC and anti- β-integrin antibodies.

    Article Snippet: Drosophila S2R+ cells were grown routinely in Schneider's Drosophila medium (GIBCO, Invitrogen) supplemented with 10% heat inactivated Fetal Bovine Serum (GIBCO, Invitrogen) at 25°C.

    Techniques: Expressing

    Specificity of the dJun-FRET biosensor. A) Time course of FL values for S2R+ cells transiently transfected with dJun-FRET biosensor and subjected to treatments with LPS (black) or Epidermal Growth Factor (EGF) (red). FLs of the donor (mCFP) were collected for 3 hours at 30 minutes intervals. In the presence of LPS, donor FL significantly decreases within 30 minutes, however, no significant shift was observed for 3 hours in the presence of EGF. B-D) S2R+ cells, serum starved for 24 hours, were plated on plastic, treated with the corresponding ligand for 2 hours, fixed and stained with phalloidin-TRITC. Untreated cells (B) show pseudopolygonal shape with peripheral accumulation of fibers and cytoplasmic spots of actin. LPS treated cells (C) present a compacted morphology and occasional thin filopodia, while EGF treated cells (D) display many highly branched short filamentous actin-rich protrusions.

    Journal: PLoS ONE

    Article Title: Integrin-Dependent Activation of the JNK Signaling Pathway by Mechanical Stress

    doi: 10.1371/journal.pone.0026182

    Figure Lengend Snippet: Specificity of the dJun-FRET biosensor. A) Time course of FL values for S2R+ cells transiently transfected with dJun-FRET biosensor and subjected to treatments with LPS (black) or Epidermal Growth Factor (EGF) (red). FLs of the donor (mCFP) were collected for 3 hours at 30 minutes intervals. In the presence of LPS, donor FL significantly decreases within 30 minutes, however, no significant shift was observed for 3 hours in the presence of EGF. B-D) S2R+ cells, serum starved for 24 hours, were plated on plastic, treated with the corresponding ligand for 2 hours, fixed and stained with phalloidin-TRITC. Untreated cells (B) show pseudopolygonal shape with peripheral accumulation of fibers and cytoplasmic spots of actin. LPS treated cells (C) present a compacted morphology and occasional thin filopodia, while EGF treated cells (D) display many highly branched short filamentous actin-rich protrusions.

    Article Snippet: Drosophila S2R+ cells were grown routinely in Schneider's Drosophila medium (GIBCO, Invitrogen) supplemented with 10% heat inactivated Fetal Bovine Serum (GIBCO, Invitrogen) at 25°C.

    Techniques: Transfection, Staining

    Mechanical stress activates the JNK pathway. S2R+ cells transiently transfected with dJun-FRET biosensor (A) or the controls mCFP alone (B) and mCFP-dJun (C) were plated on collagen-coated silicone membranes on a Stage Flexer set up. Donor mCFP FL was collected before stretching (black) and at different times after continuous static stretch (1 hour-red in A, 2 hours-blue in A, and 3 hours-green in A and red in B and C). dJun-FRET sensor activity increased upon stretching, reaching a maximum after 2 hours. FL of controls was not affected. Donor mCFP FL from a specific region of interest containing ∼15–30 cells were collected after 3 hours of mechanical stretch (D). Thereafter, the acceptor mYFP was photobleached (95%) and donor mCFP FLs were re-collected (E). Panel F shows the donor mCFP FL histograms for the dJun-FRET biosensor before (black) and after (red) acceptor (mYFP) photobleaching. Altogether, these data showed that FL changes observed after mechanical stretch can be attributed directly to energy transfer between the donor and acceptor fluorophores in the dJun-FRET biosensor.

    Journal: PLoS ONE

    Article Title: Integrin-Dependent Activation of the JNK Signaling Pathway by Mechanical Stress

    doi: 10.1371/journal.pone.0026182

    Figure Lengend Snippet: Mechanical stress activates the JNK pathway. S2R+ cells transiently transfected with dJun-FRET biosensor (A) or the controls mCFP alone (B) and mCFP-dJun (C) were plated on collagen-coated silicone membranes on a Stage Flexer set up. Donor mCFP FL was collected before stretching (black) and at different times after continuous static stretch (1 hour-red in A, 2 hours-blue in A, and 3 hours-green in A and red in B and C). dJun-FRET sensor activity increased upon stretching, reaching a maximum after 2 hours. FL of controls was not affected. Donor mCFP FL from a specific region of interest containing ∼15–30 cells were collected after 3 hours of mechanical stretch (D). Thereafter, the acceptor mYFP was photobleached (95%) and donor mCFP FLs were re-collected (E). Panel F shows the donor mCFP FL histograms for the dJun-FRET biosensor before (black) and after (red) acceptor (mYFP) photobleaching. Altogether, these data showed that FL changes observed after mechanical stretch can be attributed directly to energy transfer between the donor and acceptor fluorophores in the dJun-FRET biosensor.

    Article Snippet: Drosophila S2R+ cells were grown routinely in Schneider's Drosophila medium (GIBCO, Invitrogen) supplemented with 10% heat inactivated Fetal Bovine Serum (GIBCO, Invitrogen) at 25°C.

    Techniques: Transfection, Activity Assay

    Relish Is Displaced from the Attacin-A Promoter by the Repressosome (A) Left panel: soluble chromatin extracts were prepared from SL2 cells with (15 min or 8 h) or without (0 min) LPS/PGN treatment, and immunoprecipitated with antibodies against Relish, Jun, or Stat92E as described in Figure 3 . Right panel: double ChIP assays. The precipitates obtained from the first ChIP of cells with (15 min) or without LPS/PGN treatment were analyzed separately in a second ChIP with the antibodies indicated at the top (second ChIP). The amounts of Attacin-A promoter fragments co-precipitated with the indicated antibody are shown. (B) The transcript levels of each target gene are shown under Luciferase, Relish, Jra, Stat92E, or Dsp1 knock-down conditions with LPS/PGN treatment (10 μg/ml; 1h). The averages and standard deviations of triplicates assays are shown.

    Journal: PLoS Biology

    Article Title: Down-Regulation of NF-?B Target Genes by the AP-1 and STAT Complex during the Innate Immune Response in DrosophilaHow and Why Does a Fly Turn Its Immune System Off?

    doi: 10.1371/journal.pbio.0050238

    Figure Lengend Snippet: Relish Is Displaced from the Attacin-A Promoter by the Repressosome (A) Left panel: soluble chromatin extracts were prepared from SL2 cells with (15 min or 8 h) or without (0 min) LPS/PGN treatment, and immunoprecipitated with antibodies against Relish, Jun, or Stat92E as described in Figure 3 . Right panel: double ChIP assays. The precipitates obtained from the first ChIP of cells with (15 min) or without LPS/PGN treatment were analyzed separately in a second ChIP with the antibodies indicated at the top (second ChIP). The amounts of Attacin-A promoter fragments co-precipitated with the indicated antibody are shown. (B) The transcript levels of each target gene are shown under Luciferase, Relish, Jra, Stat92E, or Dsp1 knock-down conditions with LPS/PGN treatment (10 μg/ml; 1h). The averages and standard deviations of triplicates assays are shown.

    Article Snippet: Drosophila SL2 cells (1 × 106 ; CRL-1963, American Type Culture Collection) were washed with serum-free Drosophila medium (Welgene; http://www.welgene.com ) and treated with specific dsRNAs (20 μg) for 3 h. Serum was added back to the culture medium to 10% final concentration and the cells were incubated for an additional 72 h. The primers used for making the dsRNAs are listed in .

    Techniques: Immunoprecipitation, Chromatin Immunoprecipitation, Luciferase

    Down-Regulation of Relish Signaling by Stat92E as well as Jra in Response to LPS/PGN Real-time PCR analysis showing LPS/PGN-induced transcriptional activation in various mutant backgrounds. SL2 cells were incubated with dsRNA, as indicated in the top box, for three days. The levels of the transcripts before (-) and after (+) LPS/PGN treatment (10 μg/ml; 1hr) were measured by real time PCR. The degree of depletion of the corresponding transcript by RNAi is shown in the right panel.

    Journal: PLoS Biology

    Article Title: Down-Regulation of NF-?B Target Genes by the AP-1 and STAT Complex during the Innate Immune Response in DrosophilaHow and Why Does a Fly Turn Its Immune System Off?

    doi: 10.1371/journal.pbio.0050238

    Figure Lengend Snippet: Down-Regulation of Relish Signaling by Stat92E as well as Jra in Response to LPS/PGN Real-time PCR analysis showing LPS/PGN-induced transcriptional activation in various mutant backgrounds. SL2 cells were incubated with dsRNA, as indicated in the top box, for three days. The levels of the transcripts before (-) and after (+) LPS/PGN treatment (10 μg/ml; 1hr) were measured by real time PCR. The degree of depletion of the corresponding transcript by RNAi is shown in the right panel.

    Article Snippet: Drosophila SL2 cells (1 × 106 ; CRL-1963, American Type Culture Collection) were washed with serum-free Drosophila medium (Welgene; http://www.welgene.com ) and treated with specific dsRNAs (20 μg) for 3 h. Serum was added back to the culture medium to 10% final concentration and the cells were incubated for an additional 72 h. The primers used for making the dsRNAs are listed in .

    Techniques: Real-time Polymerase Chain Reaction, Activation Assay, Mutagenesis, Incubation

    Synergistic Binding of Jra and Stat92E to the Attacin-A Promoter ChIP assays of the transcription factors indicated below using various mutants. SL2 cells were depleted of the transcripts by dsRNA treatment, as indicated in the top box, for 3 d. Then chromatin extracts were prepared before (−) or after 30 min (+) of LPS/PGN treatment. The amounts of Attacin-A promoter fragments co-precipitated with antibodies against the transcription factors indicated below the data were measured by real-time PCR. The levels were normalized by the input used in each ChIP assay and are shown with standard deviations. These experiments were repeated independently at least three times.

    Journal: PLoS Biology

    Article Title: Down-Regulation of NF-?B Target Genes by the AP-1 and STAT Complex during the Innate Immune Response in DrosophilaHow and Why Does a Fly Turn Its Immune System Off?

    doi: 10.1371/journal.pbio.0050238

    Figure Lengend Snippet: Synergistic Binding of Jra and Stat92E to the Attacin-A Promoter ChIP assays of the transcription factors indicated below using various mutants. SL2 cells were depleted of the transcripts by dsRNA treatment, as indicated in the top box, for 3 d. Then chromatin extracts were prepared before (−) or after 30 min (+) of LPS/PGN treatment. The amounts of Attacin-A promoter fragments co-precipitated with antibodies against the transcription factors indicated below the data were measured by real-time PCR. The levels were normalized by the input used in each ChIP assay and are shown with standard deviations. These experiments were repeated independently at least three times.

    Article Snippet: Drosophila SL2 cells (1 × 106 ; CRL-1963, American Type Culture Collection) were washed with serum-free Drosophila medium (Welgene; http://www.welgene.com ) and treated with specific dsRNAs (20 μg) for 3 h. Serum was added back to the culture medium to 10% final concentration and the cells were incubated for an additional 72 h. The primers used for making the dsRNAs are listed in .

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    A Stat92E Binding Site on the Attacin-A Promoter Plays a Crucial Role in Down-Regulating Attacin-A (A) Region Y contains an NF-κB binding site and a STAT binding site. The NF-κB consensus and STAT consensus binding sequences are shown along with the wild-type sequence of region Y. The mutant forms of the NF-κB and/or the STAT binding sites of region Y are designated Relish2m, Stat92Em, and Relish2m-Stat92Em, respectively. The mutated sequences are shown in lower case and underlined. (B) Nuclear extracts of SL2 cells pre-incubated with dsRNA for Luciferase (L), Relish (R), Stat92E (S), or both Relish and Stat92E (RS) and treated with 10 μg/ml of LPS/PGN were assayed by EMSAs with 32 P-labeled double-stranded oligonucleotide probes containing Relish1 or region Y. (C) EMSAs with probes containing the wild-type region Y (double arrow), a mutation in the Relish binding site (double arrow with X on the left), a mutation in the Stat92E binding site (double arrow with X on the right) or mutations in both binding sites (double arrow with double X). Black and white arrows indicate the Relish 2 and Stat92E binding sites, respectively, and the mutations are indicated by Xs. Nuclear extracts were as in (B). (D) The dAP-1 and Stat92E promoter elements are required for down-regulation of Attacin-A . SL2 cells transfected with each reporter under the control of a mutant version of the Attacin-A promoter as indicated were treated with 10 μg/ml LPS/PGN for the time indicated on the abscissa. The mean levels of the normalized luciferase activities are shown with standard deviations. These experiments were repeated at least three times independently.

    Journal: PLoS Biology

    Article Title: Down-Regulation of NF-?B Target Genes by the AP-1 and STAT Complex during the Innate Immune Response in DrosophilaHow and Why Does a Fly Turn Its Immune System Off?

    doi: 10.1371/journal.pbio.0050238

    Figure Lengend Snippet: A Stat92E Binding Site on the Attacin-A Promoter Plays a Crucial Role in Down-Regulating Attacin-A (A) Region Y contains an NF-κB binding site and a STAT binding site. The NF-κB consensus and STAT consensus binding sequences are shown along with the wild-type sequence of region Y. The mutant forms of the NF-κB and/or the STAT binding sites of region Y are designated Relish2m, Stat92Em, and Relish2m-Stat92Em, respectively. The mutated sequences are shown in lower case and underlined. (B) Nuclear extracts of SL2 cells pre-incubated with dsRNA for Luciferase (L), Relish (R), Stat92E (S), or both Relish and Stat92E (RS) and treated with 10 μg/ml of LPS/PGN were assayed by EMSAs with 32 P-labeled double-stranded oligonucleotide probes containing Relish1 or region Y. (C) EMSAs with probes containing the wild-type region Y (double arrow), a mutation in the Relish binding site (double arrow with X on the left), a mutation in the Stat92E binding site (double arrow with X on the right) or mutations in both binding sites (double arrow with double X). Black and white arrows indicate the Relish 2 and Stat92E binding sites, respectively, and the mutations are indicated by Xs. Nuclear extracts were as in (B). (D) The dAP-1 and Stat92E promoter elements are required for down-regulation of Attacin-A . SL2 cells transfected with each reporter under the control of a mutant version of the Attacin-A promoter as indicated were treated with 10 μg/ml LPS/PGN for the time indicated on the abscissa. The mean levels of the normalized luciferase activities are shown with standard deviations. These experiments were repeated at least three times independently.

    Article Snippet: Drosophila SL2 cells (1 × 106 ; CRL-1963, American Type Culture Collection) were washed with serum-free Drosophila medium (Welgene; http://www.welgene.com ) and treated with specific dsRNAs (20 μg) for 3 h. Serum was added back to the culture medium to 10% final concentration and the cells were incubated for an additional 72 h. The primers used for making the dsRNAs are listed in .

    Techniques: Binding Assay, Sequencing, Mutagenesis, Incubation, Luciferase, Labeling, Transfection

    Dsp1 Interacts with Jra and Stat92E to Form a Repressosome Complex (A) Co-immunoprecipitation of Relish, Jra, Stat92E, and Dsp1. Nuclear extracts prepared from SL2 cells with (right panel) or without (left panel) LPS/PGN (10 μg/ml for 45 min) treatment were immunoprecipitated with the antibodies indicated at the top of the figure, and the amounts of the proteins in the pellets were measured by immunoblot analysis with the antibodies indicated on the left. For co-immunoprecipitation of Relish, Jra, and Stat92E, 10-μg aliquots of nuclear extracts were used, whereas for Dsp1, 3-μg aliquots were used. Five percent of the amount of nuclear extract used in each immunoprecipitation assay is shown as Input. (B) Regulation of Attacin-A transcription by ectopic expression of transcription factors. The N-terminal half of Relish that is competent as a transcriptional activator (Rel-ΔC), epitope-tagged Jra (S-Jra), and Stat92E (S-Stat92E) expression constructs were transfected into SL2 cells as indicated at the bottom of the figure. After induction of the recombinant proteins, the levels of Attacin-A transcripts relative to those of RpL32 were measured by real-time PCR analysis in three independent experiments. (C) ChIP assays of the Attacin-A promoter with anti-dHDAC1 antibody. SL2 cells pretreated with Luciferase dsRNA (control) or Dsp1 dsRNA (Dsp1-) were transfected with expression constructs for Rel-ΔC, S-Jra, and S-Stat92E as indicated at the bottom, and the average amounts of Attacin-A promoter fragments co-precipitated with anti-dHDAC1 antibody after induction of the transfected transcription factors were measured by real time PCR analysis in three independent experiments.

    Journal: PLoS Biology

    Article Title: Down-Regulation of NF-?B Target Genes by the AP-1 and STAT Complex during the Innate Immune Response in DrosophilaHow and Why Does a Fly Turn Its Immune System Off?

    doi: 10.1371/journal.pbio.0050238

    Figure Lengend Snippet: Dsp1 Interacts with Jra and Stat92E to Form a Repressosome Complex (A) Co-immunoprecipitation of Relish, Jra, Stat92E, and Dsp1. Nuclear extracts prepared from SL2 cells with (right panel) or without (left panel) LPS/PGN (10 μg/ml for 45 min) treatment were immunoprecipitated with the antibodies indicated at the top of the figure, and the amounts of the proteins in the pellets were measured by immunoblot analysis with the antibodies indicated on the left. For co-immunoprecipitation of Relish, Jra, and Stat92E, 10-μg aliquots of nuclear extracts were used, whereas for Dsp1, 3-μg aliquots were used. Five percent of the amount of nuclear extract used in each immunoprecipitation assay is shown as Input. (B) Regulation of Attacin-A transcription by ectopic expression of transcription factors. The N-terminal half of Relish that is competent as a transcriptional activator (Rel-ΔC), epitope-tagged Jra (S-Jra), and Stat92E (S-Stat92E) expression constructs were transfected into SL2 cells as indicated at the bottom of the figure. After induction of the recombinant proteins, the levels of Attacin-A transcripts relative to those of RpL32 were measured by real-time PCR analysis in three independent experiments. (C) ChIP assays of the Attacin-A promoter with anti-dHDAC1 antibody. SL2 cells pretreated with Luciferase dsRNA (control) or Dsp1 dsRNA (Dsp1-) were transfected with expression constructs for Rel-ΔC, S-Jra, and S-Stat92E as indicated at the bottom, and the average amounts of Attacin-A promoter fragments co-precipitated with anti-dHDAC1 antibody after induction of the transfected transcription factors were measured by real time PCR analysis in three independent experiments.

    Article Snippet: Drosophila SL2 cells (1 × 106 ; CRL-1963, American Type Culture Collection) were washed with serum-free Drosophila medium (Welgene; http://www.welgene.com ) and treated with specific dsRNAs (20 μg) for 3 h. Serum was added back to the culture medium to 10% final concentration and the cells were incubated for an additional 72 h. The primers used for making the dsRNAs are listed in .

    Techniques: Immunoprecipitation, Expressing, Construct, Transfection, Recombinant, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Luciferase

    Dsp1 Plays a Crucial Role in the Interactions between Jra and Stat92E (A) Down-regulation of Attacin-A transcripts by HMG protein. Real-time PCR analysis showing Attacin-A transcript levels after 1 h of LPS/PGN treatment of SL2 cells depleted by RNAi of the transcription factors and HMG proteins indicated below the histograms. The levels were normalized with RpL32 transcripts. The extents of depletion of the corresponding transcripts by RNAi are shown in the top panel. (B) Dsp1 is required for binding of Jra, Stat92E, and dHDAC1 to the Attacin-A promoter. SL2 cells were incubated with Luciferase or Dsp1 dsRNA for three days, then used in ChIP assays with (+) and without (−) LPS/PGN treatment (10 μg/ml for 1 h). The amounts of Attacin-A promoter fragments co-precipitated with the antibodies were normalized for the input used in each assay and are shown with standard deviations. These experiments were repeated at least three times independently. (C) Requirement for Jra and Stat92E for recruitment of Dsp1 to the Attacin-A promoter. SL2 cells were depleted of the protein indicated on the right by RNAi, then used in ChIP assays before (−) and after (+) LPS/PGN treatment. The amounts of Attacin-A promoter co-precipitated with anti-Dsp1 antibody in the ChIP assays are shown with standard deviations. These experiments were repeated at least three times independently. (D) The amounts of chromatin fragments co-precipitated with anti-Jun (solid squares), anti-Dsp1 (open triangles) or anti-Relish (solid circles) antibodies in the indicated regions of the Attacin-A promoter were measured by real-time PCR in a Roche Lightcycler, and the averages and standard deviations of three independent experiments are plotted.

    Journal: PLoS Biology

    Article Title: Down-Regulation of NF-?B Target Genes by the AP-1 and STAT Complex during the Innate Immune Response in DrosophilaHow and Why Does a Fly Turn Its Immune System Off?

    doi: 10.1371/journal.pbio.0050238

    Figure Lengend Snippet: Dsp1 Plays a Crucial Role in the Interactions between Jra and Stat92E (A) Down-regulation of Attacin-A transcripts by HMG protein. Real-time PCR analysis showing Attacin-A transcript levels after 1 h of LPS/PGN treatment of SL2 cells depleted by RNAi of the transcription factors and HMG proteins indicated below the histograms. The levels were normalized with RpL32 transcripts. The extents of depletion of the corresponding transcripts by RNAi are shown in the top panel. (B) Dsp1 is required for binding of Jra, Stat92E, and dHDAC1 to the Attacin-A promoter. SL2 cells were incubated with Luciferase or Dsp1 dsRNA for three days, then used in ChIP assays with (+) and without (−) LPS/PGN treatment (10 μg/ml for 1 h). The amounts of Attacin-A promoter fragments co-precipitated with the antibodies were normalized for the input used in each assay and are shown with standard deviations. These experiments were repeated at least three times independently. (C) Requirement for Jra and Stat92E for recruitment of Dsp1 to the Attacin-A promoter. SL2 cells were depleted of the protein indicated on the right by RNAi, then used in ChIP assays before (−) and after (+) LPS/PGN treatment. The amounts of Attacin-A promoter co-precipitated with anti-Dsp1 antibody in the ChIP assays are shown with standard deviations. These experiments were repeated at least three times independently. (D) The amounts of chromatin fragments co-precipitated with anti-Jun (solid squares), anti-Dsp1 (open triangles) or anti-Relish (solid circles) antibodies in the indicated regions of the Attacin-A promoter were measured by real-time PCR in a Roche Lightcycler, and the averages and standard deviations of three independent experiments are plotted.

    Article Snippet: Drosophila SL2 cells (1 × 106 ; CRL-1963, American Type Culture Collection) were washed with serum-free Drosophila medium (Welgene; http://www.welgene.com ) and treated with specific dsRNAs (20 μg) for 3 h. Serum was added back to the culture medium to 10% final concentration and the cells were incubated for an additional 72 h. The primers used for making the dsRNAs are listed in .

    Techniques: Real-time Polymerase Chain Reaction, Binding Assay, Incubation, Luciferase, Chromatin Immunoprecipitation

    STAT92E contributes to H1-dependent heterochromatin formation. Polytene chromosomes of salivary gland cells from L3 larvae were analyzed by indirect immunofluorescence (IF) staining with antibodies against HP1 or H1 ( red ) and STAT92E ( green ). DNA was stained with DAPI ( blue ). Scale bar represents 10 μm. (A) Polytene chromosome structure in wild type larvae ( top ), H1-depleted larvae ( middle ), and H1-depleted larvae that overexpress nonphosphorylatable STAT, STAT92E(Y704F) ( bottom ). HP1 signal is strongly enriched in a single chromocenter region in the wild type. The chromocenter is not discernable (DAPI), and HP1 staining is dispersed in multiple foci upon H1 depletion (see also Figure 1 A). The phenotype is partially rescued by STAT92E(Y704F) expression. (B) STAT92E(Y704F) overexpressed in H1-depleted larvae, co-localizes with residual H1 in polytene chromosomes ( top ). Ectopically overexpressed transgenic wild type ( WT ) STAT92E fails to restore the single chromocenter and does not co-localize with residual H1 ( bottom ).

    Journal: Epigenetics & Chromatin

    Article Title: Drosophila linker histone H1 coordinates STAT-dependent organization of heterochromatin and suppresses tumorigenesis caused by hyperactive JAK-STAT signaling

    doi: 10.1186/1756-8935-7-16

    Figure Lengend Snippet: STAT92E contributes to H1-dependent heterochromatin formation. Polytene chromosomes of salivary gland cells from L3 larvae were analyzed by indirect immunofluorescence (IF) staining with antibodies against HP1 or H1 ( red ) and STAT92E ( green ). DNA was stained with DAPI ( blue ). Scale bar represents 10 μm. (A) Polytene chromosome structure in wild type larvae ( top ), H1-depleted larvae ( middle ), and H1-depleted larvae that overexpress nonphosphorylatable STAT, STAT92E(Y704F) ( bottom ). HP1 signal is strongly enriched in a single chromocenter region in the wild type. The chromocenter is not discernable (DAPI), and HP1 staining is dispersed in multiple foci upon H1 depletion (see also Figure 1 A). The phenotype is partially rescued by STAT92E(Y704F) expression. (B) STAT92E(Y704F) overexpressed in H1-depleted larvae, co-localizes with residual H1 in polytene chromosomes ( top ). Ectopically overexpressed transgenic wild type ( WT ) STAT92E fails to restore the single chromocenter and does not co-localize with residual H1 ( bottom ).

    Article Snippet: The following antisera were used at the indicated dilutions: monoclonal mouse anti-Drosophila HP1, C1A9 (1:50, Developmental Studies Hybridoma Bank); goat anti-Drosophila STAT, dF-20 (1:50, Santa Cruz Biotechnology); affinity-purified rabbit Drosophila H1 antiserum (1:5,000) and affinity-purified rabbit anti-H3K9me2 (1:100, Abcam).

    Techniques: Immunofluorescence, Staining, Expressing, Transgenic Assay

    Distribution of STAT92E in polytene chromosomes depends on H1. Polytene chromosomes of salivary gland cells from L3 larvae were analyzed by indirect immunofluorescence (IF) staining with antibodies against H1 ( red ) and STAT92E ( green ). DNA was stained with DAPI ( blue ). Scale bars represent 10 μm. (A) Top , genome-wide localization of H1 and STAT92E in polytene chromosomes. Localization patterns of H1 and STAT92E extensively overlap in the euchromatic arms and the chromocenter of polytene chromosomes. DNA was stained with DAPI ( blue ). Bottom , higher magnification view of co-localization of H1 and STAT92E in polytene chromosome arms. Merged split image illustrates that STAT and H1 exhibit nearly identical localization patterns, which correlate with polytene bands. (B) Genome-wide localization of STAT92E in wild type , H1-depleted, Su(var)3-9 [ 1 ] /Su(var)3-9 [ 2 ] and HP1-depleted polytene chromosomes. In H1-depleted salivary glands, the polytene chromosome structure is disrupted, and STAT92E staining is strongly reduced to barely above background. Neither Su(var)3-9 mutation nor HP1 depletion substantially affects STAT92E localization. (C) The occupancy of H1 and STAT92E at regulatory regions of euchromatic ( tubulin ) and heterochromatic ( light , concertina ) genes and transposable element ZAM . The occupancy was measured by qChIP in control and H1 RNAi alleles. The ordinate indicates the amounts of specific polymerase chain reaction (PCR) products in ChIP DNA samples relative to input DNA. All qChIP experiments were performed in triplicate. Error bars, standard deviation. (D) Genome-wide localization of H1 in STAT92E-depleted and hop Tum-l mutant polytene chromosomes. The localization pattern of H1 is not affected and is similar to that in wild type chromosomes (compare to A).

    Journal: Epigenetics & Chromatin

    Article Title: Drosophila linker histone H1 coordinates STAT-dependent organization of heterochromatin and suppresses tumorigenesis caused by hyperactive JAK-STAT signaling

    doi: 10.1186/1756-8935-7-16

    Figure Lengend Snippet: Distribution of STAT92E in polytene chromosomes depends on H1. Polytene chromosomes of salivary gland cells from L3 larvae were analyzed by indirect immunofluorescence (IF) staining with antibodies against H1 ( red ) and STAT92E ( green ). DNA was stained with DAPI ( blue ). Scale bars represent 10 μm. (A) Top , genome-wide localization of H1 and STAT92E in polytene chromosomes. Localization patterns of H1 and STAT92E extensively overlap in the euchromatic arms and the chromocenter of polytene chromosomes. DNA was stained with DAPI ( blue ). Bottom , higher magnification view of co-localization of H1 and STAT92E in polytene chromosome arms. Merged split image illustrates that STAT and H1 exhibit nearly identical localization patterns, which correlate with polytene bands. (B) Genome-wide localization of STAT92E in wild type , H1-depleted, Su(var)3-9 [ 1 ] /Su(var)3-9 [ 2 ] and HP1-depleted polytene chromosomes. In H1-depleted salivary glands, the polytene chromosome structure is disrupted, and STAT92E staining is strongly reduced to barely above background. Neither Su(var)3-9 mutation nor HP1 depletion substantially affects STAT92E localization. (C) The occupancy of H1 and STAT92E at regulatory regions of euchromatic ( tubulin ) and heterochromatic ( light , concertina ) genes and transposable element ZAM . The occupancy was measured by qChIP in control and H1 RNAi alleles. The ordinate indicates the amounts of specific polymerase chain reaction (PCR) products in ChIP DNA samples relative to input DNA. All qChIP experiments were performed in triplicate. Error bars, standard deviation. (D) Genome-wide localization of H1 in STAT92E-depleted and hop Tum-l mutant polytene chromosomes. The localization pattern of H1 is not affected and is similar to that in wild type chromosomes (compare to A).

    Article Snippet: The following antisera were used at the indicated dilutions: monoclonal mouse anti-Drosophila HP1, C1A9 (1:50, Developmental Studies Hybridoma Bank); goat anti-Drosophila STAT, dF-20 (1:50, Santa Cruz Biotechnology); affinity-purified rabbit Drosophila H1 antiserum (1:5,000) and affinity-purified rabbit anti-H3K9me2 (1:100, Abcam).

    Techniques: Immunofluorescence, Staining, Genome Wide, Mutagenesis, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Standard Deviation

    H1 physically interacts with STAT92E. Protein-protein interactions between purified STAT92E and H1 were examined in vitro by GST pull-down and ChIP. (A) GST and GST fusion proteins with full-length H1, HP1 or H2A were expressed and purified from E. coli and analyzed by GST pull-down with baculovirus-expressed purified recombinant STAT92E-His 6 . The pull-down samples were examined by SDS-PAGE and Coomassie staining ( top ) or immunoblotting with anti-His 6 antibody ( bottom ). As a control, 10% of the input STAT92E-His 6 was examined. (B) Binding of STAT92E and Su(var)3-9 to reconstituted chromatin was analyzed by in vitro ChIP. Oligonucleosomes were reconstituted on supercoiled plasmid DNA with purified native core histones, with ( H1+ , dark-gray bars ) or without ( H1– , light gray bars ) purified native H1. Non-sequence specific binding to the plasmid ( DNA , white bars ) was also examined. His 6 -tagged recombinant proteins were incubated with chromatin/DNA templates, cross-linked, immunoprecipitated with anti-His 6 antibody and occupancy was measured by real-time PCR of a fragment of the plasmid. The occupancy of proteins relative to input was normalized to occupancy on naked DNA and plotted. The presence of H1 in chromatin templates strongly stimulates binding of both STAT92E and Su(var)3-9 . All ChIP experiments were performed in duplicate, and each biological sample was analyzed by PCR in triplicate. Error bars represent standard deviation of six experimental points. (C) GST and GST fusion proteins with full-length H1, H1 N-terminal domain (H1-N, amino acid residues 1–40), the globular domain (H1-G, residues 41–119) and the C-terminal domain (H1-C, 120–256) were expressed and purified from E. coli and used in GST pull-down experiments with baculovirus-expressed purified recombinant STAT92E-His 6 . The pull-down samples were examined as in (A) . Full-length polypeptides of GST fusion proteins are indicated by open triangles . STAT92E associates with GST fusions of H1 and H1-C but does not interact with GST or GST fusions of H1-N and H1-G.

    Journal: Epigenetics & Chromatin

    Article Title: Drosophila linker histone H1 coordinates STAT-dependent organization of heterochromatin and suppresses tumorigenesis caused by hyperactive JAK-STAT signaling

    doi: 10.1186/1756-8935-7-16

    Figure Lengend Snippet: H1 physically interacts with STAT92E. Protein-protein interactions between purified STAT92E and H1 were examined in vitro by GST pull-down and ChIP. (A) GST and GST fusion proteins with full-length H1, HP1 or H2A were expressed and purified from E. coli and analyzed by GST pull-down with baculovirus-expressed purified recombinant STAT92E-His 6 . The pull-down samples were examined by SDS-PAGE and Coomassie staining ( top ) or immunoblotting with anti-His 6 antibody ( bottom ). As a control, 10% of the input STAT92E-His 6 was examined. (B) Binding of STAT92E and Su(var)3-9 to reconstituted chromatin was analyzed by in vitro ChIP. Oligonucleosomes were reconstituted on supercoiled plasmid DNA with purified native core histones, with ( H1+ , dark-gray bars ) or without ( H1– , light gray bars ) purified native H1. Non-sequence specific binding to the plasmid ( DNA , white bars ) was also examined. His 6 -tagged recombinant proteins were incubated with chromatin/DNA templates, cross-linked, immunoprecipitated with anti-His 6 antibody and occupancy was measured by real-time PCR of a fragment of the plasmid. The occupancy of proteins relative to input was normalized to occupancy on naked DNA and plotted. The presence of H1 in chromatin templates strongly stimulates binding of both STAT92E and Su(var)3-9 . All ChIP experiments were performed in duplicate, and each biological sample was analyzed by PCR in triplicate. Error bars represent standard deviation of six experimental points. (C) GST and GST fusion proteins with full-length H1, H1 N-terminal domain (H1-N, amino acid residues 1–40), the globular domain (H1-G, residues 41–119) and the C-terminal domain (H1-C, 120–256) were expressed and purified from E. coli and used in GST pull-down experiments with baculovirus-expressed purified recombinant STAT92E-His 6 . The pull-down samples were examined as in (A) . Full-length polypeptides of GST fusion proteins are indicated by open triangles . STAT92E associates with GST fusions of H1 and H1-C but does not interact with GST or GST fusions of H1-N and H1-G.

    Article Snippet: The following antisera were used at the indicated dilutions: monoclonal mouse anti-Drosophila HP1, C1A9 (1:50, Developmental Studies Hybridoma Bank); goat anti-Drosophila STAT, dF-20 (1:50, Santa Cruz Biotechnology); affinity-purified rabbit Drosophila H1 antiserum (1:5,000) and affinity-purified rabbit anti-H3K9me2 (1:100, Abcam).

    Techniques: Purification, In Vitro, Chromatin Immunoprecipitation, Recombinant, SDS Page, Staining, Binding Assay, Plasmid Preparation, Sequencing, Incubation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation

    H1 depletion prevents STAT association with ectopic sites and enhances blood tumor formation induced by hyperactive JAK. Nucleosomes, H1, HP1, STAT, and JAK are represented by light-gray ovals, red rectangles, blue ovals, orange hexagons, and light-green rectangles, respectively. Hyperactive JAK is represented by an increased number of corresponding rectangles. (A) Top , in wild type chromatin, unphosphorylated STAT92E physically interacts with H1 and is recruited to ectopic loci irrespective of sequence-specific DNA recognition. The two proteins stabilize the association of HP1 with heterochromatin. Second , hyperactive JAK in hop Tum-l larvae phosphorylates a greater fraction of STAT92E, which prevents its association with H1 and HP1 at ectopic sites and destabilizes HP1 association with pericentric heterochromatin. The excess of phosphorylated STAT92E abnormally stimulates downstream transcriptional targets and leads to blood tumor formation. Third , the association of STAT92E and HP1 with heterochromatin is dependent on the presence of H1. When H1 is depleted, both STAT92E and HP1 are dissociated from chromatin. Due to limiting activity of wild type JAK, the excess of STAT92E does not activate transcription and does not cause tumorigenesis. Bottom , depleting H1 in hop Tum-l larvae leads to eviction of STAT92E from ectopic sites. The released STAT92E becomes available for phosphorylation by hyperactive JAK and enhances blood tumor formation. (B) Two independent pathways for H1-dependent heterochromatin formation. Arrows indicate physical interactions/tethering or an enzymatic reaction (H3K9 methylation). Phosphorylation by JAK prevents STAT accumulation at ectopic loci, including pericentric heterochromatin.

    Journal: Epigenetics & Chromatin

    Article Title: Drosophila linker histone H1 coordinates STAT-dependent organization of heterochromatin and suppresses tumorigenesis caused by hyperactive JAK-STAT signaling

    doi: 10.1186/1756-8935-7-16

    Figure Lengend Snippet: H1 depletion prevents STAT association with ectopic sites and enhances blood tumor formation induced by hyperactive JAK. Nucleosomes, H1, HP1, STAT, and JAK are represented by light-gray ovals, red rectangles, blue ovals, orange hexagons, and light-green rectangles, respectively. Hyperactive JAK is represented by an increased number of corresponding rectangles. (A) Top , in wild type chromatin, unphosphorylated STAT92E physically interacts with H1 and is recruited to ectopic loci irrespective of sequence-specific DNA recognition. The two proteins stabilize the association of HP1 with heterochromatin. Second , hyperactive JAK in hop Tum-l larvae phosphorylates a greater fraction of STAT92E, which prevents its association with H1 and HP1 at ectopic sites and destabilizes HP1 association with pericentric heterochromatin. The excess of phosphorylated STAT92E abnormally stimulates downstream transcriptional targets and leads to blood tumor formation. Third , the association of STAT92E and HP1 with heterochromatin is dependent on the presence of H1. When H1 is depleted, both STAT92E and HP1 are dissociated from chromatin. Due to limiting activity of wild type JAK, the excess of STAT92E does not activate transcription and does not cause tumorigenesis. Bottom , depleting H1 in hop Tum-l larvae leads to eviction of STAT92E from ectopic sites. The released STAT92E becomes available for phosphorylation by hyperactive JAK and enhances blood tumor formation. (B) Two independent pathways for H1-dependent heterochromatin formation. Arrows indicate physical interactions/tethering or an enzymatic reaction (H3K9 methylation). Phosphorylation by JAK prevents STAT accumulation at ectopic loci, including pericentric heterochromatin.

    Article Snippet: The following antisera were used at the indicated dilutions: monoclonal mouse anti-Drosophila HP1, C1A9 (1:50, Developmental Studies Hybridoma Bank); goat anti-Drosophila STAT, dF-20 (1:50, Santa Cruz Biotechnology); affinity-purified rabbit Drosophila H1 antiserum (1:5,000) and affinity-purified rabbit anti-H3K9me2 (1:100, Abcam).

    Techniques: Sequencing, Activity Assay, Methylation

    Hyperactive JAK affects chromocenter formation in Drosophila polytene chromosomes. Polytene chromosomes of salivary gland cells from L3 larvae were analyzed by indirect immunofluorescence (IF) staining with antibodies against HP1 (green) and H3K9Me 2 ( red ). DNA was stained with DAPI ( blue ). Top , wild type polytene chromosomes have a uniform regular structure of bands and interbands with a single chromocenter characterized by overlapping intense HP1 and H3K9Me 2 staining. Middle , hop Tum-l polytene chromosomes have an abnormal morphology with disrupted polytene chromosome structure and dispersed HP1 foci. A single chromocenter cannot be discerned by DAPI or HP1 staining. H3K9Me 2 staining overlaps with HP1-positive foci. Bottom , H1-depleted polytene chromosomes have an abnormal morphology with disrupted polytene chromosome structure and dispersed HP1 foci. A single chromocenter cannot be discerned by DAPI or HP1 staining. H3K9Me 2 staining is strongly reduced. Scale bar represents 10 μm.

    Journal: Epigenetics & Chromatin

    Article Title: Drosophila linker histone H1 coordinates STAT-dependent organization of heterochromatin and suppresses tumorigenesis caused by hyperactive JAK-STAT signaling

    doi: 10.1186/1756-8935-7-16

    Figure Lengend Snippet: Hyperactive JAK affects chromocenter formation in Drosophila polytene chromosomes. Polytene chromosomes of salivary gland cells from L3 larvae were analyzed by indirect immunofluorescence (IF) staining with antibodies against HP1 (green) and H3K9Me 2 ( red ). DNA was stained with DAPI ( blue ). Top , wild type polytene chromosomes have a uniform regular structure of bands and interbands with a single chromocenter characterized by overlapping intense HP1 and H3K9Me 2 staining. Middle , hop Tum-l polytene chromosomes have an abnormal morphology with disrupted polytene chromosome structure and dispersed HP1 foci. A single chromocenter cannot be discerned by DAPI or HP1 staining. H3K9Me 2 staining overlaps with HP1-positive foci. Bottom , H1-depleted polytene chromosomes have an abnormal morphology with disrupted polytene chromosome structure and dispersed HP1 foci. A single chromocenter cannot be discerned by DAPI or HP1 staining. H3K9Me 2 staining is strongly reduced. Scale bar represents 10 μm.

    Article Snippet: The following antisera were used at the indicated dilutions: monoclonal mouse anti-Drosophila HP1, C1A9 (1:50, Developmental Studies Hybridoma Bank); goat anti-Drosophila STAT, dF-20 (1:50, Santa Cruz Biotechnology); affinity-purified rabbit Drosophila H1 antiserum (1:5,000) and affinity-purified rabbit anti-H3K9me2 (1:100, Abcam).

    Techniques: Immunofluorescence, Staining

    Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Article Snippet: To further confirm the reproducibility of each method, a single SMART library and four ligation libraries (using tags TAG02, TAG04, TAG05, and TAG06) were also constructed using embryonic Drosophila melanogaster poly A+ RNA (Clontech: Cat. 636224, Lot.

    Techniques: De-Phosphorylation Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing

    Reproducibility of libraries generated by the SMART method using adult poly A+ RNA. Plots showing correlation between sequence counts of six independent replicates (A: ID01 X 02; B: ID02 X 03; C: ID03 X 04; D: ID04 X 05; E: ID05 X 06; F: ID06 X 01). The results show a very high correlation ( R = 0.99633–0.99996), indicating that the library preparation method is highly reproducible.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Reproducibility of libraries generated by the SMART method using adult poly A+ RNA. Plots showing correlation between sequence counts of six independent replicates (A: ID01 X 02; B: ID02 X 03; C: ID03 X 04; D: ID04 X 05; E: ID05 X 06; F: ID06 X 01). The results show a very high correlation ( R = 0.99633–0.99996), indicating that the library preparation method is highly reproducible.

    Article Snippet: To further confirm the reproducibility of each method, a single SMART library and four ligation libraries (using tags TAG02, TAG04, TAG05, and TAG06) were also constructed using embryonic Drosophila melanogaster poly A+ RNA (Clontech: Cat. 636224, Lot.

    Techniques: Generated, Sequencing

    Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 4949000 and 4956000 on chromosome 2L. Only the sequences mapped on the minus strand are depicted. Gene locations ( Jon25Bi , Jon25Bii , Jon25Biii , and jet ) are depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 4949000 and 4956000 on chromosome 2L. Only the sequences mapped on the minus strand are depicted. Gene locations ( Jon25Bi , Jon25Bii , Jon25Biii , and jet ) are depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.

    Article Snippet: To further confirm the reproducibility of each method, a single SMART library and four ligation libraries (using tags TAG02, TAG04, TAG05, and TAG06) were also constructed using embryonic Drosophila melanogaster poly A+ RNA (Clontech: Cat. 636224, Lot.

    Techniques: Sequencing, Ligation

    Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 8041000 and 8044000 on chromosome 2L. Only the sequences mapped on the minus strand are depicted. Gene location ( RpL36A ) is depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 8041000 and 8044000 on chromosome 2L. Only the sequences mapped on the minus strand are depicted. Gene location ( RpL36A ) is depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.

    Article Snippet: To further confirm the reproducibility of each method, a single SMART library and four ligation libraries (using tags TAG02, TAG04, TAG05, and TAG06) were also constructed using embryonic Drosophila melanogaster poly A+ RNA (Clontech: Cat. 636224, Lot.

    Techniques: Sequencing, Ligation

    Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 7574000 and 7580000 on chromosome 2L. Only the sequences mapped on the minus strand are depicted. Gene locations ( Rapgap1 and CG13791 ) are depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 7574000 and 7580000 on chromosome 2L. Only the sequences mapped on the minus strand are depicted. Gene locations ( Rapgap1 and CG13791 ) are depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.

    Article Snippet: To further confirm the reproducibility of each method, a single SMART library and four ligation libraries (using tags TAG02, TAG04, TAG05, and TAG06) were also constructed using embryonic Drosophila melanogaster poly A+ RNA (Clontech: Cat. 636224, Lot.

    Techniques: Sequencing, Ligation

    Library preparation using the ligation method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Tobacco Acid Pyrophosphatase treatment to remove the 5′ cap structure, exposing a mono-phosphate group for subsequent ligation. D) Ligation of RNA oligomers. A total of six tags ( Table 3 : TAG02, TAG04, TAG05, TAG06, TAG07, TAG12) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor, collection of biotinylated 5′ ends using beads, and sample pooling for multiplexing. H) Illumina sequencing library preparation.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Library preparation using the ligation method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Tobacco Acid Pyrophosphatase treatment to remove the 5′ cap structure, exposing a mono-phosphate group for subsequent ligation. D) Ligation of RNA oligomers. A total of six tags ( Table 3 : TAG02, TAG04, TAG05, TAG06, TAG07, TAG12) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor, collection of biotinylated 5′ ends using beads, and sample pooling for multiplexing. H) Illumina sequencing library preparation.

    Article Snippet: To further confirm the reproducibility of each method, a single SMART library and four ligation libraries (using tags TAG02, TAG04, TAG05, and TAG06) were also constructed using embryonic Drosophila melanogaster poly A+ RNA (Clontech: Cat. 636224, Lot.

    Techniques: Ligation, De-Phosphorylation Assay, Amplification, Polymerase Chain Reaction, Multiplexing, Sequencing

    Library preparation using the Non-CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Tobacco Acid Pyrophosphatase treatment to remove the 5′ cap structure, exposing a mono-phosphate group for subsequent ligation. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Library preparation using the Non-CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Tobacco Acid Pyrophosphatase treatment to remove the 5′ cap structure, exposing a mono-phosphate group for subsequent ligation. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Article Snippet: To further confirm the reproducibility of each method, a single SMART library and four ligation libraries (using tags TAG02, TAG04, TAG05, and TAG06) were also constructed using embryonic Drosophila melanogaster poly A+ RNA (Clontech: Cat. 636224, Lot.

    Techniques: De-Phosphorylation Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing

    Library preparation using the SMART method. A) The protocol used either poly A+ (0.025–0.5 µg) or total (0.05–1.0 µg) RNA. B) First-strand cDNA synthesis, together with template switching and continuous replication to the end of the oligonucleotide. C) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. D) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. E) Illumina sequencing library preparation.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Library preparation using the SMART method. A) The protocol used either poly A+ (0.025–0.5 µg) or total (0.05–1.0 µg) RNA. B) First-strand cDNA synthesis, together with template switching and continuous replication to the end of the oligonucleotide. C) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. D) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. E) Illumina sequencing library preparation.

    Article Snippet: To further confirm the reproducibility of each method, a single SMART library and four ligation libraries (using tags TAG02, TAG04, TAG05, and TAG06) were also constructed using embryonic Drosophila melanogaster poly A+ RNA (Clontech: Cat. 636224, Lot.

    Techniques: Amplification, Polymerase Chain Reaction, Sequencing

    Reproducibility of libraries generated by the ligation method using adult poly A+ RNA. Plots showing correlation between sequence counts of six independent replicates (A: TG02 X 04; B: TG04 X 05; C: TG05 X 06; D: TG06 X 07; E: TG07 X 12; F: TG12 X 02). The results show a relatively low correlation ( R = 0.09689–0.72684), indicating low reproducibility of the library preparation method.

    Journal: PLoS ONE

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    doi: 10.1371/journal.pone.0101812

    Figure Lengend Snippet: Reproducibility of libraries generated by the ligation method using adult poly A+ RNA. Plots showing correlation between sequence counts of six independent replicates (A: TG02 X 04; B: TG04 X 05; C: TG05 X 06; D: TG06 X 07; E: TG07 X 12; F: TG12 X 02). The results show a relatively low correlation ( R = 0.09689–0.72684), indicating low reproducibility of the library preparation method.

    Article Snippet: To further confirm the reproducibility of each method, a single SMART library and four ligation libraries (using tags TAG02, TAG04, TAG05, and TAG06) were also constructed using embryonic Drosophila melanogaster poly A+ RNA (Clontech: Cat. 636224, Lot.

    Techniques: Generated, Ligation, Sequencing

    Phylogenetic and principal component clustering analyses of populations from the three species: Drosophila athabasca ( WN , blue, squares), D. mahican ( EA , red, circles), and D. lenape ( EB , orange, triangles/diamonds). D. affinis was the outgroup. See text and Figure 1 for locations of abbreviations of specific locations. (a) Phylogeny inferred using the neighbor‐joining ( NJ ) method based on average pairwise Fst values across all 52 gene fragments (see Materials and Methods ). Sum of branch length = 1.7 ( MEGA 7). Minimum number of sequences allowed per population was six chromosomes. Branch lengths are shown. Phylogeny based on Dxy distances showed qualitatively similar results (data not shown). (b) A maximum likelihood consensus phylogeny with bootstrap values based on 22,261‐bp consensus sequences across populations ( D. mahican QB , MVS / MKSP , and SAIJ populations were not included due to small sample sizes). Bootstrap supports (500 replicates) are shown next to the branches. (c) Principal component analysis ( PCA ) based on covariances across a total of 1,820‐bp SNP sites across populations of each species (same populations as in panel b, except D. lenape LK population was not included due to small sample size). PC 1 explains 36%, and PC 2 explains 20% of total genetic variation across populations. Results were qualitatively similar when different nonoverlapping sets (500 bp per set) were used to generate PCA . Some sympatric locations are labeled

    Journal: Ecology and Evolution

    Article Title: Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement. Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement

    doi: 10.1002/ece3.3893

    Figure Lengend Snippet: Phylogenetic and principal component clustering analyses of populations from the three species: Drosophila athabasca ( WN , blue, squares), D. mahican ( EA , red, circles), and D. lenape ( EB , orange, triangles/diamonds). D. affinis was the outgroup. See text and Figure 1 for locations of abbreviations of specific locations. (a) Phylogeny inferred using the neighbor‐joining ( NJ ) method based on average pairwise Fst values across all 52 gene fragments (see Materials and Methods ). Sum of branch length = 1.7 ( MEGA 7). Minimum number of sequences allowed per population was six chromosomes. Branch lengths are shown. Phylogeny based on Dxy distances showed qualitatively similar results (data not shown). (b) A maximum likelihood consensus phylogeny with bootstrap values based on 22,261‐bp consensus sequences across populations ( D. mahican QB , MVS / MKSP , and SAIJ populations were not included due to small sample sizes). Bootstrap supports (500 replicates) are shown next to the branches. (c) Principal component analysis ( PCA ) based on covariances across a total of 1,820‐bp SNP sites across populations of each species (same populations as in panel b, except D. lenape LK population was not included due to small sample size). PC 1 explains 36%, and PC 2 explains 20% of total genetic variation across populations. Results were qualitatively similar when different nonoverlapping sets (500 bp per set) were used to generate PCA . Some sympatric locations are labeled

    Article Snippet: A great system to test this question is the young Drosophila athabasca species complex (obscura group: affinis subgroup; Sturtevant & Dobzhansky, ), which contains three widespread semispecies across North America: West Northern (WN), Eastern A (EA), and Eastern B (EB) (see Figure and Miller, for known ranges).

    Techniques: Labeling

    Inferred ancestry (probability of being assigned to a given species) of 281 individuals based on 4,690 total variable base pairs with software STRUCTURE (v 2.3.4) for K = 3 run. Drosophila athabasca ( WN , blue), D. mahican ( EA , red), and D. lenape ( EB , orange). Species designation for each isofemale line (represented by a sequenced individual) was established using phenotypic data (male courtship song, copulation duration, and sexual isolation) and geographical information (see Table S1 and text). Open circles represent individuals (lines) for which species designation using phenotypic/geographical data was not determined. Only individual isofemale lines that have a relatively low (70% or less) probability of being assigned to a given species are labeled

    Journal: Ecology and Evolution

    Article Title: Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement. Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement

    doi: 10.1002/ece3.3893

    Figure Lengend Snippet: Inferred ancestry (probability of being assigned to a given species) of 281 individuals based on 4,690 total variable base pairs with software STRUCTURE (v 2.3.4) for K = 3 run. Drosophila athabasca ( WN , blue), D. mahican ( EA , red), and D. lenape ( EB , orange). Species designation for each isofemale line (represented by a sequenced individual) was established using phenotypic data (male courtship song, copulation duration, and sexual isolation) and geographical information (see Table S1 and text). Open circles represent individuals (lines) for which species designation using phenotypic/geographical data was not determined. Only individual isofemale lines that have a relatively low (70% or less) probability of being assigned to a given species are labeled

    Article Snippet: A great system to test this question is the young Drosophila athabasca species complex (obscura group: affinis subgroup; Sturtevant & Dobzhansky, ), which contains three widespread semispecies across North America: West Northern (WN), Eastern A (EA), and Eastern B (EB) (see Figure and Miller, for known ranges).

    Techniques: Software, Isolation, Labeling

    Relationship between the minimum geographical distance (km) of a focal population to the closest population of the other species and its average measure of sequence divergence to all heterospecific populations. Left panels (a and d) compare Drosophila athabasca and D. mahican , central panels (b and e) compare D. athabasca and D. lenape , and right panels (c and f) compare D. mahican and D. lenape . Blue squares represent D. athabasca ( WN ), red circles represent D. mahican ( EA ), and orange triangles represent D. lenape ( EB ) focal populations in each plot. Top panels (a–c) show Dxy , and bottom panels (d–f) show Fst . Both measures of genetic divergence are averaged across all sequenced genes. Note that these relationships avoid the problem of nonindependent data points by averaging Fst or Dxy values between each focal population with all heterospecific populations. Error bars indicate 95% confidence intervals between the focal population and heterospecific populations. Only populations with greater than three individuals are considered (i.e., D. mahican QB and RIVR / LNOM and D. lenape LK populations were excluded; results did not change when these were included). R 2 values shown for each focal species ( D. lenape trendlines in center panels are not shown due to very small range of geographical data points)

    Journal: Ecology and Evolution

    Article Title: Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement. Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement

    doi: 10.1002/ece3.3893

    Figure Lengend Snippet: Relationship between the minimum geographical distance (km) of a focal population to the closest population of the other species and its average measure of sequence divergence to all heterospecific populations. Left panels (a and d) compare Drosophila athabasca and D. mahican , central panels (b and e) compare D. athabasca and D. lenape , and right panels (c and f) compare D. mahican and D. lenape . Blue squares represent D. athabasca ( WN ), red circles represent D. mahican ( EA ), and orange triangles represent D. lenape ( EB ) focal populations in each plot. Top panels (a–c) show Dxy , and bottom panels (d–f) show Fst . Both measures of genetic divergence are averaged across all sequenced genes. Note that these relationships avoid the problem of nonindependent data points by averaging Fst or Dxy values between each focal population with all heterospecific populations. Error bars indicate 95% confidence intervals between the focal population and heterospecific populations. Only populations with greater than three individuals are considered (i.e., D. mahican QB and RIVR / LNOM and D. lenape LK populations were excluded; results did not change when these were included). R 2 values shown for each focal species ( D. lenape trendlines in center panels are not shown due to very small range of geographical data points)

    Article Snippet: A great system to test this question is the young Drosophila athabasca species complex (obscura group: affinis subgroup; Sturtevant & Dobzhansky, ), which contains three widespread semispecies across North America: West Northern (WN), Eastern A (EA), and Eastern B (EB) (see Figure and Miller, for known ranges).

    Techniques: Sequencing

    Geographical range map of the Drosophila athabasca species complex: D. athabasca ( WN , blue), D. mahican ( EA , red), and D. lenape ( EB , orange) with specific locations shown as pie charts. Each pie chart represents the relative frequency of the three species in each location based on isofemale lines genotyped and/or phenotyped for species identity (sample size of identified lines used per location is shown in parentheses). See Materials and Methods for identification procedure. See Table S1 for detailed location information and description of all lines studied

    Journal: Ecology and Evolution

    Article Title: Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement. Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement

    doi: 10.1002/ece3.3893

    Figure Lengend Snippet: Geographical range map of the Drosophila athabasca species complex: D. athabasca ( WN , blue), D. mahican ( EA , red), and D. lenape ( EB , orange) with specific locations shown as pie charts. Each pie chart represents the relative frequency of the three species in each location based on isofemale lines genotyped and/or phenotyped for species identity (sample size of identified lines used per location is shown in parentheses). See Materials and Methods for identification procedure. See Table S1 for detailed location information and description of all lines studied

    Article Snippet: A great system to test this question is the young Drosophila athabasca species complex (obscura group: affinis subgroup; Sturtevant & Dobzhansky, ), which contains three widespread semispecies across North America: West Northern (WN), Eastern A (EA), and Eastern B (EB) (see Figure and Miller, for known ranges).

    Techniques:

    Sexual isolation between (panels: a–c) and within (panels: d–f) species: Drosophila athabasca ( WN ), D. mahican ( EA ), and D. lenape ( EB ). Sexual isolation indexes ( SI s) between pairwise comparisons are plotted as a function of geographical distance (km). For multiple comparisons made between the same pairwise localities, the mean SI is shown with error bars indicating 95% confidence intervals across replicates. The number of replicate tests per comparison is shown next to each data point. R 2 are shown for each plot (none of the relationships are significant). Results are based mostly on multiple‐choice and few no‐choice mating tests across multiple isofemale lines (see Table S2 and text)

    Journal: Ecology and Evolution

    Article Title: Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement. Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement

    doi: 10.1002/ece3.3893

    Figure Lengend Snippet: Sexual isolation between (panels: a–c) and within (panels: d–f) species: Drosophila athabasca ( WN ), D. mahican ( EA ), and D. lenape ( EB ). Sexual isolation indexes ( SI s) between pairwise comparisons are plotted as a function of geographical distance (km). For multiple comparisons made between the same pairwise localities, the mean SI is shown with error bars indicating 95% confidence intervals across replicates. The number of replicate tests per comparison is shown next to each data point. R 2 are shown for each plot (none of the relationships are significant). Results are based mostly on multiple‐choice and few no‐choice mating tests across multiple isofemale lines (see Table S2 and text)

    Article Snippet: A great system to test this question is the young Drosophila athabasca species complex (obscura group: affinis subgroup; Sturtevant & Dobzhansky, ), which contains three widespread semispecies across North America: West Northern (WN), Eastern A (EA), and Eastern B (EB) (see Figure and Miller, for known ranges).

    Techniques: Isolation

    Sexual isolation between and within species of Drosophila athabasca ( WN ), D. mahican ( EA ), and D. lenape ( EB ). The mean SI is shown with error bars indicating 95% confidence intervals across replicates. Number of total replicate tests per comparison shown next to each bar. Results are based mostly on multiple‐choice mating tests across multiple isofemale lines with few no‐choice tests (see Table S2 and text). See Materials and Methods on how mating tests were performed. See Table S2 for detailed description of lines used, specific matings, mating method used, and conditions for each replicate. ANOVA test: NS = not significantly different. Asterisk indicates significance with a post hoc Tukey test

    Journal: Ecology and Evolution

    Article Title: Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement. Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement

    doi: 10.1002/ece3.3893

    Figure Lengend Snippet: Sexual isolation between and within species of Drosophila athabasca ( WN ), D. mahican ( EA ), and D. lenape ( EB ). The mean SI is shown with error bars indicating 95% confidence intervals across replicates. Number of total replicate tests per comparison shown next to each bar. Results are based mostly on multiple‐choice mating tests across multiple isofemale lines with few no‐choice tests (see Table S2 and text). See Materials and Methods on how mating tests were performed. See Table S2 for detailed description of lines used, specific matings, mating method used, and conditions for each replicate. ANOVA test: NS = not significantly different. Asterisk indicates significance with a post hoc Tukey test

    Article Snippet: A great system to test this question is the young Drosophila athabasca species complex (obscura group: affinis subgroup; Sturtevant & Dobzhansky, ), which contains three widespread semispecies across North America: West Northern (WN), Eastern A (EA), and Eastern B (EB) (see Figure and Miller, for known ranges).

    Techniques: Isolation

    Genetic structure and ancestry of 281 individuals of the Drosophila athabasca species complex based on 4,690 total variable base pairs with software STRUCTURE (v 2.3.4). Species designation is as follows: D. athabasca ( WN , blue/turquoise), D. mahican ( EA , red), D. lenape ( EB , orange). K = 2–5 plots are shown with default software settings and 5,000 burn‐in period and 5,000 MCMC runs (see Materials and Methods ). Specific plot shown is the highest probability run for each K among 20 runs per K (other runs not shown). On average, K = 3 had maximal average value of Ln P ( D ) and delta K value (Evanno et al., 2005 ; Table S6 ). Samples are organized into 21 geographical populations (divided by black lines and abbreviations shown below the plots) and grouped into larger geographical regions across North America (shown above plots and designated with thick black bars)

    Journal: Ecology and Evolution

    Article Title: Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement. Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement

    doi: 10.1002/ece3.3893

    Figure Lengend Snippet: Genetic structure and ancestry of 281 individuals of the Drosophila athabasca species complex based on 4,690 total variable base pairs with software STRUCTURE (v 2.3.4). Species designation is as follows: D. athabasca ( WN , blue/turquoise), D. mahican ( EA , red), D. lenape ( EB , orange). K = 2–5 plots are shown with default software settings and 5,000 burn‐in period and 5,000 MCMC runs (see Materials and Methods ). Specific plot shown is the highest probability run for each K among 20 runs per K (other runs not shown). On average, K = 3 had maximal average value of Ln P ( D ) and delta K value (Evanno et al., 2005 ; Table S6 ). Samples are organized into 21 geographical populations (divided by black lines and abbreviations shown below the plots) and grouped into larger geographical regions across North America (shown above plots and designated with thick black bars)

    Article Snippet: A great system to test this question is the young Drosophila athabasca species complex (obscura group: affinis subgroup; Sturtevant & Dobzhansky, ), which contains three widespread semispecies across North America: West Northern (WN), Eastern A (EA), and Eastern B (EB) (see Figure and Miller, for known ranges).

    Techniques: Software

    Relationship between pairwise geographical distance (km) of conspecific populations ( x ‐axis) and average measures of sequence divergence ( y ‐axis) within each species: Drosophila athabasca ( WN , left panels, blue squares), D. mahican ( EA , center panels, red circles), and D. lenape ( EB , right panels, orange triangles). Top panels (a–c) show Dxy (absolute measure of sequence divergence), and bottom panels (d–f) show Fst (relative measure of sequence divergence). Both measures of genetic divergence are averaged across all gene fragments. Only populations with greater than six chromosomes are considered. Mantel tests were performed to determine the significance of each relationship with 1,000 permutation tests of matrix correlations between genetic divergence ( Dxy or Fst ) and geographical distance. Significant positive correlations reveal evidence of isolation by distance. Trendlines are shown only to help show patterns. Note the scale of y ‐axis

    Journal: Ecology and Evolution

    Article Title: Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement. Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement

    doi: 10.1002/ece3.3893

    Figure Lengend Snippet: Relationship between pairwise geographical distance (km) of conspecific populations ( x ‐axis) and average measures of sequence divergence ( y ‐axis) within each species: Drosophila athabasca ( WN , left panels, blue squares), D. mahican ( EA , center panels, red circles), and D. lenape ( EB , right panels, orange triangles). Top panels (a–c) show Dxy (absolute measure of sequence divergence), and bottom panels (d–f) show Fst (relative measure of sequence divergence). Both measures of genetic divergence are averaged across all gene fragments. Only populations with greater than six chromosomes are considered. Mantel tests were performed to determine the significance of each relationship with 1,000 permutation tests of matrix correlations between genetic divergence ( Dxy or Fst ) and geographical distance. Significant positive correlations reveal evidence of isolation by distance. Trendlines are shown only to help show patterns. Note the scale of y ‐axis

    Article Snippet: A great system to test this question is the young Drosophila athabasca species complex (obscura group: affinis subgroup; Sturtevant & Dobzhansky, ), which contains three widespread semispecies across North America: West Northern (WN), Eastern A (EA), and Eastern B (EB) (see Figure and Miller, for known ranges).

    Techniques: Sequencing, Isolation

    DEVDase (caspase 3-like) activity in the brain of PQ-exposed Drosophila . Graphical representation of DEVDase activity in the brain homogenate of Drosophila after their exposure to PQ for 12 and 24 h; A significant decrease in enzyme activity was observed in the brain of PQ-exposed hsp70 over-expressing strain. Data are mean ± SD (n = 3). Significance ascribed as ** p

    Journal: PLoS ONE

    Article Title: Heat Shock Protein-70 (Hsp-70) Suppresses Paraquat-Induced Neurodegeneration by Inhibiting JNK and Caspase-3 Activation in Drosophila Model of Parkinson's Disease

    doi: 10.1371/journal.pone.0098886

    Figure Lengend Snippet: DEVDase (caspase 3-like) activity in the brain of PQ-exposed Drosophila . Graphical representation of DEVDase activity in the brain homogenate of Drosophila after their exposure to PQ for 12 and 24 h; A significant decrease in enzyme activity was observed in the brain of PQ-exposed hsp70 over-expressing strain. Data are mean ± SD (n = 3). Significance ascribed as ** p

    Article Snippet: Anti-Phospho-JNK, anti-cleaved caspase-3 (1∶1000; Cell Signaling Technology, MA, USA), anti-JNK (1∶500; Santa Cruz, TX, USA), Drosophila Hsp70 monoclonal antibody (7Fb; 1∶250) and anti-β tubulin (1∶500; Developmental Studies Hybridoma Bank, IA, USA) were used as the primary antibodies.

    Techniques: Activity Assay, Expressing

    hsp70 over-expression protects PQ-exposed flies from impaired locomotor performance and poor survival. (A) Graphical representation of the negative geotaxis assay performed in PQ-exposed w 1118 , Df(hsp70) , TH-Gal4 > w 1118 , TH-Gal4 > HSP70K71E and TH-Gal4 > UAS-hsp70 flies after their exposure to PQ. (B) Survival curve representing pooled data of five independent replicate experiments. Values are mean ± SD (n = 5). Significance ascribed as * p

    Journal: PLoS ONE

    Article Title: Heat Shock Protein-70 (Hsp-70) Suppresses Paraquat-Induced Neurodegeneration by Inhibiting JNK and Caspase-3 Activation in Drosophila Model of Parkinson's Disease

    doi: 10.1371/journal.pone.0098886

    Figure Lengend Snippet: hsp70 over-expression protects PQ-exposed flies from impaired locomotor performance and poor survival. (A) Graphical representation of the negative geotaxis assay performed in PQ-exposed w 1118 , Df(hsp70) , TH-Gal4 > w 1118 , TH-Gal4 > HSP70K71E and TH-Gal4 > UAS-hsp70 flies after their exposure to PQ. (B) Survival curve representing pooled data of five independent replicate experiments. Values are mean ± SD (n = 5). Significance ascribed as * p

    Article Snippet: Anti-Phospho-JNK, anti-cleaved caspase-3 (1∶1000; Cell Signaling Technology, MA, USA), anti-JNK (1∶500; Santa Cruz, TX, USA), Drosophila Hsp70 monoclonal antibody (7Fb; 1∶250) and anti-β tubulin (1∶500; Developmental Studies Hybridoma Bank, IA, USA) were used as the primary antibodies.

    Techniques: Over Expression

    hsp70 over-expression in the dopaminergic neurons of Drosophila protects them from PQ-induced neuronal degeneration. (A) Representative confocal images of brains of control w 1118 and 20 mM PQ-exposed w 1118 , Df(hsp70) , TH-Gal4 > w 1118 , TH-Gal4 > HSP70K71E and TH-Gal4 > UAS-hsp70 fly for 24 h after anti-DTH staining. Quantitative representation of the average number of neurons in the dopaminergic clusters of adult flies that were exposed to PQ (B–D) for 12 and (C–E) 24 h. Graphical representation of (F) DA and (G) DOPAC levels in PQ-exposed w 1118 , Df(hsp70) , TH-Gal4 > w 1118 , TH-Gal4 > HSP70K71E and TH-Gal4 > UAS-hsp70 flies. Arrow head indicates PPL1 cluster. Significance ascribed as * p

    Journal: PLoS ONE

    Article Title: Heat Shock Protein-70 (Hsp-70) Suppresses Paraquat-Induced Neurodegeneration by Inhibiting JNK and Caspase-3 Activation in Drosophila Model of Parkinson's Disease

    doi: 10.1371/journal.pone.0098886

    Figure Lengend Snippet: hsp70 over-expression in the dopaminergic neurons of Drosophila protects them from PQ-induced neuronal degeneration. (A) Representative confocal images of brains of control w 1118 and 20 mM PQ-exposed w 1118 , Df(hsp70) , TH-Gal4 > w 1118 , TH-Gal4 > HSP70K71E and TH-Gal4 > UAS-hsp70 fly for 24 h after anti-DTH staining. Quantitative representation of the average number of neurons in the dopaminergic clusters of adult flies that were exposed to PQ (B–D) for 12 and (C–E) 24 h. Graphical representation of (F) DA and (G) DOPAC levels in PQ-exposed w 1118 , Df(hsp70) , TH-Gal4 > w 1118 , TH-Gal4 > HSP70K71E and TH-Gal4 > UAS-hsp70 flies. Arrow head indicates PPL1 cluster. Significance ascribed as * p

    Article Snippet: Anti-Phospho-JNK, anti-cleaved caspase-3 (1∶1000; Cell Signaling Technology, MA, USA), anti-JNK (1∶500; Santa Cruz, TX, USA), Drosophila Hsp70 monoclonal antibody (7Fb; 1∶250) and anti-β tubulin (1∶500; Developmental Studies Hybridoma Bank, IA, USA) were used as the primary antibodies.

    Techniques: Over Expression, Staining

    hsp70 over-expression in the dopaminergic neurons of Drosophila protect them from PQ-induced OS. (A) Time and dose dependent increase in superoxide (O 2 − ) in the brain of PQ-exposed w 1118 , Df(hsp70) , TH-Gal4 > w 1118 and TH-Gal4 > Hsp70K71E flies and less generation of O 2 − in exposed TH-Gal4 > UAS-hsp70 flies as compared to TH-Gal4 > w 1118 . (B) Decreased SOD activity in the brain of PQ-exposed w 1118 , Df(hsp70), TH-Gal4 > w 1118 and TH-Gal4 > Hsp70K71E flies and an increased enzyme activity in exposed TH-Gal4 > UAS-hsp70 flies. (C) Graphical representation of peroxynitrite (ONOO − ) generation using DHR 123 staining. ONOO − generation was also found to be less like O 2 − generation in PQ-exposed TH-Gal4 > UAS-hsp70 flies. (D) Histogram depicting the extent of lipid peroxidation as determined by MDA content in w 1118 , Df(hsp70) , TH-Gal4 > Hsp70K71E and TH-Gal4 > UAS-hsp70 flies. (E) Representative immuno-blot images of p-JNK, JNK and cleaved caspase-3 levels in the protein samples from brain tissues of control and PQ-exposed w 1118 and Df(hsp70) flies. (F) Densitometry analysis of data normalized against loading control tubulin. (G) Representative immuno-blotting images of TH-Gal4 > w 1118 , TH-Gal4 > HSP70K71E and TH-Gal4 > UAS-hsp70 flies. (H) Densitometry analysis of data normalized against loading control tubulin. Values are mean ± SD (n = 3). Significance were ascribed as * p

    Journal: PLoS ONE

    Article Title: Heat Shock Protein-70 (Hsp-70) Suppresses Paraquat-Induced Neurodegeneration by Inhibiting JNK and Caspase-3 Activation in Drosophila Model of Parkinson's Disease

    doi: 10.1371/journal.pone.0098886

    Figure Lengend Snippet: hsp70 over-expression in the dopaminergic neurons of Drosophila protect them from PQ-induced OS. (A) Time and dose dependent increase in superoxide (O 2 − ) in the brain of PQ-exposed w 1118 , Df(hsp70) , TH-Gal4 > w 1118 and TH-Gal4 > Hsp70K71E flies and less generation of O 2 − in exposed TH-Gal4 > UAS-hsp70 flies as compared to TH-Gal4 > w 1118 . (B) Decreased SOD activity in the brain of PQ-exposed w 1118 , Df(hsp70), TH-Gal4 > w 1118 and TH-Gal4 > Hsp70K71E flies and an increased enzyme activity in exposed TH-Gal4 > UAS-hsp70 flies. (C) Graphical representation of peroxynitrite (ONOO − ) generation using DHR 123 staining. ONOO − generation was also found to be less like O 2 − generation in PQ-exposed TH-Gal4 > UAS-hsp70 flies. (D) Histogram depicting the extent of lipid peroxidation as determined by MDA content in w 1118 , Df(hsp70) , TH-Gal4 > Hsp70K71E and TH-Gal4 > UAS-hsp70 flies. (E) Representative immuno-blot images of p-JNK, JNK and cleaved caspase-3 levels in the protein samples from brain tissues of control and PQ-exposed w 1118 and Df(hsp70) flies. (F) Densitometry analysis of data normalized against loading control tubulin. (G) Representative immuno-blotting images of TH-Gal4 > w 1118 , TH-Gal4 > HSP70K71E and TH-Gal4 > UAS-hsp70 flies. (H) Densitometry analysis of data normalized against loading control tubulin. Values are mean ± SD (n = 3). Significance were ascribed as * p

    Article Snippet: Anti-Phospho-JNK, anti-cleaved caspase-3 (1∶1000; Cell Signaling Technology, MA, USA), anti-JNK (1∶500; Santa Cruz, TX, USA), Drosophila Hsp70 monoclonal antibody (7Fb; 1∶250) and anti-β tubulin (1∶500; Developmental Studies Hybridoma Bank, IA, USA) were used as the primary antibodies.

    Techniques: Over Expression, Activity Assay, Staining, Multiple Displacement Amplification

    Schematic representation of the neuro-protective role of Hsp70 against PQ-induced PD like symptoms. PQ-induced ROS generation led to the activation of JNK and caspase-3, which eventually caused death of the dopaminergic neurons and induces PD like symptoms in the exposed organism. The observed PD like symptoms was resisted by over-expression of one of the key chaperons, Hsp70, in the dopaminergic neurons of Drosophila . Hsp70 inhibits PQ-induced activation of JNK and caspase-3 mediated neuronal cell death, thereby providing protection to the organism.

    Journal: PLoS ONE

    Article Title: Heat Shock Protein-70 (Hsp-70) Suppresses Paraquat-Induced Neurodegeneration by Inhibiting JNK and Caspase-3 Activation in Drosophila Model of Parkinson's Disease

    doi: 10.1371/journal.pone.0098886

    Figure Lengend Snippet: Schematic representation of the neuro-protective role of Hsp70 against PQ-induced PD like symptoms. PQ-induced ROS generation led to the activation of JNK and caspase-3, which eventually caused death of the dopaminergic neurons and induces PD like symptoms in the exposed organism. The observed PD like symptoms was resisted by over-expression of one of the key chaperons, Hsp70, in the dopaminergic neurons of Drosophila . Hsp70 inhibits PQ-induced activation of JNK and caspase-3 mediated neuronal cell death, thereby providing protection to the organism.

    Article Snippet: Anti-Phospho-JNK, anti-cleaved caspase-3 (1∶1000; Cell Signaling Technology, MA, USA), anti-JNK (1∶500; Santa Cruz, TX, USA), Drosophila Hsp70 monoclonal antibody (7Fb; 1∶250) and anti-β tubulin (1∶500; Developmental Studies Hybridoma Bank, IA, USA) were used as the primary antibodies.

    Techniques: Activation Assay, Over Expression

    Over-expression of a human homologue of hsp70 in the dopaminergic neurons of Drosophila provided them resistance against PQ-induced PD like symptoms. Like PQ-exposed TH-Gal4 > UAS-hsp70 flies, exposed TH-Gal4 > UAS-HSPA1L flies also exhibited reduced levels of OS as evidenced by less O 2 − generation, improved SOD activity, less formation of ONOO − and non-significant increase in MDA content. JNK phosphorylation and cleaved caspase-3 level was significantly less in the brain of PQ-exposed TH-Gal4 > UAS-HSPA1L flies in comparison to Gal4 control, as represented by immune-blotting images and densitometry analysis. Dopaminergic neuronal health depicted by representative confocal image of anti-DTH stained brain of control and PQ-exposed TH-Gal4 > UAS-HSPA1L flies and graphical representation of dopaminergic neurons in different clusters (n = 22). In the brain of PQ-exposed TH-Gal4 > UAS-HSPA1L flies, less decline in DA and lower level of DOPAC was observed. Improved locomotor performance and survival advantage were also observed in these flies. Values are mean ± SD (n = 3). Significance ascribed as * p

    Journal: PLoS ONE

    Article Title: Heat Shock Protein-70 (Hsp-70) Suppresses Paraquat-Induced Neurodegeneration by Inhibiting JNK and Caspase-3 Activation in Drosophila Model of Parkinson's Disease

    doi: 10.1371/journal.pone.0098886

    Figure Lengend Snippet: Over-expression of a human homologue of hsp70 in the dopaminergic neurons of Drosophila provided them resistance against PQ-induced PD like symptoms. Like PQ-exposed TH-Gal4 > UAS-hsp70 flies, exposed TH-Gal4 > UAS-HSPA1L flies also exhibited reduced levels of OS as evidenced by less O 2 − generation, improved SOD activity, less formation of ONOO − and non-significant increase in MDA content. JNK phosphorylation and cleaved caspase-3 level was significantly less in the brain of PQ-exposed TH-Gal4 > UAS-HSPA1L flies in comparison to Gal4 control, as represented by immune-blotting images and densitometry analysis. Dopaminergic neuronal health depicted by representative confocal image of anti-DTH stained brain of control and PQ-exposed TH-Gal4 > UAS-HSPA1L flies and graphical representation of dopaminergic neurons in different clusters (n = 22). In the brain of PQ-exposed TH-Gal4 > UAS-HSPA1L flies, less decline in DA and lower level of DOPAC was observed. Improved locomotor performance and survival advantage were also observed in these flies. Values are mean ± SD (n = 3). Significance ascribed as * p

    Article Snippet: Anti-Phospho-JNK, anti-cleaved caspase-3 (1∶1000; Cell Signaling Technology, MA, USA), anti-JNK (1∶500; Santa Cruz, TX, USA), Drosophila Hsp70 monoclonal antibody (7Fb; 1∶250) and anti-β tubulin (1∶500; Developmental Studies Hybridoma Bank, IA, USA) were used as the primary antibodies.

    Techniques: Over Expression, Activity Assay, Multiple Displacement Amplification, Staining

    E2f1 Stable acts acutely to trigger apoptosis. A–E) Detection of S phase by EdU labeling (red) and apoptosis by CC3 staining (green) in GMR-Gal4 third instar larval eye imaginal discs expressing GFP or the indicated GFP-E2f1 fusion proteins. Arrowheads indicate the position of the MF, with anterior to the left and posterior to the right. Bars = 5 µM. F) Quantification of the number of CC3 positive cells posterior to the MF. * p

    Journal: PLoS Genetics

    Article Title: S Phase-Coupled E2f1 Destruction Ensures Homeostasis in Proliferating TissuesIt's All in the Timing: Too Much E2F Is a Bad Thing

    doi: 10.1371/journal.pgen.1002831

    Figure Lengend Snippet: E2f1 Stable acts acutely to trigger apoptosis. A–E) Detection of S phase by EdU labeling (red) and apoptosis by CC3 staining (green) in GMR-Gal4 third instar larval eye imaginal discs expressing GFP or the indicated GFP-E2f1 fusion proteins. Arrowheads indicate the position of the MF, with anterior to the left and posterior to the right. Bars = 5 µM. F) Quantification of the number of CC3 positive cells posterior to the MF. * p

    Article Snippet: E2f1 protein levels were measured with affinity-purified rabbit anti-E2f1 raised against full-length Drosophila E2f1 (1∶1000) overnight at 4°C and anti-rabbit HRP secondary (1∶10,000 GE Healthcare) for 1 hour at room temperature.

    Techniques: Labeling, Staining, Expressing

    E2f1 Stable -induced apoptosis requires Rbf1 binding but not DNA binding. A–I) Detection of apoptosis via Cleaved Caspase-3 (CC3, red) staining of third instar larval wing imaginal discs expressing the indicated GFP-E2f1 (GFP, green) proteins with en-Gal4. Arrow in D indicates an example of apoptosis observed in wild type wing discs. Bars = 50 µM. J) Quantification by flow cytometry of GFP-positive apoptotic cells from trypsin-dissociated en-Gal4 wing discs expressing GFP or the indicated GFP-E2f1 fusion proteins. Error bars represent the standard error of three independent experiments. ** p

    Journal: PLoS Genetics

    Article Title: S Phase-Coupled E2f1 Destruction Ensures Homeostasis in Proliferating TissuesIt's All in the Timing: Too Much E2F Is a Bad Thing

    doi: 10.1371/journal.pgen.1002831

    Figure Lengend Snippet: E2f1 Stable -induced apoptosis requires Rbf1 binding but not DNA binding. A–I) Detection of apoptosis via Cleaved Caspase-3 (CC3, red) staining of third instar larval wing imaginal discs expressing the indicated GFP-E2f1 (GFP, green) proteins with en-Gal4. Arrow in D indicates an example of apoptosis observed in wild type wing discs. Bars = 50 µM. J) Quantification by flow cytometry of GFP-positive apoptotic cells from trypsin-dissociated en-Gal4 wing discs expressing GFP or the indicated GFP-E2f1 fusion proteins. Error bars represent the standard error of three independent experiments. ** p

    Article Snippet: E2f1 protein levels were measured with affinity-purified rabbit anti-E2f1 raised against full-length Drosophila E2f1 (1∶1000) overnight at 4°C and anti-rabbit HRP secondary (1∶10,000 GE Healthcare) for 1 hour at room temperature.

    Techniques: Binding Assay, Staining, Expressing, Flow Cytometry, Cytometry

    E2f1 Stable causes homeostasis defects and tissue hyperplasia. A–C) Detection of GFP or the indicated GFP-E2f1 proteins (green) in en-Gal4, UAS-p35 wing discs stained with DAPI (white). Scale bars indicate 50 µm. D) Quantification of morphological defects by microscopically measuring the thickness of the posterior compartment of the indicated en-Gal4 > GFP-E2f1 wing discs. Measurements were obtained by counting the number of 1 micron sections required to visualize all the way through the posterior compartment of the tissue. Bars = 50 µM. E) E2f1 Stable induces apoptosis in two ways.

    Journal: PLoS Genetics

    Article Title: S Phase-Coupled E2f1 Destruction Ensures Homeostasis in Proliferating TissuesIt's All in the Timing: Too Much E2F Is a Bad Thing

    doi: 10.1371/journal.pgen.1002831

    Figure Lengend Snippet: E2f1 Stable causes homeostasis defects and tissue hyperplasia. A–C) Detection of GFP or the indicated GFP-E2f1 proteins (green) in en-Gal4, UAS-p35 wing discs stained with DAPI (white). Scale bars indicate 50 µm. D) Quantification of morphological defects by microscopically measuring the thickness of the posterior compartment of the indicated en-Gal4 > GFP-E2f1 wing discs. Measurements were obtained by counting the number of 1 micron sections required to visualize all the way through the posterior compartment of the tissue. Bars = 50 µM. E) E2f1 Stable induces apoptosis in two ways.

    Article Snippet: E2f1 protein levels were measured with affinity-purified rabbit anti-E2f1 raised against full-length Drosophila E2f1 (1∶1000) overnight at 4°C and anti-rabbit HRP secondary (1∶10,000 GE Healthcare) for 1 hour at room temperature.

    Techniques: Staining

    E2f1 Stable causes DNA damage. A–E) Detection of DNA damage by anti-phospho-H2Av staining (red) in GMR-Gal4 third instar larval eye imaginal discs expressing GFP or the indicated GFP-E2f1 fusion proteins (green). Arrowheads indicate the position of the MF, with anterior to the bottom and posterior to the top. Bars = 10 µM. F) Quantification of the number of phospho-H2Av positive cells posterior to the MF. * p

    Journal: PLoS Genetics

    Article Title: S Phase-Coupled E2f1 Destruction Ensures Homeostasis in Proliferating TissuesIt's All in the Timing: Too Much E2F Is a Bad Thing

    doi: 10.1371/journal.pgen.1002831

    Figure Lengend Snippet: E2f1 Stable causes DNA damage. A–E) Detection of DNA damage by anti-phospho-H2Av staining (red) in GMR-Gal4 third instar larval eye imaginal discs expressing GFP or the indicated GFP-E2f1 fusion proteins (green). Arrowheads indicate the position of the MF, with anterior to the bottom and posterior to the top. Bars = 10 µM. F) Quantification of the number of phospho-H2Av positive cells posterior to the MF. * p

    Article Snippet: E2f1 protein levels were measured with affinity-purified rabbit anti-E2f1 raised against full-length Drosophila E2f1 (1∶1000) overnight at 4°C and anti-rabbit HRP secondary (1∶10,000 GE Healthcare) for 1 hour at room temperature.

    Techniques: Staining, Expressing

    Domain mutations disrupt critical E2f1 functions. A) Schematic of the experimental paradigm. B) Schematic representation of E2f1 alleles used in this study. C) qRT-PCR quantification of GFP-containing mRNA in en-Gal4 wing discs expressing GFP or the indicated GFP-E2f1 fusion proteins that lack (grey; “N

    Journal: PLoS Genetics

    Article Title: S Phase-Coupled E2f1 Destruction Ensures Homeostasis in Proliferating TissuesIt's All in the Timing: Too Much E2F Is a Bad Thing

    doi: 10.1371/journal.pgen.1002831

    Figure Lengend Snippet: Domain mutations disrupt critical E2f1 functions. A) Schematic of the experimental paradigm. B) Schematic representation of E2f1 alleles used in this study. C) qRT-PCR quantification of GFP-containing mRNA in en-Gal4 wing discs expressing GFP or the indicated GFP-E2f1 fusion proteins that lack (grey; “N") or contain (black; “Y") the PIP-3A mutation ( Figure S1A ) relative to a non-transgenic w 1118 control (Con). Error bars represent the standard error of three independent experiments. These designations will be used throughout the remaining figures. UAS-GFP expression was greater than any E2f1 construct because the UASt promoter was used rather than UASp. D) Anti-E2f1 western blot measuring GFP-E2f1 and endogenous E2f1 expression in third instar imaginal wing discs. The ratio of transgene expression to endogenous E2f1 expression is shown below. E) Quantification by flow cytometry of RFP-positive G1 cells from trypsin-dissociated en-Gal4, UAS-RFP wing discs expressing GFP or the indicated GFP-E2f1 fusion proteins. * p

    Article Snippet: E2f1 protein levels were measured with affinity-purified rabbit anti-E2f1 raised against full-length Drosophila E2f1 (1∶1000) overnight at 4°C and anti-rabbit HRP secondary (1∶10,000 GE Healthcare) for 1 hour at room temperature.

    Techniques: Quantitative RT-PCR, Expressing, Mutagenesis, Transgenic Assay, Construct, Western Blot, Flow Cytometry, Cytometry

    Induction of Apoptosis requires full-length E2f1 Stable . A) Schematic of the E2f1 336–805 mutant protein, which contains an NH 2 -terminal HA tag. B) Detection of apoptosis via Cleaved Caspase-3 (CC3, red) staining of third instar larval wing imaginal discs expressing HA-E2f1 336–805 (anti-HA, green) with en-Gal4. Bar = 50 µm. C) Anti-E2f1 western blot of third instar imaginal wing discs expressing GFP-E2f1, GFP-E2f1 Stable , or HA-E2f1 336–805 . D) Co-immunoprecipitation analysis of Myc-E2f1 and HA-Rbf1 from transiently transfected S2 cells.

    Journal: PLoS Genetics

    Article Title: S Phase-Coupled E2f1 Destruction Ensures Homeostasis in Proliferating TissuesIt's All in the Timing: Too Much E2F Is a Bad Thing

    doi: 10.1371/journal.pgen.1002831

    Figure Lengend Snippet: Induction of Apoptosis requires full-length E2f1 Stable . A) Schematic of the E2f1 336–805 mutant protein, which contains an NH 2 -terminal HA tag. B) Detection of apoptosis via Cleaved Caspase-3 (CC3, red) staining of third instar larval wing imaginal discs expressing HA-E2f1 336–805 (anti-HA, green) with en-Gal4. Bar = 50 µm. C) Anti-E2f1 western blot of third instar imaginal wing discs expressing GFP-E2f1, GFP-E2f1 Stable , or HA-E2f1 336–805 . D) Co-immunoprecipitation analysis of Myc-E2f1 and HA-Rbf1 from transiently transfected S2 cells.

    Article Snippet: E2f1 protein levels were measured with affinity-purified rabbit anti-E2f1 raised against full-length Drosophila E2f1 (1∶1000) overnight at 4°C and anti-rabbit HRP secondary (1∶10,000 GE Healthcare) for 1 hour at room temperature.

    Techniques: Mutagenesis, Hemagglutination Assay, Staining, Expressing, Western Blot, Immunoprecipitation, Transfection

    Stabilizing E2f1 during S phase induces hid expression. A, B) qRT-PCR quantification of hid mRNA (A) or rpr mRNA (B) in en-Gal4 wing discs expressing GFP or the indicated GFP-E2f1 fusion proteins that lack (grey) or contain (black) the PIP3A mutation relative to a non-transgenic w 1118 control (Con). * p

    Journal: PLoS Genetics

    Article Title: S Phase-Coupled E2f1 Destruction Ensures Homeostasis in Proliferating TissuesIt's All in the Timing: Too Much E2F Is a Bad Thing

    doi: 10.1371/journal.pgen.1002831

    Figure Lengend Snippet: Stabilizing E2f1 during S phase induces hid expression. A, B) qRT-PCR quantification of hid mRNA (A) or rpr mRNA (B) in en-Gal4 wing discs expressing GFP or the indicated GFP-E2f1 fusion proteins that lack (grey) or contain (black) the PIP3A mutation relative to a non-transgenic w 1118 control (Con). * p

    Article Snippet: E2f1 protein levels were measured with affinity-purified rabbit anti-E2f1 raised against full-length Drosophila E2f1 (1∶1000) overnight at 4°C and anti-rabbit HRP secondary (1∶10,000 GE Healthcare) for 1 hour at room temperature.

    Techniques: Expressing, Quantitative RT-PCR, Mutagenesis, Transgenic Assay

    Endogenous VPS13A is enriched at fractions containing LDs upon OA induction. Ratio of VPS13A in the LD fraction in starved (Stv) ( Figure 7A ) and OA fed cells ( Figure 7B ). The amount of VPS13A Stv is set to 1.

    Journal: eLife

    Article Title: Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility

    doi: 10.7554/eLife.43561

    Figure Lengend Snippet: Endogenous VPS13A is enriched at fractions containing LDs upon OA induction. Ratio of VPS13A in the LD fraction in starved (Stv) ( Figure 7A ) and OA fed cells ( Figure 7B ). The amount of VPS13A Stv is set to 1.

    Article Snippet: The following primary antibodies were used: anti-ATP5A (Abcam, 1:1000), anti-DRP1 (to detect total DRP) Cell Signaling 1:500), anti-p(hospho)DRP1 ser616 (Cell signaling, 1:1000), anti-GAPDH (Fitzgerald 1:10,000), anti-GFP (Clontech 1:1000), anti-GST (Santacruz Biotechnology, 1:1000), anti-EGFR (Santacruz Biotechnology, 1:1000), anti-LAMP1 (Abcam, 1:1000), anti-Myc (Enzo Life Sciences, 1:1000), anti-PLIN2 (Abcam, 1:1000), anti-Rab7 (Abcam, 1:1000), anti-TOMM20 (BD biosciences 1:1000), anti-α tubulin (Sigma, 1:5000), anti-VAP-A (Santa Cruz Biotechnology, 1:1000), anti-VAP-B (Sigma, 1:1000), anti-VPS13A (Sigma,1:1000), anti-VPS13A (H-102) (Santa Cruz Biotechnology, 1:500), Drosophila VPS13A #62 , VPS13C (Sigma).

    Techniques:

    Proposed model for VPS13A function. ( A ) Under normal growth conditions VPS13A is localized at the ER-mitochondria contact sites where it is anchored to VAP-A through its FFAT domain and via its C-terminal region it is associated with mitochondria, most likely via mitochondria specific adaptor proteins. VPS13A at this location may facilitate the transfer of lipids between ER and mitochondria and mitochondria fusion and mitophagy occur normally. ( B ) Under normal conditions VPS13A is also associated to LD, an association mediated via LD specific adaptor proteins. Via VPS13A LD are associated to the ER and VPS13A facilitate the transfer of lipids between ER and LDs. The VPS13A mediated ER-lipid connection halts LD movement. ( C ) Depletion of VPS13A leads to impaired lipid transfer between ER and mitochondria, leading to abnormal function of mitochondria which become less elongated. ( D ) Depletion of VPS13A also leads to disconnection of LD and the ER, leading to increased movement and reduced degradation of LD, resulting in increased LD numbers.

    Journal: eLife

    Article Title: Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility

    doi: 10.7554/eLife.43561

    Figure Lengend Snippet: Proposed model for VPS13A function. ( A ) Under normal growth conditions VPS13A is localized at the ER-mitochondria contact sites where it is anchored to VAP-A through its FFAT domain and via its C-terminal region it is associated with mitochondria, most likely via mitochondria specific adaptor proteins. VPS13A at this location may facilitate the transfer of lipids between ER and mitochondria and mitochondria fusion and mitophagy occur normally. ( B ) Under normal conditions VPS13A is also associated to LD, an association mediated via LD specific adaptor proteins. Via VPS13A LD are associated to the ER and VPS13A facilitate the transfer of lipids between ER and LDs. The VPS13A mediated ER-lipid connection halts LD movement. ( C ) Depletion of VPS13A leads to impaired lipid transfer between ER and mitochondria, leading to abnormal function of mitochondria which become less elongated. ( D ) Depletion of VPS13A also leads to disconnection of LD and the ER, leading to increased movement and reduced degradation of LD, resulting in increased LD numbers.

    Article Snippet: The following primary antibodies were used: anti-ATP5A (Abcam, 1:1000), anti-DRP1 (to detect total DRP) Cell Signaling 1:500), anti-p(hospho)DRP1 ser616 (Cell signaling, 1:1000), anti-GAPDH (Fitzgerald 1:10,000), anti-GFP (Clontech 1:1000), anti-GST (Santacruz Biotechnology, 1:1000), anti-EGFR (Santacruz Biotechnology, 1:1000), anti-LAMP1 (Abcam, 1:1000), anti-Myc (Enzo Life Sciences, 1:1000), anti-PLIN2 (Abcam, 1:1000), anti-Rab7 (Abcam, 1:1000), anti-TOMM20 (BD biosciences 1:1000), anti-α tubulin (Sigma, 1:5000), anti-VAP-A (Santa Cruz Biotechnology, 1:1000), anti-VAP-B (Sigma, 1:1000), anti-VPS13A (Sigma,1:1000), anti-VPS13A (H-102) (Santa Cruz Biotechnology, 1:500), Drosophila VPS13A #62 , VPS13C (Sigma).

    Techniques:

    VPS13A negatively regulates LD mobility. ( A ) WT ( A ) and VPS13A KO MRC5 cells ( A’ ) were stained with LipidTox green for LDs (green) and the nuclear marker DAPI (blue) and imaged by confocal microscopy. ( B ) Quantification of LD numbers in A. Error bars, mean ±s.e.m (n = 3), two-tailed unpaired Student’s t-test was used (*p≤0.05, **p≤0.01). ( C ) WT and VPS13A KO MRC5 cells were stained with Nile red and intensity was measured using FACS. Error bars, mean ±s.e.m (n = 3), two-tailed unpaired Student’s t-test was used (*p≤0.05, **p≤0.01). ( D ) WT and VPS13A KO MRC5 cells were exposed to 500 µM OA for 16 hr. Afterwards cells were stained with LipidTox green to visualize LDs and LD numbers were quantified. Error bars, mean ±s.e.m (n = 3), two-tailed unpaired Student’s t-test was used. ( E ) HEK293T cells were transfected with VPS13A-GFP and stained with LipidTox red to visualize LDs in vivo. Images with a time interval of 6 s were recorded of VPS13-GFP positive (cell 1) and adjacent VPS13-GFP negative (cell 2) cells. The locations of LDs at t = 0 are indicated in green, the locations of the same LDs at t = 6 s are indicated in magenta ( E ). If the LD did not move between time frames, the overlapping signal (green and magenta) is white. The VPS13A signal is shown in E’: Cell one is transfected with VPS13A-GFP; Cell two is a non-transfected cell. ( F ) Quantification of the fraction of non-moving (white) LDs compared to the total number of LDs in VPS13A-GFP positive or VPS13A-GFP negative cells. Error bars, mean ±s.e.m, two tailed unpaired Student’s t-test was used (*p≤0.05). Scale bars = 10 µm (A, A’, E,).

    Journal: eLife

    Article Title: Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility

    doi: 10.7554/eLife.43561

    Figure Lengend Snippet: VPS13A negatively regulates LD mobility. ( A ) WT ( A ) and VPS13A KO MRC5 cells ( A’ ) were stained with LipidTox green for LDs (green) and the nuclear marker DAPI (blue) and imaged by confocal microscopy. ( B ) Quantification of LD numbers in A. Error bars, mean ±s.e.m (n = 3), two-tailed unpaired Student’s t-test was used (*p≤0.05, **p≤0.01). ( C ) WT and VPS13A KO MRC5 cells were stained with Nile red and intensity was measured using FACS. Error bars, mean ±s.e.m (n = 3), two-tailed unpaired Student’s t-test was used (*p≤0.05, **p≤0.01). ( D ) WT and VPS13A KO MRC5 cells were exposed to 500 µM OA for 16 hr. Afterwards cells were stained with LipidTox green to visualize LDs and LD numbers were quantified. Error bars, mean ±s.e.m (n = 3), two-tailed unpaired Student’s t-test was used. ( E ) HEK293T cells were transfected with VPS13A-GFP and stained with LipidTox red to visualize LDs in vivo. Images with a time interval of 6 s were recorded of VPS13-GFP positive (cell 1) and adjacent VPS13-GFP negative (cell 2) cells. The locations of LDs at t = 0 are indicated in green, the locations of the same LDs at t = 6 s are indicated in magenta ( E ). If the LD did not move between time frames, the overlapping signal (green and magenta) is white. The VPS13A signal is shown in E’: Cell one is transfected with VPS13A-GFP; Cell two is a non-transfected cell. ( F ) Quantification of the fraction of non-moving (white) LDs compared to the total number of LDs in VPS13A-GFP positive or VPS13A-GFP negative cells. Error bars, mean ±s.e.m, two tailed unpaired Student’s t-test was used (*p≤0.05). Scale bars = 10 µm (A, A’, E,).

    Article Snippet: The following primary antibodies were used: anti-ATP5A (Abcam, 1:1000), anti-DRP1 (to detect total DRP) Cell Signaling 1:500), anti-p(hospho)DRP1 ser616 (Cell signaling, 1:1000), anti-GAPDH (Fitzgerald 1:10,000), anti-GFP (Clontech 1:1000), anti-GST (Santacruz Biotechnology, 1:1000), anti-EGFR (Santacruz Biotechnology, 1:1000), anti-LAMP1 (Abcam, 1:1000), anti-Myc (Enzo Life Sciences, 1:1000), anti-PLIN2 (Abcam, 1:1000), anti-Rab7 (Abcam, 1:1000), anti-TOMM20 (BD biosciences 1:1000), anti-α tubulin (Sigma, 1:5000), anti-VAP-A (Santa Cruz Biotechnology, 1:1000), anti-VAP-B (Sigma, 1:1000), anti-VPS13A (Sigma,1:1000), anti-VPS13A (H-102) (Santa Cruz Biotechnology, 1:500), Drosophila VPS13A #62 , VPS13C (Sigma).

    Techniques: Staining, Marker, Confocal Microscopy, Two Tailed Test, FACS, Transfection, In Vivo

    VPS13A interacts with VAP-A in human cells. ( A ) GFP-VPS13A constructs presented in Figure 2D–E were overexpressed in HEK293T cells for 24 hr. Cell lysates were processed for immunoblot analysis using an antibody against GFP. peGFP-C1 (GFP) expressing cells were used as a control. The stain free gel is shown as a loading control. B,C GFP-VPS13 (2003–2606) and GFP-VPS13A (2615–3174) constructs were expressed in either HEK293T cells (B/B’) or U2OS cells (C/C’). Note the differences in mitochondria morphology in both cell types. ( D ) U2OS cells expressing GFP-VPS13A (2615–3174) for 24 hr were stained with Mitotracker Red. D’ shows a higher magnification of the insert in D. Cells were co-transfected with mCherrySec61 (B/C) and BFP-Sec61 (D/D’) (not shown). Scale bars = 25 μm ( B–C’ ) and 10 μm ( D–D’ ).

    Journal: eLife

    Article Title: Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility

    doi: 10.7554/eLife.43561

    Figure Lengend Snippet: VPS13A interacts with VAP-A in human cells. ( A ) GFP-VPS13A constructs presented in Figure 2D–E were overexpressed in HEK293T cells for 24 hr. Cell lysates were processed for immunoblot analysis using an antibody against GFP. peGFP-C1 (GFP) expressing cells were used as a control. The stain free gel is shown as a loading control. B,C GFP-VPS13 (2003–2606) and GFP-VPS13A (2615–3174) constructs were expressed in either HEK293T cells (B/B’) or U2OS cells (C/C’). Note the differences in mitochondria morphology in both cell types. ( D ) U2OS cells expressing GFP-VPS13A (2615–3174) for 24 hr were stained with Mitotracker Red. D’ shows a higher magnification of the insert in D. Cells were co-transfected with mCherrySec61 (B/C) and BFP-Sec61 (D/D’) (not shown). Scale bars = 25 μm ( B–C’ ) and 10 μm ( D–D’ ).

    Article Snippet: The following primary antibodies were used: anti-ATP5A (Abcam, 1:1000), anti-DRP1 (to detect total DRP) Cell Signaling 1:500), anti-p(hospho)DRP1 ser616 (Cell signaling, 1:1000), anti-GAPDH (Fitzgerald 1:10,000), anti-GFP (Clontech 1:1000), anti-GST (Santacruz Biotechnology, 1:1000), anti-EGFR (Santacruz Biotechnology, 1:1000), anti-LAMP1 (Abcam, 1:1000), anti-Myc (Enzo Life Sciences, 1:1000), anti-PLIN2 (Abcam, 1:1000), anti-Rab7 (Abcam, 1:1000), anti-TOMM20 (BD biosciences 1:1000), anti-α tubulin (Sigma, 1:5000), anti-VAP-A (Santa Cruz Biotechnology, 1:1000), anti-VAP-B (Sigma, 1:1000), anti-VPS13A (Sigma,1:1000), anti-VPS13A (H-102) (Santa Cruz Biotechnology, 1:500), Drosophila VPS13A #62 , VPS13C (Sigma).

    Techniques: Construct, Expressing, Staining, Transfection

    Endogenous VPS13A is enriched at fractions containing LDs upon OA induction Workflow of LDs isolation and sucrose gradient fractionation. FBS starved HeLa cells were processed or were subsequently incubated for 24 hr with 500 µM OA in FBS free medium, lysed and fractionated in 5–30% sucrose density gradients. Proteins in collected fractions were concentrated by TCA precipitation and subsequently separated by SDS- PAGE.

    Journal: eLife

    Article Title: Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility

    doi: 10.7554/eLife.43561

    Figure Lengend Snippet: Endogenous VPS13A is enriched at fractions containing LDs upon OA induction Workflow of LDs isolation and sucrose gradient fractionation. FBS starved HeLa cells were processed or were subsequently incubated for 24 hr with 500 µM OA in FBS free medium, lysed and fractionated in 5–30% sucrose density gradients. Proteins in collected fractions were concentrated by TCA precipitation and subsequently separated by SDS- PAGE.

    Article Snippet: The following primary antibodies were used: anti-ATP5A (Abcam, 1:1000), anti-DRP1 (to detect total DRP) Cell Signaling 1:500), anti-p(hospho)DRP1 ser616 (Cell signaling, 1:1000), anti-GAPDH (Fitzgerald 1:10,000), anti-GFP (Clontech 1:1000), anti-GST (Santacruz Biotechnology, 1:1000), anti-EGFR (Santacruz Biotechnology, 1:1000), anti-LAMP1 (Abcam, 1:1000), anti-Myc (Enzo Life Sciences, 1:1000), anti-PLIN2 (Abcam, 1:1000), anti-Rab7 (Abcam, 1:1000), anti-TOMM20 (BD biosciences 1:1000), anti-α tubulin (Sigma, 1:5000), anti-VAP-A (Santa Cruz Biotechnology, 1:1000), anti-VAP-B (Sigma, 1:1000), anti-VPS13A (Sigma,1:1000), anti-VPS13A (H-102) (Santa Cruz Biotechnology, 1:500), Drosophila VPS13A #62 , VPS13C (Sigma).

    Techniques: Isolation, Fractionation, Incubation, TCA Precipitation, SDS Page

    Validation of the VPS13A mutant cell line (VPS13A KO). ( A, B ) Control (WT) and VPS13A KO MRC5 cells were analysed by Western blot analysis for the presence or absence of VPS13A ( A ) and VPS13C ( B ). α-Tubulin was used as a control.

    Journal: eLife

    Article Title: Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility

    doi: 10.7554/eLife.43561

    Figure Lengend Snippet: Validation of the VPS13A mutant cell line (VPS13A KO). ( A, B ) Control (WT) and VPS13A KO MRC5 cells were analysed by Western blot analysis for the presence or absence of VPS13A ( A ) and VPS13C ( B ). α-Tubulin was used as a control.

    Article Snippet: The following primary antibodies were used: anti-ATP5A (Abcam, 1:1000), anti-DRP1 (to detect total DRP) Cell Signaling 1:500), anti-p(hospho)DRP1 ser616 (Cell signaling, 1:1000), anti-GAPDH (Fitzgerald 1:10,000), anti-GFP (Clontech 1:1000), anti-GST (Santacruz Biotechnology, 1:1000), anti-EGFR (Santacruz Biotechnology, 1:1000), anti-LAMP1 (Abcam, 1:1000), anti-Myc (Enzo Life Sciences, 1:1000), anti-PLIN2 (Abcam, 1:1000), anti-Rab7 (Abcam, 1:1000), anti-TOMM20 (BD biosciences 1:1000), anti-α tubulin (Sigma, 1:5000), anti-VAP-A (Santa Cruz Biotechnology, 1:1000), anti-VAP-B (Sigma, 1:1000), anti-VPS13A (Sigma,1:1000), anti-VPS13A (H-102) (Santa Cruz Biotechnology, 1:500), Drosophila VPS13A #62 , VPS13C (Sigma).

    Techniques: Mutagenesis, Western Blot

    Endogenous VPS13A is enriched at fractions containing LDs upon OA induction. ( A ) HeLa cells grown in complete medium were fractionated on sucrose gradient and processed as described in Figure 6 . Equal amounts of proteins were detected by immunoblotting for VPS13A, LAMP1, EGFR, PLIN2, VAP-A and ATP5A. ( B ) Quantification of protein band intensities in C was performed using ImageJ and plotted as percentage of the total. B’ shows a close-up of the values of the top three fractions. Error bars, mean ±s.e.m (n = 3) ( C ) FBS starved HeLa cells were further incubated with 250 µM OA and processed as described in A. Equal amounts of proteins were processed for Western blot analysis using antibodies for VPS13A, LAMP1, EGFR, PLIN2, VAP-A and ATP5A. ( D ) Quantification of protein band intensities in E was performed using ImageJ and plotted as percentage of the total. D’ shows a close up of the values of the top three fractions. In B, D and F error bars, mean ±s.e.m (n = 3).

    Journal: eLife

    Article Title: Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility

    doi: 10.7554/eLife.43561

    Figure Lengend Snippet: Endogenous VPS13A is enriched at fractions containing LDs upon OA induction. ( A ) HeLa cells grown in complete medium were fractionated on sucrose gradient and processed as described in Figure 6 . Equal amounts of proteins were detected by immunoblotting for VPS13A, LAMP1, EGFR, PLIN2, VAP-A and ATP5A. ( B ) Quantification of protein band intensities in C was performed using ImageJ and plotted as percentage of the total. B’ shows a close-up of the values of the top three fractions. Error bars, mean ±s.e.m (n = 3) ( C ) FBS starved HeLa cells were further incubated with 250 µM OA and processed as described in A. Equal amounts of proteins were processed for Western blot analysis using antibodies for VPS13A, LAMP1, EGFR, PLIN2, VAP-A and ATP5A. ( D ) Quantification of protein band intensities in E was performed using ImageJ and plotted as percentage of the total. D’ shows a close up of the values of the top three fractions. In B, D and F error bars, mean ±s.e.m (n = 3).

    Article Snippet: The following primary antibodies were used: anti-ATP5A (Abcam, 1:1000), anti-DRP1 (to detect total DRP) Cell Signaling 1:500), anti-p(hospho)DRP1 ser616 (Cell signaling, 1:1000), anti-GAPDH (Fitzgerald 1:10,000), anti-GFP (Clontech 1:1000), anti-GST (Santacruz Biotechnology, 1:1000), anti-EGFR (Santacruz Biotechnology, 1:1000), anti-LAMP1 (Abcam, 1:1000), anti-Myc (Enzo Life Sciences, 1:1000), anti-PLIN2 (Abcam, 1:1000), anti-Rab7 (Abcam, 1:1000), anti-TOMM20 (BD biosciences 1:1000), anti-α tubulin (Sigma, 1:5000), anti-VAP-A (Santa Cruz Biotechnology, 1:1000), anti-VAP-B (Sigma, 1:1000), anti-VPS13A (Sigma,1:1000), anti-VPS13A (H-102) (Santa Cruz Biotechnology, 1:500), Drosophila VPS13A #62 , VPS13C (Sigma).

    Techniques: Incubation, Western Blot

    VPS13A colocalizes with mitochondria but not with the endocytic compartment. ( A ) 48 hours after transfection with either VPS13A-Myc or VPS13A-GFP, HEK293T cells were processed for immunoblotting using antibodies against VPS13A and a -Tubulin. peGFP-C1 transfected or non-transfected (NT) cells were used as controls. Note the enrichment of VPS13A in both VPS13A-Myc or VPS13A-GFP lanes. ( B ) Quantification of protein bands detected with anti-VPS13A antibody in A. The ratio of VPS13A to a -tubulin was normalized to NT cells. Error bars, mean ± s.e.m (n=3), two-tailed unpaired Student’s t-test was used (*P ≤ 0.05, **P≤0.01). C-F HEK293T cells were co-transfected with VPS13A-Myc and with GFP-Rab5 Q79L ( C ), GFP-Rab7 Q67L ( D ), LAMP1-GFP ( E ) or mCherry FYCO1 ( F ). Cells were stained with anti-myc (C-E, red; F, green) and DAPI (blue). Bottom panels (C’-F’ show a magnification of the inset in top panels. Scale bars = 10 µm ( C–F’ ).

    Journal: eLife

    Article Title: Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility

    doi: 10.7554/eLife.43561

    Figure Lengend Snippet: VPS13A colocalizes with mitochondria but not with the endocytic compartment. ( A ) 48 hours after transfection with either VPS13A-Myc or VPS13A-GFP, HEK293T cells were processed for immunoblotting using antibodies against VPS13A and a -Tubulin. peGFP-C1 transfected or non-transfected (NT) cells were used as controls. Note the enrichment of VPS13A in both VPS13A-Myc or VPS13A-GFP lanes. ( B ) Quantification of protein bands detected with anti-VPS13A antibody in A. The ratio of VPS13A to a -tubulin was normalized to NT cells. Error bars, mean ± s.e.m (n=3), two-tailed unpaired Student’s t-test was used (*P ≤ 0.05, **P≤0.01). C-F HEK293T cells were co-transfected with VPS13A-Myc and with GFP-Rab5 Q79L ( C ), GFP-Rab7 Q67L ( D ), LAMP1-GFP ( E ) or mCherry FYCO1 ( F ). Cells were stained with anti-myc (C-E, red; F, green) and DAPI (blue). Bottom panels (C’-F’ show a magnification of the inset in top panels. Scale bars = 10 µm ( C–F’ ).

    Article Snippet: The following primary antibodies were used: anti-ATP5A (Abcam, 1:1000), anti-DRP1 (to detect total DRP) Cell Signaling 1:500), anti-p(hospho)DRP1 ser616 (Cell signaling, 1:1000), anti-GAPDH (Fitzgerald 1:10,000), anti-GFP (Clontech 1:1000), anti-GST (Santacruz Biotechnology, 1:1000), anti-EGFR (Santacruz Biotechnology, 1:1000), anti-LAMP1 (Abcam, 1:1000), anti-Myc (Enzo Life Sciences, 1:1000), anti-PLIN2 (Abcam, 1:1000), anti-Rab7 (Abcam, 1:1000), anti-TOMM20 (BD biosciences 1:1000), anti-α tubulin (Sigma, 1:5000), anti-VAP-A (Santa Cruz Biotechnology, 1:1000), anti-VAP-B (Sigma, 1:1000), anti-VPS13A (Sigma,1:1000), anti-VPS13A (H-102) (Santa Cruz Biotechnology, 1:500), Drosophila VPS13A #62 , VPS13C (Sigma).

    Techniques: Transfection, Two Tailed Test, Staining

    VPS13A is enriched in fractions of the outer mitochondria membrane. ( A ) Crude mitochondria, cytosolic and microsomal fractions (pellet) were isolated from HeLa cells. Equal amounts of proteins were processed for Western blot analysis and detected with antibodies against indicated proteins. PNS = post nuclear supernatant. For all blots ( A–C ) the total protein is also shown with the ‘stain free’ gel.”Stain free’: to this gel a trihalo compound is added which binds to tryptophan amino acids and enhances fluorescence’s when exposed to UV-light. ( B ) The crude mitochondria fraction isolated from HeLa cells was treated with Na 2 CO 3 to extract peripheral membrane proteins. Soluble and insoluble fractions were separated by centrifugation. Equal amounts of proteins were processed for Western blot analysis and detected with antibodies against indicated proteins. ( C ) The crude mitochondria fraction described in A was treated with different concentrations of Proteinase K. Equal amounts of proteins were processed for Western blot analysis and detected with antibodies against indicated proteins. ( D–F ) Quantification of protein band intensities in A-C was performed using ImageJ and plotted as percentage of the total. For Figure ( C ) only the VPS13A band was quantified. Error bars, mean ±s.e.m (n = 3).

    Journal: eLife

    Article Title: Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility

    doi: 10.7554/eLife.43561

    Figure Lengend Snippet: VPS13A is enriched in fractions of the outer mitochondria membrane. ( A ) Crude mitochondria, cytosolic and microsomal fractions (pellet) were isolated from HeLa cells. Equal amounts of proteins were processed for Western blot analysis and detected with antibodies against indicated proteins. PNS = post nuclear supernatant. For all blots ( A–C ) the total protein is also shown with the ‘stain free’ gel.”Stain free’: to this gel a trihalo compound is added which binds to tryptophan amino acids and enhances fluorescence’s when exposed to UV-light. ( B ) The crude mitochondria fraction isolated from HeLa cells was treated with Na 2 CO 3 to extract peripheral membrane proteins. Soluble and insoluble fractions were separated by centrifugation. Equal amounts of proteins were processed for Western blot analysis and detected with antibodies against indicated proteins. ( C ) The crude mitochondria fraction described in A was treated with different concentrations of Proteinase K. Equal amounts of proteins were processed for Western blot analysis and detected with antibodies against indicated proteins. ( D–F ) Quantification of protein band intensities in A-C was performed using ImageJ and plotted as percentage of the total. For Figure ( C ) only the VPS13A band was quantified. Error bars, mean ±s.e.m (n = 3).

    Article Snippet: The following primary antibodies were used: anti-ATP5A (Abcam, 1:1000), anti-DRP1 (to detect total DRP) Cell Signaling 1:500), anti-p(hospho)DRP1 ser616 (Cell signaling, 1:1000), anti-GAPDH (Fitzgerald 1:10,000), anti-GFP (Clontech 1:1000), anti-GST (Santacruz Biotechnology, 1:1000), anti-EGFR (Santacruz Biotechnology, 1:1000), anti-LAMP1 (Abcam, 1:1000), anti-Myc (Enzo Life Sciences, 1:1000), anti-PLIN2 (Abcam, 1:1000), anti-Rab7 (Abcam, 1:1000), anti-TOMM20 (BD biosciences 1:1000), anti-α tubulin (Sigma, 1:5000), anti-VAP-A (Santa Cruz Biotechnology, 1:1000), anti-VAP-B (Sigma, 1:1000), anti-VPS13A (Sigma,1:1000), anti-VPS13A (H-102) (Santa Cruz Biotechnology, 1:500), Drosophila VPS13A #62 , VPS13C (Sigma).

    Techniques: Isolation, Western Blot, Staining, Fluorescence, Centrifugation

    VPS13A interacts with VAP-A. ( A ) GST-fusion proteins of VPS13A fragments expressed in E.Coli were enriched on Sepharose beads and incubated with equal amounts of HeLa cell lysate. GST alone was used as a control. Samples were immunoblotted against VAP-A, GST and N-terminal VPS13A (H-102). ( B ) Full length VPS13A-GFP ( B ) or VPS13A ∆FFAT -GFP ( B’ ) were expressed in HEK293T cells and mitochondria were marked using an antibody against TOMM20. The yellow signal in the overlay represents sites of close association between mitochondria and the VPS13A-GFP. Cells were co-transfected with BFP-Sec61 (not shown). ( C ) HEK293T cells were transfected with VPS13A-GFP or VPS13A ∆FFAT -GFP and stained for TOMM20. The fraction of the GFP signal overlapping the TOMM20 signal was quantified with ImageJ using the JACoP plugin. ( D ) HEK293T cells were transfected with VPS13A-GFP or VPS13A ∆FFAT -GFP and mCherry-VAP-A. The fraction of the GFP signal overlapping the mCherry signal was quantified with ImageJ using the JACoP plugin. Error bars ( C, D ), mean ±s.e.m (n = 3), two-tailed unpaired Student’s t-test was used (**p≤0.01). ( E ) Full length VPS13A-GFP, VPS13A ∆FFAT -GFP or peGFP-C1 (as a control) were expressed in HEK293T cells and immunoprecipitated using a GFP-trap assay. Samples from Figure 4F were co-analysed for the presence of VAP-B. Scale bar = 10 µm (B/B’).

    Journal: eLife

    Article Title: Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility

    doi: 10.7554/eLife.43561

    Figure Lengend Snippet: VPS13A interacts with VAP-A. ( A ) GST-fusion proteins of VPS13A fragments expressed in E.Coli were enriched on Sepharose beads and incubated with equal amounts of HeLa cell lysate. GST alone was used as a control. Samples were immunoblotted against VAP-A, GST and N-terminal VPS13A (H-102). ( B ) Full length VPS13A-GFP ( B ) or VPS13A ∆FFAT -GFP ( B’ ) were expressed in HEK293T cells and mitochondria were marked using an antibody against TOMM20. The yellow signal in the overlay represents sites of close association between mitochondria and the VPS13A-GFP. Cells were co-transfected with BFP-Sec61 (not shown). ( C ) HEK293T cells were transfected with VPS13A-GFP or VPS13A ∆FFAT -GFP and stained for TOMM20. The fraction of the GFP signal overlapping the TOMM20 signal was quantified with ImageJ using the JACoP plugin. ( D ) HEK293T cells were transfected with VPS13A-GFP or VPS13A ∆FFAT -GFP and mCherry-VAP-A. The fraction of the GFP signal overlapping the mCherry signal was quantified with ImageJ using the JACoP plugin. Error bars ( C, D ), mean ±s.e.m (n = 3), two-tailed unpaired Student’s t-test was used (**p≤0.01). ( E ) Full length VPS13A-GFP, VPS13A ∆FFAT -GFP or peGFP-C1 (as a control) were expressed in HEK293T cells and immunoprecipitated using a GFP-trap assay. Samples from Figure 4F were co-analysed for the presence of VAP-B. Scale bar = 10 µm (B/B’).

    Article Snippet: The following primary antibodies were used: anti-ATP5A (Abcam, 1:1000), anti-DRP1 (to detect total DRP) Cell Signaling 1:500), anti-p(hospho)DRP1 ser616 (Cell signaling, 1:1000), anti-GAPDH (Fitzgerald 1:10,000), anti-GFP (Clontech 1:1000), anti-GST (Santacruz Biotechnology, 1:1000), anti-EGFR (Santacruz Biotechnology, 1:1000), anti-LAMP1 (Abcam, 1:1000), anti-Myc (Enzo Life Sciences, 1:1000), anti-PLIN2 (Abcam, 1:1000), anti-Rab7 (Abcam, 1:1000), anti-TOMM20 (BD biosciences 1:1000), anti-α tubulin (Sigma, 1:5000), anti-VAP-A (Santa Cruz Biotechnology, 1:1000), anti-VAP-B (Sigma, 1:1000), anti-VPS13A (Sigma,1:1000), anti-VPS13A (H-102) (Santa Cruz Biotechnology, 1:500), Drosophila VPS13A #62 , VPS13C (Sigma).

    Techniques: Incubation, Transfection, Staining, Two Tailed Test, Immunoprecipitation, TRAP Assay

    P-TEFb and the Super Elongation Complex (SEC) display similar phenotypes. ( A ) RNA in situ hybridization using a digoxigenin-labeled Serendipity-α (Sry-α) probe demonstrates decreased levels of Sry-α mRNA in embryos depleted of maternal Cdk9 (ii) or Cyclin T (iii) compared to wild-type (i). ( B ) DIC micrographs of wild-type (wt, i), or embryos derived from females with germline clones of the SEC component dAFF4/Lilliputian ( lilli -/- , ii) or females expressing TubGal4 and a shmiRNA targeting the SEC subunit dEll (dELLi, iii) show the same posterior phenotype as in P-TEFb embryos. A close up of the same embryos is shown below (iv-vi). ( C ) Expression of the cellularization genes bottleneck ( bnk , i, ii), slow-as-molasses ( slam , iii, iv), and nullo (v, vi) is comparable between wild-type and embryos depleted of maternal Cdk9. ( D ) Quantification of cellularization gene expression by RT-qPCR. Columns show average values in 2–4h embryos with S.E.M. (n = 5–6) and control values were set to 100%. Relative expression was normalized to a mean of four reference genes ( beta-tubulin , GAPDH , RpL32 , and 28SrRNA ). *indicates P

    Journal: PLoS Genetics

    Article Title: P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early Drosophila Embryo Development

    doi: 10.1371/journal.pgen.1004971

    Figure Lengend Snippet: P-TEFb and the Super Elongation Complex (SEC) display similar phenotypes. ( A ) RNA in situ hybridization using a digoxigenin-labeled Serendipity-α (Sry-α) probe demonstrates decreased levels of Sry-α mRNA in embryos depleted of maternal Cdk9 (ii) or Cyclin T (iii) compared to wild-type (i). ( B ) DIC micrographs of wild-type (wt, i), or embryos derived from females with germline clones of the SEC component dAFF4/Lilliputian ( lilli -/- , ii) or females expressing TubGal4 and a shmiRNA targeting the SEC subunit dEll (dELLi, iii) show the same posterior phenotype as in P-TEFb embryos. A close up of the same embryos is shown below (iv-vi). ( C ) Expression of the cellularization genes bottleneck ( bnk , i, ii), slow-as-molasses ( slam , iii, iv), and nullo (v, vi) is comparable between wild-type and embryos depleted of maternal Cdk9. ( D ) Quantification of cellularization gene expression by RT-qPCR. Columns show average values in 2–4h embryos with S.E.M. (n = 5–6) and control values were set to 100%. Relative expression was normalized to a mean of four reference genes ( beta-tubulin , GAPDH , RpL32 , and 28SrRNA ). *indicates P

    Article Snippet: A dounce homogenizer was used to grind 2–4h old wild-type or Cdk9 Drosophila embryos and a Diagenode Bioruptor was used to sonicate the material before IP.

    Techniques: Size-exclusion Chromatography, RNA In Situ Hybridization, Labeling, Derivative Assay, Clone Assay, Expressing, Quantitative RT-PCR

    Maternal knockdown of P-TEFb disrupts embryo development. ( A ) Embryos were collected from females containing the maternal α-Tubulin-Gal4-VP16 (TubGal4) driver alone (i), TubGal4 and a shmiRNA targeting Cdk9 (ii, iii) or CycT (v, vi), or TubGal4, Cdk9 shmiRNA and an miRNA-resistant Cdk9 transgene (iv). Dark-field micrographs of embryo cuticle preparations show patterning defects. ( B ) Quantitative RT-PCR of Cdk9 and CycT expression relative four reference genes in 2–4 hour old maternally depleted embryos derived from females with TubGal4 (tub) crossed to wild-type ( w 1118 , control), or to shmiRNAs targeting Cdk9 or CycT. Expression in control embryos was set to 100%. Error bars denote S.E.M. n = 6. ( C ) Western blot showing CycT levels in wild-type 2–4 h embryos and in embryos depleted of maternal CycT or Cdk9. HDAC3 was used as a loading control. Molecular weight markers are indicated to the left, and arrowheads point to the predicted 118 and 94 kDa CycT isoforms. ( D ) Western blot of FLAG epitope-tagged Cdk9 expressed from its endogenous promoter demonstrates reduced Cdk9 protein in Cdk9 miRNA depleted 2–4 h embryos. Histone H3 was used as a loading control. ( E ) Western blot of FLAG-tagged Cdk9 transgenes in adult flies with either the native DNA sequence or an miRNA resistant sequence shows comparable levels of protein. Histone H3 was used as a loading control. ( F ) A schematic drawing of the Cdk9 locus. The sequence targeted by the miRNA is underlined, and the nucleotide changes in the miRNA resistant transgene highlighted in bold and italic. The arrowheads indicate the positions of the primers used to amplify the genomic region used in the transgenes.

    Journal: PLoS Genetics

    Article Title: P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early Drosophila Embryo Development

    doi: 10.1371/journal.pgen.1004971

    Figure Lengend Snippet: Maternal knockdown of P-TEFb disrupts embryo development. ( A ) Embryos were collected from females containing the maternal α-Tubulin-Gal4-VP16 (TubGal4) driver alone (i), TubGal4 and a shmiRNA targeting Cdk9 (ii, iii) or CycT (v, vi), or TubGal4, Cdk9 shmiRNA and an miRNA-resistant Cdk9 transgene (iv). Dark-field micrographs of embryo cuticle preparations show patterning defects. ( B ) Quantitative RT-PCR of Cdk9 and CycT expression relative four reference genes in 2–4 hour old maternally depleted embryos derived from females with TubGal4 (tub) crossed to wild-type ( w 1118 , control), or to shmiRNAs targeting Cdk9 or CycT. Expression in control embryos was set to 100%. Error bars denote S.E.M. n = 6. ( C ) Western blot showing CycT levels in wild-type 2–4 h embryos and in embryos depleted of maternal CycT or Cdk9. HDAC3 was used as a loading control. Molecular weight markers are indicated to the left, and arrowheads point to the predicted 118 and 94 kDa CycT isoforms. ( D ) Western blot of FLAG epitope-tagged Cdk9 expressed from its endogenous promoter demonstrates reduced Cdk9 protein in Cdk9 miRNA depleted 2–4 h embryos. Histone H3 was used as a loading control. ( E ) Western blot of FLAG-tagged Cdk9 transgenes in adult flies with either the native DNA sequence or an miRNA resistant sequence shows comparable levels of protein. Histone H3 was used as a loading control. ( F ) A schematic drawing of the Cdk9 locus. The sequence targeted by the miRNA is underlined, and the nucleotide changes in the miRNA resistant transgene highlighted in bold and italic. The arrowheads indicate the positions of the primers used to amplify the genomic region used in the transgenes.

    Article Snippet: A dounce homogenizer was used to grind 2–4h old wild-type or Cdk9 Drosophila embryos and a Diagenode Bioruptor was used to sonicate the material before IP.

    Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Western Blot, Molecular Weight, FLAG-tag, Sequencing

    Pol II occupancy is reduced at genes affected by P-TEFb depletion. ( A ) Confocal images of wild-type embryos (i, iii) and embryos depleted of maternal Cdk9 (ii, iv) stained with an antibody recognizing phosphorylated Pol II CTD Ser2 (Ser2-P, Abcam ab5095). In pre-cellular embryos, an elevated Ser2-P signal was observed in the cytoplasm in Cdk9 embryos (ii) compared to wild-type (i), indicating that the maternal contribution of Ser2 phosphorylated Pol II was increased in Cdk9 embryos. In cellularizing embryos, less Ser2-P was detected in nuclei of Cdk9 embryos (iv) compared to wild-type nuclei (iii). ( B ) Western blot with extracts from 0–5h old embryos show a decrease in Ser2-P in embryos depleted of maternal Cdk9 or CycT. The monoclonal antibody 8WG16 recognizing the Pol II CTD was used as a loading control. The ratio of Ser2-P to CTD signal was quantified from 3 biological replicates. ( C-F ) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) of 2–4h wild-type or Cdk9 embryo extracts using antibodies recognizing the Pol II CTD, Pol II Ser2 phosphorylation, and Pol II Ser5 phosphorylation. ( C) Pol II occupancy plotted as CTD enrichment relative the intergenic locus IG2c. Less Pol II associates with Sry-α and CG7271 in Cdk9 embryos. ( D ) Less Ser2-P per CTD was observed in Cdk9 embryos compared to wild-type. ( E ) The Ser5-P/CTD ratio was comparable in wild-type and Cdk9 embryos. ( F ) The ratio of Pol II Ser-5 signal at the 5’ end versus the 3’ end. No increase at the 5’ end was detected in Cdk9 embryos. Error bars show standard error of the mean (n = 3–5). * indicates P

    Journal: PLoS Genetics

    Article Title: P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early Drosophila Embryo Development

    doi: 10.1371/journal.pgen.1004971

    Figure Lengend Snippet: Pol II occupancy is reduced at genes affected by P-TEFb depletion. ( A ) Confocal images of wild-type embryos (i, iii) and embryos depleted of maternal Cdk9 (ii, iv) stained with an antibody recognizing phosphorylated Pol II CTD Ser2 (Ser2-P, Abcam ab5095). In pre-cellular embryos, an elevated Ser2-P signal was observed in the cytoplasm in Cdk9 embryos (ii) compared to wild-type (i), indicating that the maternal contribution of Ser2 phosphorylated Pol II was increased in Cdk9 embryos. In cellularizing embryos, less Ser2-P was detected in nuclei of Cdk9 embryos (iv) compared to wild-type nuclei (iii). ( B ) Western blot with extracts from 0–5h old embryos show a decrease in Ser2-P in embryos depleted of maternal Cdk9 or CycT. The monoclonal antibody 8WG16 recognizing the Pol II CTD was used as a loading control. The ratio of Ser2-P to CTD signal was quantified from 3 biological replicates. ( C-F ) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) of 2–4h wild-type or Cdk9 embryo extracts using antibodies recognizing the Pol II CTD, Pol II Ser2 phosphorylation, and Pol II Ser5 phosphorylation. ( C) Pol II occupancy plotted as CTD enrichment relative the intergenic locus IG2c. Less Pol II associates with Sry-α and CG7271 in Cdk9 embryos. ( D ) Less Ser2-P per CTD was observed in Cdk9 embryos compared to wild-type. ( E ) The Ser5-P/CTD ratio was comparable in wild-type and Cdk9 embryos. ( F ) The ratio of Pol II Ser-5 signal at the 5’ end versus the 3’ end. No increase at the 5’ end was detected in Cdk9 embryos. Error bars show standard error of the mean (n = 3–5). * indicates P

    Article Snippet: A dounce homogenizer was used to grind 2–4h old wild-type or Cdk9 Drosophila embryos and a Diagenode Bioruptor was used to sonicate the material before IP.

    Techniques: Staining, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    The P-TEFb and SEC-regulated gene terminus (term) is essential in early embryos. ( A ) In situ hybridization showing term/CG7271 expression in wild-type during different embryo stages (i-vi). The transcripts become concentrated to the posterior in cellularized embryos (iv and v) ( B ) Expression of term/CG7271 is severely reduced in embryos depleted of maternal Cdk9 (ii) compared to wild-type (i), and rescued by the miRNA-resistant transgene in Cdk9 embryos (iii). Greatly diminished term/CG7271 levels are also observed in embryos depleted of maternal CycT (iv) or dEll (v), and in embryos from lilli germline clones (vi). ( C ) Knockdown of zygotic term by crossing TubGal4 females with Term shmiRNA males (ii), eliminating Term and CG7271 by deletion in embryos derived from the deficiency Df(3L)BSC416 (iii), or over-expressing Term by crossing TubGal4 females with UAS- term males (iv) results in severe morphological defects in early embryos, including a failure to form a cellular blastoderm. A wild-type cellularizing embryo (i) is shown for comparison.

    Journal: PLoS Genetics

    Article Title: P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early Drosophila Embryo Development

    doi: 10.1371/journal.pgen.1004971

    Figure Lengend Snippet: The P-TEFb and SEC-regulated gene terminus (term) is essential in early embryos. ( A ) In situ hybridization showing term/CG7271 expression in wild-type during different embryo stages (i-vi). The transcripts become concentrated to the posterior in cellularized embryos (iv and v) ( B ) Expression of term/CG7271 is severely reduced in embryos depleted of maternal Cdk9 (ii) compared to wild-type (i), and rescued by the miRNA-resistant transgene in Cdk9 embryos (iii). Greatly diminished term/CG7271 levels are also observed in embryos depleted of maternal CycT (iv) or dEll (v), and in embryos from lilli germline clones (vi). ( C ) Knockdown of zygotic term by crossing TubGal4 females with Term shmiRNA males (ii), eliminating Term and CG7271 by deletion in embryos derived from the deficiency Df(3L)BSC416 (iii), or over-expressing Term by crossing TubGal4 females with UAS- term males (iv) results in severe morphological defects in early embryos, including a failure to form a cellular blastoderm. A wild-type cellularizing embryo (i) is shown for comparison.

    Article Snippet: A dounce homogenizer was used to grind 2–4h old wild-type or Cdk9 Drosophila embryos and a Diagenode Bioruptor was used to sonicate the material before IP.

    Techniques: Size-exclusion Chromatography, In Situ, Hybridization, Expressing, Derivative Assay

    The expression of many patterning genes is relatively normal in P-TEFb embryos. Wild-type embryos ( A, C, E, G, I, K ) and embryos depleted of maternal Cdk9 ( B, D, F, H, J, L ) were hybridized with probes detecting nanos (nos) , polar granule component (pgc) , tailless (tll) , even-skipped (eve) , fushi-tarazu (ftz) , and rhomboid (rho) RNA. Relatively normal expression patterns were observed in Cdk9 embryos. ( M ) Expression of tll , eve , ftz , and rho in 2–4h P-TEFb embryos quantified by RT-qPCR (n = 4–6). Relative expression was normalized to a mean of four reference genes as described in Fig. 3 . *indicates P

    Journal: PLoS Genetics

    Article Title: P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early Drosophila Embryo Development

    doi: 10.1371/journal.pgen.1004971

    Figure Lengend Snippet: The expression of many patterning genes is relatively normal in P-TEFb embryos. Wild-type embryos ( A, C, E, G, I, K ) and embryos depleted of maternal Cdk9 ( B, D, F, H, J, L ) were hybridized with probes detecting nanos (nos) , polar granule component (pgc) , tailless (tll) , even-skipped (eve) , fushi-tarazu (ftz) , and rhomboid (rho) RNA. Relatively normal expression patterns were observed in Cdk9 embryos. ( M ) Expression of tll , eve , ftz , and rho in 2–4h P-TEFb embryos quantified by RT-qPCR (n = 4–6). Relative expression was normalized to a mean of four reference genes as described in Fig. 3 . *indicates P

    Article Snippet: A dounce homogenizer was used to grind 2–4h old wild-type or Cdk9 Drosophila embryos and a Diagenode Bioruptor was used to sonicate the material before IP.

    Techniques: Expressing, Pyrolysis Gas Chromatography, Quantitative RT-PCR

    P-TEFb embryos show cellularization defects and lose cells in the embryo posterior. ( A ) Differential interference contrast (DIC) microscopy reveals a posterior loss of cells that appear prior to gastrulation in P-TEFb depleted embryos. Cellularizing P-TEFb embryos look normal (ii and iii), whereas cellularized embryos are missing cells in the posterior (v and vi). Compare to wild-type (wt) embryos in (i) and (iv). ( B, C ) Confocal images of wild-type (Bi, Ci, iii, v) and P-TEFb embryos immunostained with anti-phosphotyrosine (pTYR) antibody that marks the plasma membrane (green) and anti-Histone 4 marking the nuclei (red). Cells are lost from the posterior in P-TEFb embryos (Bii and iii). In addition, cell shape changes as well as multi-nucleated cells are evident in Cdk9 depleted embryos (Cii), the nuclei do not elongate as in wild-type (compare Ciii with iv), and some nuclei are present outside the basal side of cells in Cdk9 embryos (yellow arrowheads in C iv and vi). In cellularized P-TEFb embryos, mesodermal cells are not properly arranged (Bii, iii, and C vi). Embryos were generated as in Fig. 1 , and are oriented with anterior to the left and dorsal side up in A and B.

    Journal: PLoS Genetics

    Article Title: P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early Drosophila Embryo Development

    doi: 10.1371/journal.pgen.1004971

    Figure Lengend Snippet: P-TEFb embryos show cellularization defects and lose cells in the embryo posterior. ( A ) Differential interference contrast (DIC) microscopy reveals a posterior loss of cells that appear prior to gastrulation in P-TEFb depleted embryos. Cellularizing P-TEFb embryos look normal (ii and iii), whereas cellularized embryos are missing cells in the posterior (v and vi). Compare to wild-type (wt) embryos in (i) and (iv). ( B, C ) Confocal images of wild-type (Bi, Ci, iii, v) and P-TEFb embryos immunostained with anti-phosphotyrosine (pTYR) antibody that marks the plasma membrane (green) and anti-Histone 4 marking the nuclei (red). Cells are lost from the posterior in P-TEFb embryos (Bii and iii). In addition, cell shape changes as well as multi-nucleated cells are evident in Cdk9 depleted embryos (Cii), the nuclei do not elongate as in wild-type (compare Ciii with iv), and some nuclei are present outside the basal side of cells in Cdk9 embryos (yellow arrowheads in C iv and vi). In cellularized P-TEFb embryos, mesodermal cells are not properly arranged (Bii, iii, and C vi). Embryos were generated as in Fig. 1 , and are oriented with anterior to the left and dorsal side up in A and B.

    Article Snippet: A dounce homogenizer was used to grind 2–4h old wild-type or Cdk9 Drosophila embryos and a Diagenode Bioruptor was used to sonicate the material before IP.

    Techniques: Microscopy, Generated

    Dissemination phenotypes induced by four- and five-hit models. ( a – d ) Mmp1 staining of control and ras G12V p53 Ri pten Ri apc Ri hindguts. ( b , d ) Mmp1 channel only. ( e – l ) Cross-section views of control and ras G12V p53 Ri pten Ri apc Ri hindguts. ( f , h , j , l ) Laminin channel only; arrows indicate reduced/absent laminin staining. ( m – s ) Examples of dissemination phenotype. Arrows indicate GFP-positive foci inside the abdominal cavity ( m ), underneath the abdomen epidermis ( n ), ovaries ( o ), head ( p ) and legs ( q , r ) (f, fat body, n, nephrocyte; t, trachea). ( m ) Inset: close-up view showing close association of GFP foci with tracheal branches. ( s ) Live confocal image of a multicellular GFP cluster inside the abdominal cavity. Nuclei are visualized by a nuclear dsRed transgene (nls-dsRed). ( t ) Quantification of dissemination into the abdominal cavity. Each animal is dissected and assigned into one of the following categories based on the number of disseminated foci inside the abdominal cavity: none, no dissemination; weak, 1–3 GFP-positive foci inside the abdominal cavity; moderate, 4–10 GFP-positive foci; strong, > 10 GFP-positive foci ( n =2 replicates, 20–30 flies per replicate; error bars: s.e.m.). Scale bars, 25 mm.

    Journal: Nature Communications

    Article Title: Functional exploration of colorectal cancer genomes using Drosophila

    doi: 10.1038/ncomms13615

    Figure Lengend Snippet: Dissemination phenotypes induced by four- and five-hit models. ( a – d ) Mmp1 staining of control and ras G12V p53 Ri pten Ri apc Ri hindguts. ( b , d ) Mmp1 channel only. ( e – l ) Cross-section views of control and ras G12V p53 Ri pten Ri apc Ri hindguts. ( f , h , j , l ) Laminin channel only; arrows indicate reduced/absent laminin staining. ( m – s ) Examples of dissemination phenotype. Arrows indicate GFP-positive foci inside the abdominal cavity ( m ), underneath the abdomen epidermis ( n ), ovaries ( o ), head ( p ) and legs ( q , r ) (f, fat body, n, nephrocyte; t, trachea). ( m ) Inset: close-up view showing close association of GFP foci with tracheal branches. ( s ) Live confocal image of a multicellular GFP cluster inside the abdominal cavity. Nuclei are visualized by a nuclear dsRed transgene (nls-dsRed). ( t ) Quantification of dissemination into the abdominal cavity. Each animal is dissected and assigned into one of the following categories based on the number of disseminated foci inside the abdominal cavity: none, no dissemination; weak, 1–3 GFP-positive foci inside the abdominal cavity; moderate, 4–10 GFP-positive foci; strong, > 10 GFP-positive foci ( n =2 replicates, 20–30 flies per replicate; error bars: s.e.m.). Scale bars, 25 mm.

    Article Snippet: Primary antibodies were as follows: rabbit anti Drosophila phospho-AKT (p-Ser505; Drosophila equivalent of mammalian AKT p-Ser473, 1:1,000, Cell Signaling, catalogue number 4054S), rabbit anti-mouse AKT (1:1,000, Cell Signaling, catalogue number 4691S), rabbit anti-mouse phospho-4EBP (Thr37/46, 1:1,000, Cell Signaling, catalogue number 2855S), rabbit anti-human phospho-AKT (Ser473, 1:1,000, Cell Signaling, catalogue number 4060S), mouse anti-chicken α-actin (1:1,000, DSHB catalogue number JLA20-s), rabbit anti-GFP (1:2,000, Sigma, catalogue number G1544), rabbit anti Drosophila P53 (1:1,000, DSHB, catalogue number H3), rabbit anti Drosophila Pten (1:1,000, gift from A. Wodarz ), mouse anti-dpERK (Thr183/Tyr185, 1:2,000, Sigma, catalogue number M8159), rabbit anti human phospho-S6 (Ser 235/236, 1:1,000, Cell Signaling, catalogue number 2211) and mouse anti Drosophila Syntaxin (1:1,000, DSHB, catalogue number 8C3).

    Techniques: Staining

    Bortezomib promotes sensitivity to PI3K pathway inhibition. ( a ) Western blot analysis of PI3K pathway activity in ras G12V p53 Ri pten Ri apc Ri hindguts after 1 day feeding of bortezomib at indicated doses. Each data point represents the average response of two to five biological replicates with ten hindguts per replicate. Error bars: s.e.m. ( b ) Quantification of dissemination in ras G12V p53 Ri pten Ri apc Ri animals after sequential treatment with BEZ235 and indicated doses of bortezomib. ( c ) Quantification of dissemination in ras G12V p53 Ri pten Ri apc Ri animals after a 1-day/2-day alternating treatment schedule of bortezomib/BEZ235 and each drug alone. ( d ) Western blot analysis of PI3K pathway activity in ras G12V p53 Ri pten Ri apc Ri hindguts with and without raptor knockdown treated with 5 μm bortezomib for 1 day. ( e ) Quantification of dissemination in ras G12V p53 Ri pten Ri apc Ri raptor Ri animals after sequential treatment with indicated doses of bortezomib followed by BEZ235. ( f ) Schematic illustration of the mechanism by which the two-step therapy overcomes resistance to BEZ235: elevating mTORC1 activity increases subsequent sensitivity to BEZ235. ( b , c , e ) n =2 replicates, 30 flies per replicate; error bars: s.e.m. * P

    Journal: Nature Communications

    Article Title: Functional exploration of colorectal cancer genomes using Drosophila

    doi: 10.1038/ncomms13615

    Figure Lengend Snippet: Bortezomib promotes sensitivity to PI3K pathway inhibition. ( a ) Western blot analysis of PI3K pathway activity in ras G12V p53 Ri pten Ri apc Ri hindguts after 1 day feeding of bortezomib at indicated doses. Each data point represents the average response of two to five biological replicates with ten hindguts per replicate. Error bars: s.e.m. ( b ) Quantification of dissemination in ras G12V p53 Ri pten Ri apc Ri animals after sequential treatment with BEZ235 and indicated doses of bortezomib. ( c ) Quantification of dissemination in ras G12V p53 Ri pten Ri apc Ri animals after a 1-day/2-day alternating treatment schedule of bortezomib/BEZ235 and each drug alone. ( d ) Western blot analysis of PI3K pathway activity in ras G12V p53 Ri pten Ri apc Ri hindguts with and without raptor knockdown treated with 5 μm bortezomib for 1 day. ( e ) Quantification of dissemination in ras G12V p53 Ri pten Ri apc Ri raptor Ri animals after sequential treatment with indicated doses of bortezomib followed by BEZ235. ( f ) Schematic illustration of the mechanism by which the two-step therapy overcomes resistance to BEZ235: elevating mTORC1 activity increases subsequent sensitivity to BEZ235. ( b , c , e ) n =2 replicates, 30 flies per replicate; error bars: s.e.m. * P

    Article Snippet: Primary antibodies were as follows: rabbit anti Drosophila phospho-AKT (p-Ser505; Drosophila equivalent of mammalian AKT p-Ser473, 1:1,000, Cell Signaling, catalogue number 4054S), rabbit anti-mouse AKT (1:1,000, Cell Signaling, catalogue number 4691S), rabbit anti-mouse phospho-4EBP (Thr37/46, 1:1,000, Cell Signaling, catalogue number 2855S), rabbit anti-human phospho-AKT (Ser473, 1:1,000, Cell Signaling, catalogue number 4060S), mouse anti-chicken α-actin (1:1,000, DSHB catalogue number JLA20-s), rabbit anti-GFP (1:2,000, Sigma, catalogue number G1544), rabbit anti Drosophila P53 (1:1,000, DSHB, catalogue number H3), rabbit anti Drosophila Pten (1:1,000, gift from A. Wodarz ), mouse anti-dpERK (Thr183/Tyr185, 1:2,000, Sigma, catalogue number M8159), rabbit anti human phospho-S6 (Ser 235/236, 1:1,000, Cell Signaling, catalogue number 2211) and mouse anti Drosophila Syntaxin (1:1,000, DSHB, catalogue number 8C3).

    Techniques: Inhibition, Western Blot, Activity Assay

    Targeting quadruple combinations to the adult hindgut. ( a ) The adult Drosophila digestive track. Hindgut cells are visualized with byn > GFP ; nuclei are in red. ( b – i ) Control ( byn > GFP,dcr2 ) and ras G12V p53 Ri pten Ri apc Ri hindguts 7 and 21 days after induction. Asterisks in c and e indicate regions of multilayering. Longitudinal optical sections ( f , g ) and pylorus regions ( h , i ) are shown. ( j – o ) Control ( j ) and ras G12V p53 Ri pten Ri apc Ri ( k – o ) ilea; arrows indicate migrating cells. ( l ) Close-up view view of k . ( m – o ) Apical-to-basal confocal sections of a migrating cell (asterisk). ( p , q ) Surface views of ras G12V p53 Ri smad4 Ri apc Ri hindguts with cells migrating on top of the muscle layer. ( p ) Inset: laminin (grey) and GFP channels only to highlight trachea (arrows). ( r , s ) Phospho-Src staining of control and ras G12V p53 Ri pten Ri apc Ri hindguts.cr, crop; h, hindgut; i, ileum; m, midgut; mp, malphigian tubules; p, pylorus; r, rectum; t, trachea. Scale bars, 250 mm ( a – e ) and 25 mm ( f – s ).

    Journal: Nature Communications

    Article Title: Functional exploration of colorectal cancer genomes using Drosophila

    doi: 10.1038/ncomms13615

    Figure Lengend Snippet: Targeting quadruple combinations to the adult hindgut. ( a ) The adult Drosophila digestive track. Hindgut cells are visualized with byn > GFP ; nuclei are in red. ( b – i ) Control ( byn > GFP,dcr2 ) and ras G12V p53 Ri pten Ri apc Ri hindguts 7 and 21 days after induction. Asterisks in c and e indicate regions of multilayering. Longitudinal optical sections ( f , g ) and pylorus regions ( h , i ) are shown. ( j – o ) Control ( j ) and ras G12V p53 Ri pten Ri apc Ri ( k – o ) ilea; arrows indicate migrating cells. ( l ) Close-up view view of k . ( m – o ) Apical-to-basal confocal sections of a migrating cell (asterisk). ( p , q ) Surface views of ras G12V p53 Ri smad4 Ri apc Ri hindguts with cells migrating on top of the muscle layer. ( p ) Inset: laminin (grey) and GFP channels only to highlight trachea (arrows). ( r , s ) Phospho-Src staining of control and ras G12V p53 Ri pten Ri apc Ri hindguts.cr, crop; h, hindgut; i, ileum; m, midgut; mp, malphigian tubules; p, pylorus; r, rectum; t, trachea. Scale bars, 250 mm ( a – e ) and 25 mm ( f – s ).

    Article Snippet: Primary antibodies were as follows: rabbit anti Drosophila phospho-AKT (p-Ser505; Drosophila equivalent of mammalian AKT p-Ser473, 1:1,000, Cell Signaling, catalogue number 4054S), rabbit anti-mouse AKT (1:1,000, Cell Signaling, catalogue number 4691S), rabbit anti-mouse phospho-4EBP (Thr37/46, 1:1,000, Cell Signaling, catalogue number 2855S), rabbit anti-human phospho-AKT (Ser473, 1:1,000, Cell Signaling, catalogue number 4060S), mouse anti-chicken α-actin (1:1,000, DSHB catalogue number JLA20-s), rabbit anti-GFP (1:2,000, Sigma, catalogue number G1544), rabbit anti Drosophila P53 (1:1,000, DSHB, catalogue number H3), rabbit anti Drosophila Pten (1:1,000, gift from A. Wodarz ), mouse anti-dpERK (Thr183/Tyr185, 1:2,000, Sigma, catalogue number M8159), rabbit anti human phospho-S6 (Ser 235/236, 1:1,000, Cell Signaling, catalogue number 2211) and mouse anti Drosophila Syntaxin (1:1,000, DSHB, catalogue number 8C3).

    Techniques: Staining

    Drug resistance as an emergent feature of increased genetic complexity. ( a , b ) Plot of P -values indicating significance of compound rescue ( a ) and summary of compound response ( b ) of ras G12V and ras G12V p53 Ri pten Ri apc Ri animals. P -values ( a ) were obtained by comparing the dissemination phenotype after compound feeding to dimethyl sulfoxide (DMSO) fed flies (dissemination plots can be found in Supplementary Fig. 3a,b ). Blue dots represent statistically significant results. ( c ) Quantification of dissemination in ras G12V pten Ri and ras G12V p53 Ri apc Ri animals treated with BEZ235. ( d ) Western blot analysis of PI3K pathway output from hindguts with indicated genotypes 7 days after induction of transgenes and quantification. Syn, Syntaxin (loading control). ( e ) Time-course analysis of PI3K pathway activation status in control and ras G12V p53 Ri pten Ri apc Ri hindguts. ( f ) Western blot analysis of the biochemical response by ras G12V and ras G12V p53 Ri pten Ri apc Ri animals to PI3K pathway inhibitors. ( g ) Quantification of dissemination in indicated genotypes treated with BEZ235 or DMSO. ( h ) Schematic illustration of the mechanism of resistance to BEZ235: genetically activating mTORC1 promotes BEZ235 sensitivity. ( d , e ) Each data point represents the average response of two to five biological replicates with ten hindguts per replicate; error bars: s.e.m. ( a – c , g ) n =2 replicates, 30 flies per replicate; error bars: s.e.m. * P

    Journal: Nature Communications

    Article Title: Functional exploration of colorectal cancer genomes using Drosophila

    doi: 10.1038/ncomms13615

    Figure Lengend Snippet: Drug resistance as an emergent feature of increased genetic complexity. ( a , b ) Plot of P -values indicating significance of compound rescue ( a ) and summary of compound response ( b ) of ras G12V and ras G12V p53 Ri pten Ri apc Ri animals. P -values ( a ) were obtained by comparing the dissemination phenotype after compound feeding to dimethyl sulfoxide (DMSO) fed flies (dissemination plots can be found in Supplementary Fig. 3a,b ). Blue dots represent statistically significant results. ( c ) Quantification of dissemination in ras G12V pten Ri and ras G12V p53 Ri apc Ri animals treated with BEZ235. ( d ) Western blot analysis of PI3K pathway output from hindguts with indicated genotypes 7 days after induction of transgenes and quantification. Syn, Syntaxin (loading control). ( e ) Time-course analysis of PI3K pathway activation status in control and ras G12V p53 Ri pten Ri apc Ri hindguts. ( f ) Western blot analysis of the biochemical response by ras G12V and ras G12V p53 Ri pten Ri apc Ri animals to PI3K pathway inhibitors. ( g ) Quantification of dissemination in indicated genotypes treated with BEZ235 or DMSO. ( h ) Schematic illustration of the mechanism of resistance to BEZ235: genetically activating mTORC1 promotes BEZ235 sensitivity. ( d , e ) Each data point represents the average response of two to five biological replicates with ten hindguts per replicate; error bars: s.e.m. ( a – c , g ) n =2 replicates, 30 flies per replicate; error bars: s.e.m. * P

    Article Snippet: Primary antibodies were as follows: rabbit anti Drosophila phospho-AKT (p-Ser505; Drosophila equivalent of mammalian AKT p-Ser473, 1:1,000, Cell Signaling, catalogue number 4054S), rabbit anti-mouse AKT (1:1,000, Cell Signaling, catalogue number 4691S), rabbit anti-mouse phospho-4EBP (Thr37/46, 1:1,000, Cell Signaling, catalogue number 2855S), rabbit anti-human phospho-AKT (Ser473, 1:1,000, Cell Signaling, catalogue number 4060S), mouse anti-chicken α-actin (1:1,000, DSHB catalogue number JLA20-s), rabbit anti-GFP (1:2,000, Sigma, catalogue number G1544), rabbit anti Drosophila P53 (1:1,000, DSHB, catalogue number H3), rabbit anti Drosophila Pten (1:1,000, gift from A. Wodarz ), mouse anti-dpERK (Thr183/Tyr185, 1:2,000, Sigma, catalogue number M8159), rabbit anti human phospho-S6 (Ser 235/236, 1:1,000, Cell Signaling, catalogue number 2211) and mouse anti Drosophila Syntaxin (1:1,000, DSHB, catalogue number 8C3).

    Techniques: Western Blot, Activation Assay

    AKT activator SC79 promotes sensitivity to PI3K pathway inhibition. ( a ) Western blot analysis of PI3K signalling pathway output in ras G12V and ras G12V p53 Ri pten Ri apc Ri hindguts after 1 day feeding of SC79 at indicated doses. Syn, Syntaxin (loading control);ten hindguts per replicate. ( b ) Quantification of dissemination in ras G12V and ras G12V p53 Ri pten Ri apc Ri animals after sequential treatment with BEZ235 and indicated doses of SC79. ( c ) Quantification of dissemination in ras G12V and ras G12V p53 Ri pten Ri apc Ri animals after two different treatment schedules of SC79/BEZ235 and each drug alone. ( b , c ) n =2 replicates, 30 flies per replicate; error bars: s.e.m. * P

    Journal: Nature Communications

    Article Title: Functional exploration of colorectal cancer genomes using Drosophila

    doi: 10.1038/ncomms13615

    Figure Lengend Snippet: AKT activator SC79 promotes sensitivity to PI3K pathway inhibition. ( a ) Western blot analysis of PI3K signalling pathway output in ras G12V and ras G12V p53 Ri pten Ri apc Ri hindguts after 1 day feeding of SC79 at indicated doses. Syn, Syntaxin (loading control);ten hindguts per replicate. ( b ) Quantification of dissemination in ras G12V and ras G12V p53 Ri pten Ri apc Ri animals after sequential treatment with BEZ235 and indicated doses of SC79. ( c ) Quantification of dissemination in ras G12V and ras G12V p53 Ri pten Ri apc Ri animals after two different treatment schedules of SC79/BEZ235 and each drug alone. ( b , c ) n =2 replicates, 30 flies per replicate; error bars: s.e.m. * P

    Article Snippet: Primary antibodies were as follows: rabbit anti Drosophila phospho-AKT (p-Ser505; Drosophila equivalent of mammalian AKT p-Ser473, 1:1,000, Cell Signaling, catalogue number 4054S), rabbit anti-mouse AKT (1:1,000, Cell Signaling, catalogue number 4691S), rabbit anti-mouse phospho-4EBP (Thr37/46, 1:1,000, Cell Signaling, catalogue number 2855S), rabbit anti-human phospho-AKT (Ser473, 1:1,000, Cell Signaling, catalogue number 4060S), mouse anti-chicken α-actin (1:1,000, DSHB catalogue number JLA20-s), rabbit anti-GFP (1:2,000, Sigma, catalogue number G1544), rabbit anti Drosophila P53 (1:1,000, DSHB, catalogue number H3), rabbit anti Drosophila Pten (1:1,000, gift from A. Wodarz ), mouse anti-dpERK (Thr183/Tyr185, 1:2,000, Sigma, catalogue number M8159), rabbit anti human phospho-S6 (Ser 235/236, 1:1,000, Cell Signaling, catalogue number 2211) and mouse anti Drosophila Syntaxin (1:1,000, DSHB, catalogue number 8C3).

    Techniques: Inhibition, Western Blot

    Notopleural  is required for embryonic tracheal gas filling and encodes a serine protease related to human matriptase. (A-D) Bright field light microscopic images of stage 17 wild-type (A),  btl- Gal4; UAS-RNAi- GD13443  (B),  Np P6 /Np C2  mutant (C) and  Np P6 , btl -Gal4/ Np C2 ,UAS- Np  mutant (D) embryos. Wild-type embryos show gas filled tracheal tubes at the end of embryogenesis (arrow in A). RNAi-mediated tracheal knock-down of  CG34350  ( Np ) leads to lack of tracheal gas filling (arrow in B).  Np P6 /Np C2  mutant embryos lack gas filling (arrow in C) while  Np  mutant embryos with tracheal expression of  Np  show normal gas filling of the tracheal system (arrow in D). (E) Schema showing the protein domain organisations of  Drosophila  Np and human matriptase. The transmembrane domains (yellow), the SEA ( s ea urchin sperm protein/ e nteropeptidase/ a grin), CUB ( C ls/Clr,  u rchin embryonic growth factor,  b one morphogenetic protein-1), LDLa ( l ow- d ensity  l ipoprotein receptor class  A ) and the catalytic protease domains are shown. Conserved disulphide bridges (-S-S-) and zymogen activation cleavage sites (V) are indicated. (F-J”) Confocal LSM images of whole-mount antibody stainings of  Np ::GFP embryos at stage 16 (F-F”, H-J”) and stage 17 (G-G”) stained with anti-Spectrin (magenta) and anti-GFP (green, Np::GFP) antibodies. Np::GFP is expressed in the tracheal system (F-G”), the hindgut (H-H”), the epidermis (I-I”) and the salivary glands (J-J”). Np::GFP is localized in the tracheal lumen during stage 16 (arrow in F’) and 17 (arrow in G’) and localizes to the apical membrane of tracheal cells during stage 17 (arrowheads in G’). In the hindgut and epidermis, Np::GFP is localized exclusively at the apical cell membranes (arrowheads in H’ and I’). In the salivary glands, Np::GFP is localized exclusively in the lumen (arrow in J’). Scale bars correspond to 10 μm.

    Journal: PLoS Genetics

    Article Title: Conserved function of the matriptase-prostasin proteolytic cascade during epithelial morphogenesis

    doi: 10.1371/journal.pgen.1007882

    Figure Lengend Snippet: Notopleural is required for embryonic tracheal gas filling and encodes a serine protease related to human matriptase. (A-D) Bright field light microscopic images of stage 17 wild-type (A), btl- Gal4; UAS-RNAi- GD13443 (B), Np P6 /Np C2 mutant (C) and Np P6 , btl -Gal4/ Np C2 ,UAS- Np mutant (D) embryos. Wild-type embryos show gas filled tracheal tubes at the end of embryogenesis (arrow in A). RNAi-mediated tracheal knock-down of CG34350 ( Np ) leads to lack of tracheal gas filling (arrow in B). Np P6 /Np C2 mutant embryos lack gas filling (arrow in C) while Np mutant embryos with tracheal expression of Np show normal gas filling of the tracheal system (arrow in D). (E) Schema showing the protein domain organisations of Drosophila Np and human matriptase. The transmembrane domains (yellow), the SEA ( s ea urchin sperm protein/ e nteropeptidase/ a grin), CUB ( C ls/Clr, u rchin embryonic growth factor, b one morphogenetic protein-1), LDLa ( l ow- d ensity l ipoprotein receptor class A ) and the catalytic protease domains are shown. Conserved disulphide bridges (-S-S-) and zymogen activation cleavage sites (V) are indicated. (F-J”) Confocal LSM images of whole-mount antibody stainings of Np ::GFP embryos at stage 16 (F-F”, H-J”) and stage 17 (G-G”) stained with anti-Spectrin (magenta) and anti-GFP (green, Np::GFP) antibodies. Np::GFP is expressed in the tracheal system (F-G”), the hindgut (H-H”), the epidermis (I-I”) and the salivary glands (J-J”). Np::GFP is localized in the tracheal lumen during stage 16 (arrow in F’) and 17 (arrow in G’) and localizes to the apical membrane of tracheal cells during stage 17 (arrowheads in G’). In the hindgut and epidermis, Np::GFP is localized exclusively at the apical cell membranes (arrowheads in H’ and I’). In the salivary glands, Np::GFP is localized exclusively in the lumen (arrow in J’). Scale bars correspond to 10 μm.

    Article Snippet: Stage 17 Drosophila embryos were dechorionated, transferred to a 150 μm specimen planchette (Engineering Office M. Wohlwend GmbH), and frozen with a Leica HBM 100 high-pressure freezer (Leica Microsystems).

    Techniques: Mutagenesis, Expressing, Activation Assay, Staining

    Notopleural is required for epithelial barrier function and aECM formation. (A-B’) Confocal LSM images of tracheal dorsal trunks of wild-type (A) and  Np P6 /Np C2  mutant (B) stage 17 embryos after Texas Red-labelled 10 kDa dextran injection into the haemocoel. Texas Red dextran (red) is not found in the dorsal trunk lumen of wild-type embryos (arrow in A), but is detectable in the dorsal trunk lumen (arrow in B) and the tracheal paracellular space (arrowheads in B’) of  Np P6 /Np C2  mutant embryos. (B’) shows the tracheal dorsal trunk with increased contrast to visualize the paracellular space (arrowheads in B’). (C, D) Confocal LSM images of whole-mount stainings of stage 16 wild-type (C) and  Np P6 /Np C2  mutant (D) embryos with FITC labelled chitin-binding probe (CBP). CBP binds the luminal chitin matrix and outlines the tracheal network during embryogenesis. The tracheal network formation of  Np  mutant embryos (D) is indistinguishable from wild-type embryos (C). (E, F) Confocal LSM images of tracheal dorsal trunks of wild-type (E) and  Np P6 /Np C2  mutant (F) stage 16 embryos stained with CBP (green), anti-Uif (magenta) and anti-Mega (cyan) antibodies. (G) Schema of tracheal aECM maturation and liquid clearance. The aECM (green) in the tracheal lumen is degraded after mid-stage 16 and the mature taenidial folds (green spiral) form at the apical side of tracheal cells (magenta) during mid-stage 17. Liquid (blue) is cleared from the tracheal lumen during late-stage 17. Time data refer to embryonic development at 22°C. (H-K) Confocal LSM optical sections (H, I) and sagittal z-stack projection images (J, K) of dorsal trunks of wild-type (H, J) and  Np P6 /Np C2  mutant (I, K) stage 17 embryos stained with CBP. (L-M’) Dark field microscopy images of stage 17 wild-type (L, L’) and  Np P6 /Np C2  mutant (M, M’) cuticle preparations. Denticle belts (L’, M`) develop only rudimentarily in  Np  mutant embryos (arrowhead in M’). Scale bars correspond to 10 μm in (A, B, E, F), to 50 μm in (C, D) and to 5 μm in (H-K).

    Journal: PLoS Genetics

    Article Title: Conserved function of the matriptase-prostasin proteolytic cascade during epithelial morphogenesis

    doi: 10.1371/journal.pgen.1007882

    Figure Lengend Snippet: Notopleural is required for epithelial barrier function and aECM formation. (A-B’) Confocal LSM images of tracheal dorsal trunks of wild-type (A) and Np P6 /Np C2 mutant (B) stage 17 embryos after Texas Red-labelled 10 kDa dextran injection into the haemocoel. Texas Red dextran (red) is not found in the dorsal trunk lumen of wild-type embryos (arrow in A), but is detectable in the dorsal trunk lumen (arrow in B) and the tracheal paracellular space (arrowheads in B’) of Np P6 /Np C2 mutant embryos. (B’) shows the tracheal dorsal trunk with increased contrast to visualize the paracellular space (arrowheads in B’). (C, D) Confocal LSM images of whole-mount stainings of stage 16 wild-type (C) and Np P6 /Np C2 mutant (D) embryos with FITC labelled chitin-binding probe (CBP). CBP binds the luminal chitin matrix and outlines the tracheal network during embryogenesis. The tracheal network formation of Np mutant embryos (D) is indistinguishable from wild-type embryos (C). (E, F) Confocal LSM images of tracheal dorsal trunks of wild-type (E) and Np P6 /Np C2 mutant (F) stage 16 embryos stained with CBP (green), anti-Uif (magenta) and anti-Mega (cyan) antibodies. (G) Schema of tracheal aECM maturation and liquid clearance. The aECM (green) in the tracheal lumen is degraded after mid-stage 16 and the mature taenidial folds (green spiral) form at the apical side of tracheal cells (magenta) during mid-stage 17. Liquid (blue) is cleared from the tracheal lumen during late-stage 17. Time data refer to embryonic development at 22°C. (H-K) Confocal LSM optical sections (H, I) and sagittal z-stack projection images (J, K) of dorsal trunks of wild-type (H, J) and Np P6 /Np C2 mutant (I, K) stage 17 embryos stained with CBP. (L-M’) Dark field microscopy images of stage 17 wild-type (L, L’) and Np P6 /Np C2 mutant (M, M’) cuticle preparations. Denticle belts (L’, M`) develop only rudimentarily in Np mutant embryos (arrowhead in M’). Scale bars correspond to 10 μm in (A, B, E, F), to 50 μm in (C, D) and to 5 μm in (H-K).

    Article Snippet: Stage 17 Drosophila embryos were dechorionated, transferred to a 150 μm specimen planchette (Engineering Office M. Wohlwend GmbH), and frozen with a Leica HBM 100 high-pressure freezer (Leica Microsystems).

    Techniques: Mutagenesis, Injection, Binding Assay, Staining, Microscopy

    Drosophila  Tracheal-prostasin and human prostasin show functional similarity. (A) Schema showing the protein domain organization of  Drosophila  Tpr and human prostasin. The signal peptides (sp; blue), conserved disulfide bridges (S-S), the activation cleavage sites (V), the catalytic protease domains (green) and the GPI anchor (red) are indicated. (B, C) Bright field light microscopic images of stage 17  tpr D1 /tpr D1  (B) and  tpr D1 , btl -Gal4/ tpr D1 ,UAS- tpr  (C) mutant embryos. (D, E) Whole-mount antibody stainings of stage 16  tpr ::RFP embryos stained with anti-RFP (D, E) and anti-Spectrin (E) antibodies. Tpr::RFP (green) is restricted to the tracheal system (D) and localized in the tracheal lumen (E). Spectrin (magenta) outlines the tracheal cells (E). (F, G) Transmission electron microscopic images of stage 17 wild-type (F) and  tpr D1 /tpr D1  mutant (G) tracheal aECM. The taenidial folds of the tracheal aECM are associated with the apical side of tracheal cells in wild-type (F), while the taenidial folds are detached from tracheal cells in  tpr  mutant embryos (asterisks in G). (H, I) Whole-mount antibody stainings of  dpy ::YFP/ dpy ::YFP (H) and  tpr D1 , dpy ::YFP/ tpr D1 , dpy ::YFP mutant (I) embryos at stage 17 stained with FITC labelled CBP (green) and anti-GFP antibodies (magenta). Chitin is cleared from the tracheal lumen of wild-type (H) and  tpr  mutant (I) embryos. Dpy::YFP is cleared from the tracheal lumen of wild-type embryos (H), while Dpy::YFP degradation is incomplete in  tpr  mutant embryos (I). (J-M) Bright field light microscopic images of stage 17 (J, L) and confocal LSM images of whole mount antibody stainings of stage 16 (K, M)  tpr D1 , btl -Gal4/ tpr D1 ,UAS-prostasin (J, K) and  tpr D1 , btl -Gal4/ tpr D1 ,UAS-prostasin; UAS-HAI-2/+ (L, M) mutant embryos with anti-prostasin (green) and anti-Spectrin (magenta) antibodies.  tpr  mutant embryos expressing prostasin in the trachea lack tracheal gas filling (arrow in J) and show prostasin localization in tracheal cells (green in K). In contrast, tracheal co-expression of prostasin and HAI-2 in  tpr  mutant embryos facilitates gas filling of the tracheal system (arrow in L) and prostasin localizes to the tracheal lumen (green in M). (N) Purified Tpr-Flag or zymogen locked Tpr ZL -Flag were incubated with buffer, Np-Strep or matriptase-Strep and samples were analysed by western blotting and immunostained using anti-Flag antibody. Tpr-Flag is detectable as a fragment of approximately 60 kDa that is processed to a fragment of approximately 40 kDa by incubation with purified Np-Strep or purified matriptase-Strep. Proteolytic processing occurs at the zymogen activation site, since zymogen locked Tpr ZL -Flag is resistant to cleavage by Np-Strep and matriptase-Strep. Scale bars correspond to 1 μm in (F, G) to 10 μm in (E, H, I, K, M) and to 50 μm in (D).

    Journal: PLoS Genetics

    Article Title: Conserved function of the matriptase-prostasin proteolytic cascade during epithelial morphogenesis

    doi: 10.1371/journal.pgen.1007882

    Figure Lengend Snippet: Drosophila Tracheal-prostasin and human prostasin show functional similarity. (A) Schema showing the protein domain organization of Drosophila Tpr and human prostasin. The signal peptides (sp; blue), conserved disulfide bridges (S-S), the activation cleavage sites (V), the catalytic protease domains (green) and the GPI anchor (red) are indicated. (B, C) Bright field light microscopic images of stage 17 tpr D1 /tpr D1 (B) and tpr D1 , btl -Gal4/ tpr D1 ,UAS- tpr (C) mutant embryos. (D, E) Whole-mount antibody stainings of stage 16 tpr ::RFP embryos stained with anti-RFP (D, E) and anti-Spectrin (E) antibodies. Tpr::RFP (green) is restricted to the tracheal system (D) and localized in the tracheal lumen (E). Spectrin (magenta) outlines the tracheal cells (E). (F, G) Transmission electron microscopic images of stage 17 wild-type (F) and tpr D1 /tpr D1 mutant (G) tracheal aECM. The taenidial folds of the tracheal aECM are associated with the apical side of tracheal cells in wild-type (F), while the taenidial folds are detached from tracheal cells in tpr mutant embryos (asterisks in G). (H, I) Whole-mount antibody stainings of dpy ::YFP/ dpy ::YFP (H) and tpr D1 , dpy ::YFP/ tpr D1 , dpy ::YFP mutant (I) embryos at stage 17 stained with FITC labelled CBP (green) and anti-GFP antibodies (magenta). Chitin is cleared from the tracheal lumen of wild-type (H) and tpr mutant (I) embryos. Dpy::YFP is cleared from the tracheal lumen of wild-type embryos (H), while Dpy::YFP degradation is incomplete in tpr mutant embryos (I). (J-M) Bright field light microscopic images of stage 17 (J, L) and confocal LSM images of whole mount antibody stainings of stage 16 (K, M) tpr D1 , btl -Gal4/ tpr D1 ,UAS-prostasin (J, K) and tpr D1 , btl -Gal4/ tpr D1 ,UAS-prostasin; UAS-HAI-2/+ (L, M) mutant embryos with anti-prostasin (green) and anti-Spectrin (magenta) antibodies. tpr mutant embryos expressing prostasin in the trachea lack tracheal gas filling (arrow in J) and show prostasin localization in tracheal cells (green in K). In contrast, tracheal co-expression of prostasin and HAI-2 in tpr mutant embryos facilitates gas filling of the tracheal system (arrow in L) and prostasin localizes to the tracheal lumen (green in M). (N) Purified Tpr-Flag or zymogen locked Tpr ZL -Flag were incubated with buffer, Np-Strep or matriptase-Strep and samples were analysed by western blotting and immunostained using anti-Flag antibody. Tpr-Flag is detectable as a fragment of approximately 60 kDa that is processed to a fragment of approximately 40 kDa by incubation with purified Np-Strep or purified matriptase-Strep. Proteolytic processing occurs at the zymogen activation site, since zymogen locked Tpr ZL -Flag is resistant to cleavage by Np-Strep and matriptase-Strep. Scale bars correspond to 1 μm in (F, G) to 10 μm in (E, H, I, K, M) and to 50 μm in (D).

    Article Snippet: Stage 17 Drosophila embryos were dechorionated, transferred to a 150 μm specimen planchette (Engineering Office M. Wohlwend GmbH), and frozen with a Leica HBM 100 high-pressure freezer (Leica Microsystems).

    Techniques: Functional Assay, Activation Assay, Mutagenesis, Staining, Transmission Assay, Expressing, Purification, Incubation, Western Blot

    Notopleural  is required in the trachea for luminal Dumpy degradation. Confocal LSM images of whole-mount antibody stainings of  dpy ::YFP/ dpy ::YFP (A-C”) and  Np P6 , dpy ::YFP/  Np P6 , dpy ::YFP mutant (D-F”) embryos at late-stage 16 (A-A”, D-D”), early-stage 17 (B-B”, E-E”) and mid-stage 17 (C-C”, F-F”) stained with CBP and anti-GFP antibody. During late-stage 16 tracheal luminal Dpy::YFP (magenta) forms a central core (arrowhead in A”) and a peripheral “shell” layer (arrow in A”; see also [  22 ]) in  dpy ::YFP embryos. In  Np  mutant embryos the luminal Dpy::YFP core (arrowhead in D”) and “shell” (arrow in D”) are also formed normally. Dpy::YFP and luminal chitin (green) condense at early-stage 17 (arrowhead and arrow in B”) and during mid-stage 17 the tracheal lumen is cleared from luminal chitin and Dpy (C’, C”) in  dpy ::YFP embryos.  Np  mutant embryos show no Dpy::YFP condensation during early stage 17 (arrowhead and arrow in E”; compare with B”) and no luminal clearance of Dpy::YFP during mid-stage 17 (F”; compare with C”). Note: Chitin is cleared normally from the tracheal lumen in  Np  mutant embryos during mid-stage 17 (F’) as found in  dpy ::YFP embryos (C’). Scale bars correspond to 10 μm.

    Journal: PLoS Genetics

    Article Title: Conserved function of the matriptase-prostasin proteolytic cascade during epithelial morphogenesis

    doi: 10.1371/journal.pgen.1007882

    Figure Lengend Snippet: Notopleural is required in the trachea for luminal Dumpy degradation. Confocal LSM images of whole-mount antibody stainings of dpy ::YFP/ dpy ::YFP (A-C”) and Np P6 , dpy ::YFP/ Np P6 , dpy ::YFP mutant (D-F”) embryos at late-stage 16 (A-A”, D-D”), early-stage 17 (B-B”, E-E”) and mid-stage 17 (C-C”, F-F”) stained with CBP and anti-GFP antibody. During late-stage 16 tracheal luminal Dpy::YFP (magenta) forms a central core (arrowhead in A”) and a peripheral “shell” layer (arrow in A”; see also [ 22 ]) in dpy ::YFP embryos. In Np mutant embryos the luminal Dpy::YFP core (arrowhead in D”) and “shell” (arrow in D”) are also formed normally. Dpy::YFP and luminal chitin (green) condense at early-stage 17 (arrowhead and arrow in B”) and during mid-stage 17 the tracheal lumen is cleared from luminal chitin and Dpy (C’, C”) in dpy ::YFP embryos. Np mutant embryos show no Dpy::YFP condensation during early stage 17 (arrowhead and arrow in E”; compare with B”) and no luminal clearance of Dpy::YFP during mid-stage 17 (F”; compare with C”). Note: Chitin is cleared normally from the tracheal lumen in Np mutant embryos during mid-stage 17 (F’) as found in dpy ::YFP embryos (C’). Scale bars correspond to 10 μm.

    Article Snippet: Stage 17 Drosophila embryos were dechorionated, transferred to a 150 μm specimen planchette (Engineering Office M. Wohlwend GmbH), and frozen with a Leica HBM 100 high-pressure freezer (Leica Microsystems).

    Techniques: Mutagenesis, Staining

    Notopleural and human matriptase are functional homologues. (A-I) Confocal LSM images of dorsal trunks of  Np P6 /Np C2  mutant embryos rescued by  btl- Gal4/+ (A, control), or  btl -Gal4/UAS- Np ::GFP (B, F), or  btl -Gal4/UAS- Np S990A  (C), or  btl -Gal4/UAS-matriptase (D, G), or btl-Gal4/UAS-lint (E), or btl-Gal4/UAS-matriptase,UAS-HAI-1 (H), or btl-Gal4/UAS-matriptase,UAS-HAI-2 (I), stained with CBP (A-E), or anti-Spectrin (magenta) and anti-GFP (green) antibodies (F), or anti-Spectrin (magenta) and anti-matriptase (green) antibodies (G-I). Tracheal dorsal trunks of stage 17 embryos (A-E) and stage 16 embryos (F-I) are shown. Asterisks in (F) and (I) indicate matriptase (green) and Np::GFP (green) localization in the tracheal lumen. (J) Quantification of chitin matrix organisation (green bars) and tracheal gas filling (red bars). The UAS-reporter lines are driven by  btl -Gal4 in  Np P6 /Np C2  mutant embryos. For each genotype: n = 120 for LC analysis; n = 40 for aECM formation (for details see Materials and Methods). (K) Quantification of number and diameter of stage 17 dorsal trunk chitin strands. Data points represent mean values for each genotype (n = 10) and error bars represent standard deviation (for details see Materials and Methods). (L) Schema showing the Piopio protein domain organization. Identified Np (see M) and matriptase (see N) protease cleavage site (pcs) is indicated. Furin pcs, signal peptide (blue), transmembrane helix (yellow) and position of Flag-tag (green) are shown. (M, N) Purified Pio-Flag or Pio R196A -Flag were incubated with buffer, Np-Strep, matriptase-Strep, Np S990A -Strep or matriptase S805A -Strep. Samples were analyzed by western blotting and immunostained using anti-Flag, anti-Strep or anti-matriptase antibodies. Pio-Flag is detectable in two fragments at approximately 80 and 55 kDa, presumably due to cleavage by Furin. After incubation of Pio-Flag with Np-Strep (M) or matriptase-Strep (N), a 30 kDa Pio-Flag fragment is detectable that indicates cleavage. Pio-Flag is not cleaved by catalytically inactive Np S990A -Strep (M) or matriptase S805A -Strep (N) and a single amino acid substitution in the Pio ZP domain (Pio R196A -Flag) is sufficient to establish cleavage resistance to Np and matriptase (M, N). Note that purified Np-Strep (M) and matriptase-Strep (N) are detected as approximately 35 kDa fragments, consistent with the predicted sizes of the catalytic protease domains and, thus, indicating zymogen activation. Purified catalytically inactive Np S990A -Strep (M) and matriptase S805A -Strep (N), are detected as 150–200 kDa and 90–120 kDa zymogens, respectively. These results indicate autocatalytic zymogen activation for both proteases. Scale bars correspond to 5 μm.

    Journal: PLoS Genetics

    Article Title: Conserved function of the matriptase-prostasin proteolytic cascade during epithelial morphogenesis

    doi: 10.1371/journal.pgen.1007882

    Figure Lengend Snippet: Notopleural and human matriptase are functional homologues. (A-I) Confocal LSM images of dorsal trunks of Np P6 /Np C2 mutant embryos rescued by btl- Gal4/+ (A, control), or btl -Gal4/UAS- Np ::GFP (B, F), or btl -Gal4/UAS- Np S990A (C), or btl -Gal4/UAS-matriptase (D, G), or btl-Gal4/UAS-lint (E), or btl-Gal4/UAS-matriptase,UAS-HAI-1 (H), or btl-Gal4/UAS-matriptase,UAS-HAI-2 (I), stained with CBP (A-E), or anti-Spectrin (magenta) and anti-GFP (green) antibodies (F), or anti-Spectrin (magenta) and anti-matriptase (green) antibodies (G-I). Tracheal dorsal trunks of stage 17 embryos (A-E) and stage 16 embryos (F-I) are shown. Asterisks in (F) and (I) indicate matriptase (green) and Np::GFP (green) localization in the tracheal lumen. (J) Quantification of chitin matrix organisation (green bars) and tracheal gas filling (red bars). The UAS-reporter lines are driven by btl -Gal4 in Np P6 /Np C2 mutant embryos. For each genotype: n = 120 for LC analysis; n = 40 for aECM formation (for details see Materials and Methods). (K) Quantification of number and diameter of stage 17 dorsal trunk chitin strands. Data points represent mean values for each genotype (n = 10) and error bars represent standard deviation (for details see Materials and Methods). (L) Schema showing the Piopio protein domain organization. Identified Np (see M) and matriptase (see N) protease cleavage site (pcs) is indicated. Furin pcs, signal peptide (blue), transmembrane helix (yellow) and position of Flag-tag (green) are shown. (M, N) Purified Pio-Flag or Pio R196A -Flag were incubated with buffer, Np-Strep, matriptase-Strep, Np S990A -Strep or matriptase S805A -Strep. Samples were analyzed by western blotting and immunostained using anti-Flag, anti-Strep or anti-matriptase antibodies. Pio-Flag is detectable in two fragments at approximately 80 and 55 kDa, presumably due to cleavage by Furin. After incubation of Pio-Flag with Np-Strep (M) or matriptase-Strep (N), a 30 kDa Pio-Flag fragment is detectable that indicates cleavage. Pio-Flag is not cleaved by catalytically inactive Np S990A -Strep (M) or matriptase S805A -Strep (N) and a single amino acid substitution in the Pio ZP domain (Pio R196A -Flag) is sufficient to establish cleavage resistance to Np and matriptase (M, N). Note that purified Np-Strep (M) and matriptase-Strep (N) are detected as approximately 35 kDa fragments, consistent with the predicted sizes of the catalytic protease domains and, thus, indicating zymogen activation. Purified catalytically inactive Np S990A -Strep (M) and matriptase S805A -Strep (N), are detected as 150–200 kDa and 90–120 kDa zymogens, respectively. These results indicate autocatalytic zymogen activation for both proteases. Scale bars correspond to 5 μm.

    Article Snippet: Stage 17 Drosophila embryos were dechorionated, transferred to a 150 μm specimen planchette (Engineering Office M. Wohlwend GmbH), and frozen with a Leica HBM 100 high-pressure freezer (Leica Microsystems).

    Techniques: Functional Assay, Mutagenesis, Staining, Liquid Chromatography, Standard Deviation, FLAG-tag, Purification, Incubation, Western Blot, Activation Assay

    Notopleural  mutants display defects in taenidial folds formation and maintenance of the transepithelial barrier. (A-D) Transmission electron microscopy images of stage 17 (21–22 hours AEL) wild-type (A, C) and  Np P6 /Np C2  mutant (B, D) tracheal aECM (A, B) and lateral tracheal cell membranes (C, D). Arrow in (A) indicates the electron-dense envelope of the taenidial folds and the arrowhead indicates the procuticle. Note the accumulation of electron-dense material in the tracheal lumen of  Np  mutant embryos (asterisks in B) as compared to wild-type embryos (asterisks in A). The ladder-like structure of SJs in wild-type (arrow in C) and  Np  mutant (arrow in D) tracheal cells is detectable. (E, F) Tracheal transepithelial barrier function proofed by 10 kDa (E) and 70 kDa (F) dextran. Dextran injections into the haemocoel of embryos reveal a tracheal barrier function (indicated by the lack of dextran diffusion into the tracheal lumen) or a defective barrier function (indicated by dextran diffusion into the tracheal system). For each indicated stage: n = 8 for  mega ; n = 15 for  Np  and control. For details see   S6 Fig . Scale bars correspond to 1 μm in (A, B) and to 0.1 μm in (C, D).

    Journal: PLoS Genetics

    Article Title: Conserved function of the matriptase-prostasin proteolytic cascade during epithelial morphogenesis

    doi: 10.1371/journal.pgen.1007882

    Figure Lengend Snippet: Notopleural mutants display defects in taenidial folds formation and maintenance of the transepithelial barrier. (A-D) Transmission electron microscopy images of stage 17 (21–22 hours AEL) wild-type (A, C) and Np P6 /Np C2 mutant (B, D) tracheal aECM (A, B) and lateral tracheal cell membranes (C, D). Arrow in (A) indicates the electron-dense envelope of the taenidial folds and the arrowhead indicates the procuticle. Note the accumulation of electron-dense material in the tracheal lumen of Np mutant embryos (asterisks in B) as compared to wild-type embryos (asterisks in A). The ladder-like structure of SJs in wild-type (arrow in C) and Np mutant (arrow in D) tracheal cells is detectable. (E, F) Tracheal transepithelial barrier function proofed by 10 kDa (E) and 70 kDa (F) dextran. Dextran injections into the haemocoel of embryos reveal a tracheal barrier function (indicated by the lack of dextran diffusion into the tracheal lumen) or a defective barrier function (indicated by dextran diffusion into the tracheal system). For each indicated stage: n = 8 for mega ; n = 15 for Np and control. For details see S6 Fig . Scale bars correspond to 1 μm in (A, B) and to 0.1 μm in (C, D).

    Article Snippet: Stage 17 Drosophila embryos were dechorionated, transferred to a 150 μm specimen planchette (Engineering Office M. Wohlwend GmbH), and frozen with a Leica HBM 100 high-pressure freezer (Leica Microsystems).

    Techniques: Transmission Assay, Electron Microscopy, Mutagenesis, Diffusion-based Assay

    Dynamics of human CDK8 A-loop during the 50-ns molecular dynamic simulation

    Journal:

    Article Title: All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

    doi: 10.1016/j.compbiolchem.2014.03.003

    Figure Lengend Snippet: Dynamics of human CDK8 A-loop during the 50-ns molecular dynamic simulation

    Article Snippet: We have cloned Drosophila CDK8 (dCDK8) and CycC (dCycC) into pGBKT7 and pGADT7, yeast two-hybrid vectors (Clontech, Mountain View, CA, USA), to investigate physically interactions between pGBKT7 and pGADT7 in yeast cells.

    Techniques:

    The molecular dynamics of CDK8 D173A, D189N, T196A and T196D structures

    Journal:

    Article Title: All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

    doi: 10.1016/j.compbiolchem.2014.03.003

    Figure Lengend Snippet: The molecular dynamics of CDK8 D173A, D189N, T196A and T196D structures

    Article Snippet: We have cloned Drosophila CDK8 (dCDK8) and CycC (dCycC) into pGBKT7 and pGADT7, yeast two-hybrid vectors (Clontech, Mountain View, CA, USA), to investigate physically interactions between pGBKT7 and pGADT7 in yeast cells.

    Techniques:

    Hydrogen bond analysis of the residues in human CDK8 A-loop during the 50-ns MD simulation

    Journal:

    Article Title: All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

    doi: 10.1016/j.compbiolchem.2014.03.003

    Figure Lengend Snippet: Hydrogen bond analysis of the residues in human CDK8 A-loop during the 50-ns MD simulation

    Article Snippet: We have cloned Drosophila CDK8 (dCDK8) and CycC (dCycC) into pGBKT7 and pGADT7, yeast two-hybrid vectors (Clontech, Mountain View, CA, USA), to investigate physically interactions between pGBKT7 and pGADT7 in yeast cells.

    Techniques:

    The theoretical structure of human CDK8-CycC complex with the lowest potential energy in solution during the molecular dynamic simulation

    Journal:

    Article Title: All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

    doi: 10.1016/j.compbiolchem.2014.03.003

    Figure Lengend Snippet: The theoretical structure of human CDK8-CycC complex with the lowest potential energy in solution during the molecular dynamic simulation

    Article Snippet: We have cloned Drosophila CDK8 (dCDK8) and CycC (dCycC) into pGBKT7 and pGADT7, yeast two-hybrid vectors (Clontech, Mountain View, CA, USA), to investigate physically interactions between pGBKT7 and pGADT7 in yeast cells.

    Techniques:

    The theoretical structure of human CDK8 based on molecular modeling and 50-ns molecular dynamics simulation

    Journal:

    Article Title: All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

    doi: 10.1016/j.compbiolchem.2014.03.003

    Figure Lengend Snippet: The theoretical structure of human CDK8 based on molecular modeling and 50-ns molecular dynamics simulation

    Article Snippet: We have cloned Drosophila CDK8 (dCDK8) and CycC (dCycC) into pGBKT7 and pGADT7, yeast two-hybrid vectors (Clontech, Mountain View, CA, USA), to investigate physically interactions between pGBKT7 and pGADT7 in yeast cells.

    Techniques:

    The molecular dynamic study of human CDK8

    Journal:

    Article Title: All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

    doi: 10.1016/j.compbiolchem.2014.03.003

    Figure Lengend Snippet: The molecular dynamic study of human CDK8

    Article Snippet: We have cloned Drosophila CDK8 (dCDK8) and CycC (dCycC) into pGBKT7 and pGADT7, yeast two-hybrid vectors (Clontech, Mountain View, CA, USA), to investigate physically interactions between pGBKT7 and pGADT7 in yeast cells.

    Techniques:

    Comparison of the CDK8 crystal and MD Structures

    Journal:

    Article Title: All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

    doi: 10.1016/j.compbiolchem.2014.03.003

    Figure Lengend Snippet: Comparison of the CDK8 crystal and MD Structures

    Article Snippet: We have cloned Drosophila CDK8 (dCDK8) and CycC (dCycC) into pGBKT7 and pGADT7, yeast two-hybrid vectors (Clontech, Mountain View, CA, USA), to investigate physically interactions between pGBKT7 and pGADT7 in yeast cells.

    Techniques:

    Hydrogen bond dynamics of human CDK8 A-loop during the 50-ns MD simulation

    Journal:

    Article Title: All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

    doi: 10.1016/j.compbiolchem.2014.03.003

    Figure Lengend Snippet: Hydrogen bond dynamics of human CDK8 A-loop during the 50-ns MD simulation

    Article Snippet: We have cloned Drosophila CDK8 (dCDK8) and CycC (dCycC) into pGBKT7 and pGADT7, yeast two-hybrid vectors (Clontech, Mountain View, CA, USA), to investigate physically interactions between pGBKT7 and pGADT7 in yeast cells.

    Techniques:

    Molecular dynamic simulation of CDK8-CycC complex

    Journal:

    Article Title: All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

    doi: 10.1016/j.compbiolchem.2014.03.003

    Figure Lengend Snippet: Molecular dynamic simulation of CDK8-CycC complex

    Article Snippet: We have cloned Drosophila CDK8 (dCDK8) and CycC (dCycC) into pGBKT7 and pGADT7, yeast two-hybrid vectors (Clontech, Mountain View, CA, USA), to investigate physically interactions between pGBKT7 and pGADT7 in yeast cells.

    Techniques:

    The sequence alignment of human and drosophila CDK8 and human CDK7 (PDB code: IUA2, A chain)

    Journal:

    Article Title: All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

    doi: 10.1016/j.compbiolchem.2014.03.003

    Figure Lengend Snippet: The sequence alignment of human and drosophila CDK8 and human CDK7 (PDB code: IUA2, A chain)

    Article Snippet: We have cloned Drosophila CDK8 (dCDK8) and CycC (dCycC) into pGBKT7 and pGADT7, yeast two-hybrid vectors (Clontech, Mountain View, CA, USA), to investigate physically interactions between pGBKT7 and pGADT7 in yeast cells.

    Techniques: Sequencing

    Endosomal acidification is necessary for Wg–DFz2 interaction and signaling. ( A–C ) Confocal images of wing discs, surface labeled on ice with A568 anti-Wg and A647 anti-DFz2 ( A ), or surface labeled and pulsed for 3–5 min followed

    Journal:

    Article Title: Endocytosis of Wingless via a dynamin-independent pathway is necessary for signaling in Drosophila wing discs

    doi: 10.1073/pnas.1610565113

    Figure Lengend Snippet: Endosomal acidification is necessary for Wg–DFz2 interaction and signaling. ( A–C ) Confocal images of wing discs, surface labeled on ice with A568 anti-Wg and A647 anti-DFz2 ( A ), or surface labeled and pulsed for 3–5 min followed

    Article Snippet: Monoclonal antibody (1A3G4) was raised against the N terminus of Drosophila DFz2 (22–114 aa; Custom Monoclonal Antibody Development, Genscript).

    Techniques: Labeling

    Wg signal transduction components are localized to endosomes (supplementary to ). ( A and B ) Confocal sections of control ( A ; C5GAL4/+) or C5GAL4 driving Vps34 RNAi ( B ) wing discs pulsed for 5 min and chased for 10 min to visualize Wg and DFz2 endosomes.

    Journal:

    Article Title: Endocytosis of Wingless via a dynamin-independent pathway is necessary for signaling in Drosophila wing discs

    doi: 10.1073/pnas.1610565113

    Figure Lengend Snippet: Wg signal transduction components are localized to endosomes (supplementary to ). ( A and B ) Confocal sections of control ( A ; C5GAL4/+) or C5GAL4 driving Vps34 RNAi ( B ) wing discs pulsed for 5 min and chased for 10 min to visualize Wg and DFz2 endosomes.

    Article Snippet: Monoclonal antibody (1A3G4) was raised against the N terminus of Drosophila DFz2 (22–114 aa; Custom Monoclonal Antibody Development, Genscript).

    Techniques: Transduction

    Extracellular distribution and endocytosis of Wg from the apical surface devoid of its signaling receptor, DFz2 (supplementary to ). ( A and B ) Characterization of 1A3G4 monoclonal antibody against extra cellular DFz2. Western blot ( A ) shows the

    Journal:

    Article Title: Endocytosis of Wingless via a dynamin-independent pathway is necessary for signaling in Drosophila wing discs

    doi: 10.1073/pnas.1610565113

    Figure Lengend Snippet: Extracellular distribution and endocytosis of Wg from the apical surface devoid of its signaling receptor, DFz2 (supplementary to ). ( A and B ) Characterization of 1A3G4 monoclonal antibody against extra cellular DFz2. Western blot ( A ) shows the

    Article Snippet: Monoclonal antibody (1A3G4) was raised against the N terminus of Drosophila DFz2 (22–114 aa; Custom Monoclonal Antibody Development, Genscript).

    Techniques: Western Blot

    Endosomal acidification is important for Wg–DFz2 interaction (supplementary to ). ( A ) Standard curve for endosomal pH showing the ratio of fluorescence of FITC/TMR from the ratiometric pH indicator (pH-sensitive FITC and pH-insensitive TMR

    Journal:

    Article Title: Endocytosis of Wingless via a dynamin-independent pathway is necessary for signaling in Drosophila wing discs

    doi: 10.1073/pnas.1610565113

    Figure Lengend Snippet: Endosomal acidification is important for Wg–DFz2 interaction (supplementary to ). ( A ) Standard curve for endosomal pH showing the ratio of fluorescence of FITC/TMR from the ratiometric pH indicator (pH-sensitive FITC and pH-insensitive TMR

    Article Snippet: Monoclonal antibody (1A3G4) was raised against the N terminus of Drosophila DFz2 (22–114 aa; Custom Monoclonal Antibody Development, Genscript).

    Techniques: Fluorescence

    Wg is endocytosed apically devoid of its signaling receptor, DFz2. ( A ) Endocytic assay: Surface distribution of Wg and DFz2 evaluated by incubating fluorescently labeled primary antibodies (Ab*) A568-anti-Wg, A647-anti-DFz2 on ice (step 1) ().

    Journal:

    Article Title: Endocytosis of Wingless via a dynamin-independent pathway is necessary for signaling in Drosophila wing discs

    doi: 10.1073/pnas.1610565113

    Figure Lengend Snippet: Wg is endocytosed apically devoid of its signaling receptor, DFz2. ( A ) Endocytic assay: Surface distribution of Wg and DFz2 evaluated by incubating fluorescently labeled primary antibodies (Ab*) A568-anti-Wg, A647-anti-DFz2 on ice (step 1) ().

    Article Snippet: Monoclonal antibody (1A3G4) was raised against the N terminus of Drosophila DFz2 (22–114 aa; Custom Monoclonal Antibody Development, Genscript).

    Techniques: Labeling

    Model for interaction of Wg and DFz2 in the wing disc Wg and DFz2 are internalized from different surfaces of the polarized wing epithelium via distinct endocytic pathways. Wg interacting with its putative receptor at the apical surface is directed toward

    Journal:

    Article Title: Endocytosis of Wingless via a dynamin-independent pathway is necessary for signaling in Drosophila wing discs

    doi: 10.1073/pnas.1610565113

    Figure Lengend Snippet: Model for interaction of Wg and DFz2 in the wing disc Wg and DFz2 are internalized from different surfaces of the polarized wing epithelium via distinct endocytic pathways. Wg interacting with its putative receptor at the apical surface is directed toward

    Article Snippet: Monoclonal antibody (1A3G4) was raised against the N terminus of Drosophila DFz2 (22–114 aa; Custom Monoclonal Antibody Development, Genscript).

    Techniques: