rhodamine-conjugated phalloidin Search Results


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  • 99
    Thermo Fisher rhodamine conjugated phalloidin
    The effects of LAMC1 siRNAs on cell adhesion and actin cytoskeleton in HUVECs. A: The expression level of LAMC1 protein was examined by western blot analysis. B: Cell adhesion rate were analyzed in different groups at 4h after subculture. C: HUVECs were stained with the Rhodamine-conjugated <t>phalloidin</t> (red) and FAK (green) at 48 h after transfection with NC siRNA or LAMC1 si-1. The representative images show alteration of actin cytoskeleton. Scale bar, 25 μm *p
    Rhodamine Conjugated Phalloidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4009 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore rhodamine conjugated phalloidin
    Recombinant moesin reconstitutes GTPγS- induced effects. Cells were permeabilized according to protocol 2, and incubated with 50 nM (saturating) recombinant moesin and 50 μM GTPγS ( a–f ); additionally, in c and d 0.1 nM C3 was present and in e and f 1 nM N17rac. The cells shown in g and h were incubated with 50 nM truncated moesin and 50 μM GTPγS. ( a , c , e , and g ) <t>Phalloidin</t> staining for F-actin; ( b , d , f , and h ) monoclonal antibody against vinculin. Bar, 30 μm.
    Rhodamine Conjugated Phalloidin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cytoskeleton Inc rhodamine conjugated phalloidin
    Functional mapping of tdc2-Gal4 neurons by targeted laser lesion. ( A ) Expression of nuclear LacZ in the tdc2-Gal4 neurons in the central nervous system of tdc2-Gal4/ UAS -nlsLacZ larvae. The tissue is counterstained with <t>phalloidin.</t> ( Inset )The distribution of tdc2-Gal4 neurons in three SOG compartments (SOG1–3). Five VUM1 and four VPM1 neurons (arrows) in the SOG1; six VUM2 and two VPM2 neurons (arrowheads) in the SOG2; five VUM3 and two VPM3 neurons (asterisks) in the SOG3. (Scale bar, 50 μm.) ( B ) Expression of TβH in the larval brain lobes and ventral nerve cord. No TβH-positive somata were detected in the brain lobes. (Scale bar, 50 μm.) ( C ) Expression of a nuclear GFP in the central nervous system of tdc2-Gal4/tsh-Gal80/UAS-nlsGFP larvae (lateral view). The GFP expression in the OA neurons from the thoracic and ventral ganglia is blocked by tsh-Gal80 . The numbers denote SOG1 to 3, respectively. (Scale bar, 50 μm.) ( D ) At 30 °C, fed larvae expressing tdc2-Gal4 /UAS - shi ts1 /tsh-Gal80 remained deficient in hunger-driven feeding response; ** P
    Rhodamine Conjugated Phalloidin, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 93/100, based on 203 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore tetramethyl rhodamine isothiocyanate tritc conjugated phalloidin
    Effect of ectopic expression of dominant negative mutants of p38MAPK, ROCK-1, and Smad4 on TGF-β1–induced actin reorganization. PC-3U cells were starved in 1% FBS for 12 h and stimulated with 10 ng/ml TGF-β1 for 30 min or 48 h before and after transient transfection with plasmids encoding flag-tagged DNp38MAPK, EGFP-tagged DNROCK-1, or flag-tagged DNSmad4. Filamentous actin was visualized by <t>TRITC-labeled</t> <t>phalloidin.</t> Flag-tagged proteins were visualized by a flag-specific antibody followed by FITC-labeled anti-mouse antibody. Bar, 20 μm.
    Tetramethyl Rhodamine Isothiocyanate Tritc Conjugated Phalloidin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rhodamine phalloidin conjugate
    Pten deletion in excitatory cortical neurons results in increased soma size. a Brain sections obtained from P0 wild type (WT) mice in the NEX- Pten colony were processed for immunofluorescence using Cre antibodies. A low magnification confocal image shows widespread expression in the cerebral cortex (CX) and hippocampus (HC), but not in the midbrain (MB) or cerebellum (CB). b Western blot analysis of the forebrain and cerebellum of newborn wild type (WT), heterozygous (Het), and homozygous (Hom) NEX- Pten littermates. The blot, which is representative of data obtained from 3 sets of littermates, was probed with Pten antibodies and then reprobed with actin antibodies to ensure equal protein loading. Pten expression is progressively reduced in the forebrain of heterozygous and homozygous mutants. c, d Pten immunohistochemistry staining (brown) of brain sections obtained from WT and Hom NEX- Pten littermates. Sections were counterstained with cresyl violet. Brightfield images of the upper cortical layers show that the majority of cells are Pten-positive in wild type and Pten-negative in homozygous mutants. Pten-negative cells appeared enlarged, and some protrude in the marginal zone near the pial surface. e, f Confocal images of representative dissociated cortical neurons cultured for 15 DIV and triple-labeled with <t>rhodamine-phalloidin</t> (red), Pten antibodies (green) and DAPI (blue). g Soma size comparison between Pten-positive ( n = 35) and Pten-negative cultured neurons ( n =22). The difference is statistically significant ( p
    Rhodamine Phalloidin Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 207 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore tetramethylrhodamine b isothiocyanate conjugated phalloidin
    The F-actin bundling activity of DdVASP is required for filopodium formation in vivo . ( a ) 3D reconstructions of <t>tetramethylrhodamine</t> B <t>isothiocyanate–phalloidin-labeled</t> knockout and reconstituted cells as indicated. (Scale bar: 5 μm.)
    Tetramethylrhodamine B Isothiocyanate Conjugated Phalloidin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Biotium rhodamine conjugated phalloidin
    PKA activation inhibits EphA2-dependent cell retraction. (A) Representative images of <t>phalloidin-labeled</t> PC3 cells stimulated for 12 min with 0.5 μg/ml control Fc or ephrin-A1 Fc or pretreated for 40 min with 20 μM forskolin or 200 μM of the PKA agonist 6-Benz-cAMP before ephrin-A1 Fc stimulation. Scale bar, 50 μm. (B) Histogram showing average cell areas ± SE under the different conditions (693 cells/condition from three experiments in each of which 77 cells/well from three wells were counted). **** p
    Rhodamine Conjugated Phalloidin, supplied by Biotium, used in various techniques. Bioz Stars score: 93/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology rhodamine conjugated phalloidin
    The in vitro assessments of FGM biocompatibility. Notes: ( A ) Cell metabolic activity measured by the Alamar Blue assay. ( B ) Vital cell density determined by a DAPI assay. ( C ) Immunofluorescence images of MSCs seeded on the control and PDLLA nanofiber-coated cell culture dishes after 1 day. F-actin was labeled by rhodamine-conjugated <t>phalloidin</t> (red), and nuclei was labeled by DAPI (blue). Magnification: 400×. Scale bar: 50 μm (* P
    Rhodamine Conjugated Phalloidin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore phalloidin conjugated to rhodamine
    ( A ) Portion of a kelch neo stage 10 egg chamber stained with rhodamine-conjugated <t>phalloidin</t> and examined by confocal microscopy. A number of striated actin bundles can be seen ( arrowheads ). Of interest here are the distorted regions characterized by angular intersections of relatively straight bundles ( arrows ). Similar distortions can be seen in wild-type egg chambers, especially where the actin bundles contact the nuclear envelope (e.g., Fig. 2 b ). This is a 3-μm optical section. ( B ) Thin-section through the portion of the nurse cell cytoplasm from a kelch neo stage 11 egg follicle. Of interest is that the cable appears broken. This occurs at the point of overlap of two modules. Bars: ( A ) 5 μm; ( B ) 0.1 μm.
    Phalloidin Conjugated To Rhodamine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Beyotime rhodamine conjugated phalloidin
    JARID2 significantly promotes invasion and metastasis of HCC cells in vitro and in vivo ( A ) Knockdown of JARID2 expression inhibited HCCLM3 ( A1 ) and MHCC97-H ( A2 ) cells migration, whereas overexpressed JARID2 in HepG2 cells promoted cell migration ( A3 ) in wound-healing assays. ( B ) The transwell assays showed that knockdown of JARID2 expression inhibited HCCLM3 ( B1 ) and MHCC97-H ( B2 ) cells invasion, but JARID2 overexpression enhanced HepG2 cells ( B3 ) invasion. ( C ) Immunofluorescence assays of cytoskeleton of HCCLM3 control and HCCLM3 shJARID2-1 ( C1 ), MHCC97-H control and MHCC97-H shJARID2-1 ( C2 ), HepG2 Vector and HepG2 JARID2 cells ( C3 ). F-actin filaments were visualized in cells using <t>rhodamine-phalloidin.</t> ( D ) The HCC metastatic mouse model was constructed by using HCCLM3 and MHCC97-H cells transfected with shJARID2-1 or control vector, and HepG2 cells transfected with JARID2 or control vector as described in the Materials and Methods. The size of liver tumors in each group was calculated and compared in HCCLM3 ( D1 ), MHCC97-H ( D2 ) and HepG2 ( D3 ) cells. Scale bar, 1 cm. ( E ) Representative pictures for intrahepatic ( E1 ) and lung metastasis ( E2 ). ( F ) The number of metastatic nodules per liver or lung was calculated and compared between HCCLM3 control and HCCLM3 shJARID2-1 ( F1 ), MHCC97-H control and MHCC97-H shJARID2-1 ( F2 ), HepG2 Vector and HepG2 JARID2 cells group ( F3 , F4 ). * P
    Rhodamine Conjugated Phalloidin, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific rhodamine conjugated phalloidin
    Organization of the serotonin-like immunoreactive nervous system in a 24-day-old larva. Micrographs of a live animal (A), Z-projections (B-I) of larvae after mono- and double staining with antibodies against 5-HT (serotonin) (green), as well as staining with <t>phalloidin</t> (grey), and Hoechst (violet), and 3-D reconstructions (J-K). ( A ) General view of a live larva showing the apical plate (ap), the border of the preoral coelom (bc1), vestibulum (v), esophagus (eso), stomach (st), midgut (mg), proctodaeum (pr), the border of the trunk coelom (bc3), tentacle (t), telotroch (tt), and primordium of the metasomal sac (pms). Lateral view; apical is to the top; ventral is to the right. ( B ) Nervous system with all major elements: apical organ (ao), median neurite bundle (men), frontal organ (fo), oral nerve ring (or), tentacular neurite bundle (tn) with dorsal commissure (dc), and nerve ring of the telotroch (nt). Lateral view; apical is to the top, ventral to the right. ( C ) Dorsal view of branched proximal ends of the tentacular neurite bundle (br) and apical organ, which is composed of monopolar (mo) and bipolar or multipolar perikarya. The latter forms the larger group on the left (lgn), which rises above the neuropil (np). Anterior is to the upper right. ( D ) Overview of the musculature and serotonin-like immunoreactive nervous system showing apical organ (ao), tentacular neurite bundle (tn) which branches (br) on the dorsal side, thin trunk neurites (trn), and nerve ring of the telotroch (nt). Ventral view; apical is to the top; preoral lobe bends backward. ( E ) Dorso-lateral view of the preoral lobe showing apical organ (ao), median neurite bundle (men), and branches (br) of the tentacular neurite bundle (tn). Anterior is to the right. ( F ) Left portion of the apical organ showing monopolar perikarya (mo) and the left group of bipolar or multipolar perikarya (lgn), which gives rise to the left branch of the tentacular neurite bundle (ltn) and median neurite bundle (mn) of the preoral lobe. Lateral view; apical is to the top; anterior of the preoral lobe is to the right. ( G ) Details of a monopolar perikaryon; lateral view showing perikaryon (mo) and basal projection (bp) that passes from the median part of the cell to the neuropil (np). ( H ) Lateral view of the preoral lobe showing anterior part of the apical organ (ao), median neurite bundle (mn), and frontal organ (fo), which is composed of several weakly stained perikarya. Anterior of the preoral lobe is to the right. ( I ) Dorsal commissure (dc) between two dorso-lateral branches of left (ltn) and right (rtn) branches of the tentacular neurite bundle. Dorsal view; apical is to the top. ( J ) Posterior view of the apical organ that is composed of two groups of bipolar or multipolar perikarya (light blue), which give rise to two dorso-lateral branches of the tentacular neurite bundle (light pink) and median neurite bundle (magenta). Monopolar perikarya form the upper large neuropil (golden). Anterior is to the right; left is to the bottom. ( K ) Top view of the apical organ showing large neuropil and monopolar perikarya (golden), neuropil and bipolar or multipolar perikarya (light blue), tentacular neurite bundle (light pink), median neurite bundle (magenta), and oral nerve ring (pale blue).
    Rhodamine Conjugated Phalloidin, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The effects of LAMC1 siRNAs on cell adhesion and actin cytoskeleton in HUVECs. A: The expression level of LAMC1 protein was examined by western blot analysis. B: Cell adhesion rate were analyzed in different groups at 4h after subculture. C: HUVECs were stained with the Rhodamine-conjugated phalloidin (red) and FAK (green) at 48 h after transfection with NC siRNA or LAMC1 si-1. The representative images show alteration of actin cytoskeleton. Scale bar, 25 μm *p

    Journal: Chinese Journal of Traumatology

    Article Title: The subsequent biological effects of simulated microgravity on endothelial cell growth in HUVECs

    doi: 10.1016/j.cjtee.2018.04.004

    Figure Lengend Snippet: The effects of LAMC1 siRNAs on cell adhesion and actin cytoskeleton in HUVECs. A: The expression level of LAMC1 protein was examined by western blot analysis. B: Cell adhesion rate were analyzed in different groups at 4h after subculture. C: HUVECs were stained with the Rhodamine-conjugated phalloidin (red) and FAK (green) at 48 h after transfection with NC siRNA or LAMC1 si-1. The representative images show alteration of actin cytoskeleton. Scale bar, 25 μm *p

    Article Snippet: F-actin was labeled with the Rhodamine-conjugated phalloidin (Invitrogen).

    Techniques: Expressing, Western Blot, Staining, Transfection

    Cellular changes during subsequent culturing after SMG treatment. A: Cell growth curve. B: Cell adhesion rate. C: Actin stress fibers were stained with the Rhodamine-conjugated phalloidin. Scale bar, 25 μm. D: Number of actin stress fibers was presented. *p

    Journal: Chinese Journal of Traumatology

    Article Title: The subsequent biological effects of simulated microgravity on endothelial cell growth in HUVECs

    doi: 10.1016/j.cjtee.2018.04.004

    Figure Lengend Snippet: Cellular changes during subsequent culturing after SMG treatment. A: Cell growth curve. B: Cell adhesion rate. C: Actin stress fibers were stained with the Rhodamine-conjugated phalloidin. Scale bar, 25 μm. D: Number of actin stress fibers was presented. *p

    Article Snippet: F-actin was labeled with the Rhodamine-conjugated phalloidin (Invitrogen).

    Techniques: Staining

    P-cadherin signaling in response to laminin involves FAK/Src activation Analysis of integrin downstream signaling molecules in breast cancer cell lines was performed after adhesion to laminin-332 (20 min for MDA-MB-468 and 30 min for BT-20) (A); The number of stress fibers (F-actin was stained with phalloidin-rhodamine) and focal adhesions/contacts (stained with pFAK Tyr397 – Alexa 488) is reduced by P-cadherin knockdown in MDAB-MB-468 cells grown on top of laminin (B). The same result was found for BT-20 cell line.

    Journal: Oncotarget

    Article Title: P-cadherin signals through the laminin receptor ?6?4 integrin to induce stem cell and invasive properties in basal-like breast cancer cells

    doi:

    Figure Lengend Snippet: P-cadherin signaling in response to laminin involves FAK/Src activation Analysis of integrin downstream signaling molecules in breast cancer cell lines was performed after adhesion to laminin-332 (20 min for MDA-MB-468 and 30 min for BT-20) (A); The number of stress fibers (F-actin was stained with phalloidin-rhodamine) and focal adhesions/contacts (stained with pFAK Tyr397 – Alexa 488) is reduced by P-cadherin knockdown in MDAB-MB-468 cells grown on top of laminin (B). The same result was found for BT-20 cell line.

    Article Snippet: F-actin was detected by staining with phalloidin conjugated to rhodamine (Invitrogen) at a dilution of 1:1000.

    Techniques: Activation Assay, Multiple Displacement Amplification, Staining

    a , b Quantification (branch length and number of intersection points) of capillary-like structure formation in A549 ( a ) and H1299 ( b ) cells plated on matrigel and treated with 20 μM CPTH6 for 18 h. The results represent the average ± SD of two independent experiments. c Representative images of immunofluorescence conducted in A549 and H1299 cells control or treated with 50 μM CPTH6 for 24 h using rhodamine-conjugated phalloidin to visualize F-actin fibers or for vinculin expression and localization. Blue fluorescence represents DAPI stained nuclei. d Representative images and relative quantification of migration of A549 cells control or treated with 50 μM CPTH6 for 48 h and subjected to time-lapse videorecording. The images were recorded every 15 min, and were taken at the starting point (0 h), after 24 and 36 h. The red outlines show the gap area. The migration rates of two different conditions (control and CPTH6) were determined as the percentage of wound closure or the percentage of area reduction. Magnification 10X. Scale bar, 100 μm. a , b , d p values were calculated between control and CPTH6-treated cells. * p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Inhibition of lysine acetyltransferases impairs tumor angiogenesis acting on both endothelial and tumor cells

    doi: 10.1186/s13046-020-01604-z

    Figure Lengend Snippet: a , b Quantification (branch length and number of intersection points) of capillary-like structure formation in A549 ( a ) and H1299 ( b ) cells plated on matrigel and treated with 20 μM CPTH6 for 18 h. The results represent the average ± SD of two independent experiments. c Representative images of immunofluorescence conducted in A549 and H1299 cells control or treated with 50 μM CPTH6 for 24 h using rhodamine-conjugated phalloidin to visualize F-actin fibers or for vinculin expression and localization. Blue fluorescence represents DAPI stained nuclei. d Representative images and relative quantification of migration of A549 cells control or treated with 50 μM CPTH6 for 48 h and subjected to time-lapse videorecording. The images were recorded every 15 min, and were taken at the starting point (0 h), after 24 and 36 h. The red outlines show the gap area. The migration rates of two different conditions (control and CPTH6) were determined as the percentage of wound closure or the percentage of area reduction. Magnification 10X. Scale bar, 100 μm. a , b , d p values were calculated between control and CPTH6-treated cells. * p

    Article Snippet: For analysis of F-actin filament, cells were incubated with phalloidin conjugated-rhodamine (Thermo Fisher Scientific).

    Techniques: Immunofluorescence, Expressing, Fluorescence, Staining, Migration

    Cadm1 ablation enhances bone-resorbing activity of osteoclasts. (A) BMMs from wild-type or Cadm1 KO mouse were cultured in the presence of M-CSF (50 ng/ml) and RANKL (25 ng/ml) for 4 days, stained with rhodamine phalloidin to visualize actin protein, and observed under a fluorescence microscope. Bar, 100 μm. (B) Whole cell lysates from wild-type or Cadm1 KO osteoclasts were subjected to western blotting using antibodies against FAK, Pyk2 and Src. Actin protein expression served as an internal control. Protein levels relative to actin were quantified by densitometry and are shown below.

    Journal: PLoS ONE

    Article Title: Negative feedback loop of bone resorption by NFATc1-dependent induction of Cadm1

    doi: 10.1371/journal.pone.0175632

    Figure Lengend Snippet: Cadm1 ablation enhances bone-resorbing activity of osteoclasts. (A) BMMs from wild-type or Cadm1 KO mouse were cultured in the presence of M-CSF (50 ng/ml) and RANKL (25 ng/ml) for 4 days, stained with rhodamine phalloidin to visualize actin protein, and observed under a fluorescence microscope. Bar, 100 μm. (B) Whole cell lysates from wild-type or Cadm1 KO osteoclasts were subjected to western blotting using antibodies against FAK, Pyk2 and Src. Actin protein expression served as an internal control. Protein levels relative to actin were quantified by densitometry and are shown below.

    Article Snippet: To detect actin rings, cells were incubated for 30 min with rhodamine-conjugated phalloidin solution (Molecular Probes, Inc., Eugene, OR, USA) and observed under a fluorescence microscope (BZ-8100, Keyence).

    Techniques: Activity Assay, Cell Culture, Staining, Fluorescence, Microscopy, Western Blot, Expressing

    Eye morphogenesis in Tribolium eyg knockdown pupae . (a, b) Confocal images of lateral head of phalloidin labeled 24 h old Tribolium pupa of wild type (a) and eyg knockdown (b) specimens. Numbers indicate vertical columns of differentiating ommatidial precursor clusters. Arrowheads point at midline area of the anterior differentiating retina. ant = antenna, gen = gena.

    Journal: EvoDevo

    Article Title: The Pax gene eyegone facilitates repression of eye development in Tribolium

    doi: 10.1186/2041-9139-2-8

    Figure Lengend Snippet: Eye morphogenesis in Tribolium eyg knockdown pupae . (a, b) Confocal images of lateral head of phalloidin labeled 24 h old Tribolium pupa of wild type (a) and eyg knockdown (b) specimens. Numbers indicate vertical columns of differentiating ommatidial precursor clusters. Arrowheads point at midline area of the anterior differentiating retina. ant = antenna, gen = gena.

    Article Snippet: Fixed tissues were washed three times in PBT followed by overnight incubation at 4°C in PBT containing rhodamin-conjugated phalloidin (Molecular Probes, Eugene, Oregon, USA) at 1:30 dilution.

    Techniques: Labeling

    Suppressive effects of OPG on F-actin ring formation in duck embryo osteoclasts. Duck embryo bone marrow cells were suspended in α-MEM with FBS (v/v 10%) and then seeded in 12-well culture plates. After incubating for 24 h, the α-MEM was replaced with serum-free medium containing M-CSF (25 ng/mL) and RANKL (30 ng/mL). The cells were cultured for up to 5 days. Next, 0, 10, 20, 50, and 100 ng/mL OPG were added to the different groups of cells in the presence of M-CSF and RANKL. The cells were incubated for another 3 days. At the end of the cultivation period, the cells were fixed and stained for TRITC-phalloidin. The F-actin rings were observed using an inverted phase contrast fluorescence microscope with appropriate filters. (A) control, (B) 10 ng/mL OPG, (C) 20 ng/mL OPG, (D) 50 ng/mL OPG, (E) 100 ng/mL OPG. ×400. Scale bars = 100 µm (A~E).

    Journal: Journal of Veterinary Science

    Article Title: Inhibitory effects of osteoprotegerin on osteoclast formation and function under serum-free conditions

    doi: 10.4142/jvs.2013.14.4.405

    Figure Lengend Snippet: Suppressive effects of OPG on F-actin ring formation in duck embryo osteoclasts. Duck embryo bone marrow cells were suspended in α-MEM with FBS (v/v 10%) and then seeded in 12-well culture plates. After incubating for 24 h, the α-MEM was replaced with serum-free medium containing M-CSF (25 ng/mL) and RANKL (30 ng/mL). The cells were cultured for up to 5 days. Next, 0, 10, 20, 50, and 100 ng/mL OPG were added to the different groups of cells in the presence of M-CSF and RANKL. The cells were incubated for another 3 days. At the end of the cultivation period, the cells were fixed and stained for TRITC-phalloidin. The F-actin rings were observed using an inverted phase contrast fluorescence microscope with appropriate filters. (A) control, (B) 10 ng/mL OPG, (C) 20 ng/mL OPG, (D) 50 ng/mL OPG, (E) 100 ng/mL OPG. ×400. Scale bars = 100 µm (A~E).

    Article Snippet: F-actin staining To visualize the actin cytoskeleton, tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin (Invitrogen, USA) was used.

    Techniques: Cell Culture, Incubation, Staining, Fluorescence, Microscopy

    Gomesin accumulated on the cell membrane and permeabilized B16F10-Nex2 tumor cells. Cells were cultivated on round glass coverslips, treated with 5 µM Gm for 10 minutes, fixed, and incubated with anti- Gm polyclonal monospecific antibody (B), or murine antitubulin antibody (C), both revealed with FITC-conjugated secondary antibodies (green). The fluorescence was analyzed by confocal microscopy as described in Materials and Methods. Red , phalloidin-rhodamine; blue , DAPI staining. (A) Single optical section through control cells treated with phalloidin-rhodamine, DAPI, and antitubulin in the absence of Gm ; (B, C) maximum pixel value projections of serial optical sections. Scale bars, 20 µm.

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

    Article Title: Effective Topical Treatment of Subcutaneous Murine B16F10-Nex2 Melanoma By the Antimicrobial Peptide Gomesin 1

    doi:

    Figure Lengend Snippet: Gomesin accumulated on the cell membrane and permeabilized B16F10-Nex2 tumor cells. Cells were cultivated on round glass coverslips, treated with 5 µM Gm for 10 minutes, fixed, and incubated with anti- Gm polyclonal monospecific antibody (B), or murine antitubulin antibody (C), both revealed with FITC-conjugated secondary antibodies (green). The fluorescence was analyzed by confocal microscopy as described in Materials and Methods. Red , phalloidin-rhodamine; blue , DAPI staining. (A) Single optical section through control cells treated with phalloidin-rhodamine, DAPI, and antitubulin in the absence of Gm ; (B, C) maximum pixel value projections of serial optical sections. Scale bars, 20 µm.

    Article Snippet: Staining of actin filaments and nuclei were performed with 0.3 µg/ml phalloidin-rhodamine conjugate (Invitrogen) and 50 µg/ml DAPI (Invitrogen), respectively, for 1 hour at room temperature.

    Techniques: Incubation, Fluorescence, Confocal Microscopy, Staining

    Recombinant moesin reconstitutes GTPγS- induced effects. Cells were permeabilized according to protocol 2, and incubated with 50 nM (saturating) recombinant moesin and 50 μM GTPγS ( a–f ); additionally, in c and d 0.1 nM C3 was present and in e and f 1 nM N17rac. The cells shown in g and h were incubated with 50 nM truncated moesin and 50 μM GTPγS. ( a , c , e , and g ) Phalloidin staining for F-actin; ( b , d , f , and h ) monoclonal antibody against vinculin. Bar, 30 μm.

    Journal: The Journal of Cell Biology

    Article Title: Rho- and Rac-dependent Assembly of Focal Adhesion Complexes and Actin Filaments in Permeabilized Fibroblasts: An Essential Role for Ezrin/Radixin/Moesin Proteins

    doi:

    Figure Lengend Snippet: Recombinant moesin reconstitutes GTPγS- induced effects. Cells were permeabilized according to protocol 2, and incubated with 50 nM (saturating) recombinant moesin and 50 μM GTPγS ( a–f ); additionally, in c and d 0.1 nM C3 was present and in e and f 1 nM N17rac. The cells shown in g and h were incubated with 50 nM truncated moesin and 50 μM GTPγS. ( a , c , e , and g ) Phalloidin staining for F-actin; ( b , d , f , and h ) monoclonal antibody against vinculin. Bar, 30 μm.

    Article Snippet: The final antibody treatment also contained rhodamine-conjugated phalloidin and Hoechst dye 33342 (both at 0.1 μg/ml; Sigma Chemical Co. ).

    Techniques: Recombinant, Incubation, Staining

    Recombinant Rho stimulates formation of focal adhesions and stress fibers in permeabilized cells. Cells were permeabilized (protocol 1) in the presence of 1 μM recombinant V14Rho. ( a ) Phalloidin staining for F-actin; ( b ) monoclonal antibody against vinculin. Bar, 30 μm.

    Journal: The Journal of Cell Biology

    Article Title: Rho- and Rac-dependent Assembly of Focal Adhesion Complexes and Actin Filaments in Permeabilized Fibroblasts: An Essential Role for Ezrin/Radixin/Moesin Proteins

    doi:

    Figure Lengend Snippet: Recombinant Rho stimulates formation of focal adhesions and stress fibers in permeabilized cells. Cells were permeabilized (protocol 1) in the presence of 1 μM recombinant V14Rho. ( a ) Phalloidin staining for F-actin; ( b ) monoclonal antibody against vinculin. Bar, 30 μm.

    Article Snippet: The final antibody treatment also contained rhodamine-conjugated phalloidin and Hoechst dye 33342 (both at 0.1 μg/ml; Sigma Chemical Co. ).

    Techniques: Recombinant, Staining

    Reconstitution of Rho-induced effects after extended permeabilization using a pig brain cytosolic extract. Cells were permeabilized according to protocol 2 (i.e., permeabilized for 6 min in the presence of digitonin before addition of stimulus; see Materials and Methods) in the presence of ( a and b ) 25 μg/ml V14Rho, ( c and d ) 2 mg/ml pig brain extract, ( e and f ) 25 μg/ml V14Rho plus 2 mg/ml pig brain extract. F-actin in the permeabilized cells was visualized using rhodamine-conjugated phalloidin ( a , c , and e ) and focal adhesions with anti-vinculin antiserum ( b , d , and f ). Bar, 30 μm.

    Journal: The Journal of Cell Biology

    Article Title: Rho- and Rac-dependent Assembly of Focal Adhesion Complexes and Actin Filaments in Permeabilized Fibroblasts: An Essential Role for Ezrin/Radixin/Moesin Proteins

    doi:

    Figure Lengend Snippet: Reconstitution of Rho-induced effects after extended permeabilization using a pig brain cytosolic extract. Cells were permeabilized according to protocol 2 (i.e., permeabilized for 6 min in the presence of digitonin before addition of stimulus; see Materials and Methods) in the presence of ( a and b ) 25 μg/ml V14Rho, ( c and d ) 2 mg/ml pig brain extract, ( e and f ) 25 μg/ml V14Rho plus 2 mg/ml pig brain extract. F-actin in the permeabilized cells was visualized using rhodamine-conjugated phalloidin ( a , c , and e ) and focal adhesions with anti-vinculin antiserum ( b , d , and f ). Bar, 30 μm.

    Article Snippet: The final antibody treatment also contained rhodamine-conjugated phalloidin and Hoechst dye 33342 (both at 0.1 μg/ml; Sigma Chemical Co. ).

    Techniques:

    Permeabilization of quiescent Swiss 3T3 cells in the presence of GTPγS. ( A ) Permeabilization (protocol 1) was performed in the absence of stimulus ( left ) or in the presence of 50 μM GTPγS ( right ). Cellular F-actin was visualized using rhodamine-conjugated phalloidin. ( B ) Permeabilization (protocol 1) was performed in the absence of stimulus ( a and b ), in the presence of 50 μM GTPγS ( c and d ); 50 μM GTPγS with 0.1 nM C3 transferase ( e and f ); 50 μM GTPγS with 1 nM N17Rac ( g and h ). After 20 min at 37°C, cells were fixed and F-actin visualized with rhodamine-phalloidin ( a , c , e , and g ) and vinculin visualized with a monoclonal antibody ( b , d , f , and h ). In c and d , arrowheads show termini of bundled actin filaments decorated with focal adhesions, while arrows mark regions of peripheral actin polymerization decorated with linear arrays of focal complexes. Bars: ( A ) 150 μm; ( B ) 30 μm.

    Journal: The Journal of Cell Biology

    Article Title: Rho- and Rac-dependent Assembly of Focal Adhesion Complexes and Actin Filaments in Permeabilized Fibroblasts: An Essential Role for Ezrin/Radixin/Moesin Proteins

    doi:

    Figure Lengend Snippet: Permeabilization of quiescent Swiss 3T3 cells in the presence of GTPγS. ( A ) Permeabilization (protocol 1) was performed in the absence of stimulus ( left ) or in the presence of 50 μM GTPγS ( right ). Cellular F-actin was visualized using rhodamine-conjugated phalloidin. ( B ) Permeabilization (protocol 1) was performed in the absence of stimulus ( a and b ), in the presence of 50 μM GTPγS ( c and d ); 50 μM GTPγS with 0.1 nM C3 transferase ( e and f ); 50 μM GTPγS with 1 nM N17Rac ( g and h ). After 20 min at 37°C, cells were fixed and F-actin visualized with rhodamine-phalloidin ( a , c , e , and g ) and vinculin visualized with a monoclonal antibody ( b , d , f , and h ). In c and d , arrowheads show termini of bundled actin filaments decorated with focal adhesions, while arrows mark regions of peripheral actin polymerization decorated with linear arrays of focal complexes. Bars: ( A ) 150 μm; ( B ) 30 μm.

    Article Snippet: The final antibody treatment also contained rhodamine-conjugated phalloidin and Hoechst dye 33342 (both at 0.1 μg/ml; Sigma Chemical Co. ).

    Techniques:

    Functional mapping of tdc2-Gal4 neurons by targeted laser lesion. ( A ) Expression of nuclear LacZ in the tdc2-Gal4 neurons in the central nervous system of tdc2-Gal4/ UAS -nlsLacZ larvae. The tissue is counterstained with phalloidin. ( Inset )The distribution of tdc2-Gal4 neurons in three SOG compartments (SOG1–3). Five VUM1 and four VPM1 neurons (arrows) in the SOG1; six VUM2 and two VPM2 neurons (arrowheads) in the SOG2; five VUM3 and two VPM3 neurons (asterisks) in the SOG3. (Scale bar, 50 μm.) ( B ) Expression of TβH in the larval brain lobes and ventral nerve cord. No TβH-positive somata were detected in the brain lobes. (Scale bar, 50 μm.) ( C ) Expression of a nuclear GFP in the central nervous system of tdc2-Gal4/tsh-Gal80/UAS-nlsGFP larvae (lateral view). The GFP expression in the OA neurons from the thoracic and ventral ganglia is blocked by tsh-Gal80 . The numbers denote SOG1 to 3, respectively. (Scale bar, 50 μm.) ( D ) At 30 °C, fed larvae expressing tdc2-Gal4 /UAS - shi ts1 /tsh-Gal80 remained deficient in hunger-driven feeding response; ** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Octopamine-mediated circuit mechanism underlying controlled appetite for palatable food in Drosophila

    doi: 10.1073/pnas.1308816110

    Figure Lengend Snippet: Functional mapping of tdc2-Gal4 neurons by targeted laser lesion. ( A ) Expression of nuclear LacZ in the tdc2-Gal4 neurons in the central nervous system of tdc2-Gal4/ UAS -nlsLacZ larvae. The tissue is counterstained with phalloidin. ( Inset )The distribution of tdc2-Gal4 neurons in three SOG compartments (SOG1–3). Five VUM1 and four VPM1 neurons (arrows) in the SOG1; six VUM2 and two VPM2 neurons (arrowheads) in the SOG2; five VUM3 and two VPM3 neurons (asterisks) in the SOG3. (Scale bar, 50 μm.) ( B ) Expression of TβH in the larval brain lobes and ventral nerve cord. No TβH-positive somata were detected in the brain lobes. (Scale bar, 50 μm.) ( C ) Expression of a nuclear GFP in the central nervous system of tdc2-Gal4/tsh-Gal80/UAS-nlsGFP larvae (lateral view). The GFP expression in the OA neurons from the thoracic and ventral ganglia is blocked by tsh-Gal80 . The numbers denote SOG1 to 3, respectively. (Scale bar, 50 μm.) ( D ) At 30 °C, fed larvae expressing tdc2-Gal4 /UAS - shi ts1 /tsh-Gal80 remained deficient in hunger-driven feeding response; ** P

    Article Snippet: Brains from larvae 76 h after egg lay were dissected out and the immunostaining were performed as previously described ( ) by using Chicken anti-GFP (1:1,000; Invitrogen), rabbit anti-ßgal (1:1,000; Promega), rabbit anti-tyromine-β-hydroxylase (1:500; gift from Vivian Budnik, University of Massachusetts Medical School, Worcester, MA), Alexa 488-goat anti-chicken (1:2,000; Invitrogen), Alexa Fluor-568 goat anti-rabbit (1:2,000; Invitrogen), and rhodamine conjugated phalloidin (1:1,000; Cytoskeleton).

    Techniques: Functional Assay, Expressing

    Effect of ectopic expression of dominant negative mutants of p38MAPK, ROCK-1, and Smad4 on TGF-β1–induced actin reorganization. PC-3U cells were starved in 1% FBS for 12 h and stimulated with 10 ng/ml TGF-β1 for 30 min or 48 h before and after transient transfection with plasmids encoding flag-tagged DNp38MAPK, EGFP-tagged DNROCK-1, or flag-tagged DNSmad4. Filamentous actin was visualized by TRITC-labeled phalloidin. Flag-tagged proteins were visualized by a flag-specific antibody followed by FITC-labeled anti-mouse antibody. Bar, 20 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Transforming Growth Factor-?-induced Mobilization of Actin Cytoskeleton Requires Signaling by Small GTPases Cdc42 and RhoA

    doi: 10.1091/mbc.01-08-0398

    Figure Lengend Snippet: Effect of ectopic expression of dominant negative mutants of p38MAPK, ROCK-1, and Smad4 on TGF-β1–induced actin reorganization. PC-3U cells were starved in 1% FBS for 12 h and stimulated with 10 ng/ml TGF-β1 for 30 min or 48 h before and after transient transfection with plasmids encoding flag-tagged DNp38MAPK, EGFP-tagged DNROCK-1, or flag-tagged DNSmad4. Filamentous actin was visualized by TRITC-labeled phalloidin. Flag-tagged proteins were visualized by a flag-specific antibody followed by FITC-labeled anti-mouse antibody. Bar, 20 μm.

    Article Snippet: Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin was from Sigma Chemical.

    Techniques: Expressing, Dominant Negative Mutation, Transfection, Labeling

    TGF-β1–induced actin reorganization and membrane ruffling in PC-3U cells. PC-3U cells were starved in 1% FBS for 12 h and stimulated with 10 ng/ml TGF-β1 for 5, 15, 30, and 60 min (A) and 2, 12, 24, and 48 h (B). Filamentous actin was visualized by TRITC-labeled phalloidin. Bar, 20 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Transforming Growth Factor-?-induced Mobilization of Actin Cytoskeleton Requires Signaling by Small GTPases Cdc42 and RhoA

    doi: 10.1091/mbc.01-08-0398

    Figure Lengend Snippet: TGF-β1–induced actin reorganization and membrane ruffling in PC-3U cells. PC-3U cells were starved in 1% FBS for 12 h and stimulated with 10 ng/ml TGF-β1 for 5, 15, 30, and 60 min (A) and 2, 12, 24, and 48 h (B). Filamentous actin was visualized by TRITC-labeled phalloidin. Bar, 20 μm.

    Article Snippet: Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin was from Sigma Chemical.

    Techniques: Labeling

    TGF-β1–induced actin reorganization: dependence on protein synthesis and comparison with the effect of HGF. PC-3U cells were starved in 1% FBS for 12 h and stimulated with 10 ng/ml TGF-β1 for 30 min or 48 h (A), 25 ng/ml HGF for 30 min or 48 h (B), or pretreated with 10 μg/ml cycloheximide for 1 h before TGF-β1 stimulation (C). Filamentous actin was visualized by TRITC-labeled phalloidin. Bar, 20 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Transforming Growth Factor-?-induced Mobilization of Actin Cytoskeleton Requires Signaling by Small GTPases Cdc42 and RhoA

    doi: 10.1091/mbc.01-08-0398

    Figure Lengend Snippet: TGF-β1–induced actin reorganization: dependence on protein synthesis and comparison with the effect of HGF. PC-3U cells were starved in 1% FBS for 12 h and stimulated with 10 ng/ml TGF-β1 for 30 min or 48 h (A), 25 ng/ml HGF for 30 min or 48 h (B), or pretreated with 10 μg/ml cycloheximide for 1 h before TGF-β1 stimulation (C). Filamentous actin was visualized by TRITC-labeled phalloidin. Bar, 20 μm.

    Article Snippet: Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin was from Sigma Chemical.

    Techniques: Labeling

    Effect of ectopic expression of dominant negative Rac1, Cdc42, and RhoA on TGF-β1–induced actin reorganization. PC-3U cells were starved in 1% FBS for 12 h and stimulated with 10 ng/ml TGF-β1 for 30 min or 48 h before and after transient transfection with pRK5mycN17Rac1, pRK5mycN17Cdc42, or pRK5mycN19RhoA. Filamentous actin was visualized by TRITC-labeled phalloidin. Myc-tagged GTPases were visualized by a myc-specific antibody followed by FITC-labeled anti-mouse antibody. Bar, 20 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Transforming Growth Factor-?-induced Mobilization of Actin Cytoskeleton Requires Signaling by Small GTPases Cdc42 and RhoA

    doi: 10.1091/mbc.01-08-0398

    Figure Lengend Snippet: Effect of ectopic expression of dominant negative Rac1, Cdc42, and RhoA on TGF-β1–induced actin reorganization. PC-3U cells were starved in 1% FBS for 12 h and stimulated with 10 ng/ml TGF-β1 for 30 min or 48 h before and after transient transfection with pRK5mycN17Rac1, pRK5mycN17Cdc42, or pRK5mycN19RhoA. Filamentous actin was visualized by TRITC-labeled phalloidin. Myc-tagged GTPases were visualized by a myc-specific antibody followed by FITC-labeled anti-mouse antibody. Bar, 20 μm.

    Article Snippet: Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin was from Sigma Chemical.

    Techniques: Expressing, Dominant Negative Mutation, Transfection, Labeling

    TGF-β1–induced actin reorganization and membrane ruffling in RBL-2H3 cells. (A) RBL-2H3 cells were serum-starved for 12 h and treated with 25 ng/ml TGF-β1 for 5, 15, 30, and 60 min. FcεRI activation was induced by addition of the cross-linking antibody BC4. (B) Monoclonal mouse anti-TGF-β1-3 neutralizing antibody was added to RBL-2H3 cells at a concentration of 25 μg/ml 1 h before stimulation with 25 ng/ml TGF-β1 for 15 min. Filamentous actin was visualized by TRITC-labeled phalloidin. Bar, 20 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Transforming Growth Factor-?-induced Mobilization of Actin Cytoskeleton Requires Signaling by Small GTPases Cdc42 and RhoA

    doi: 10.1091/mbc.01-08-0398

    Figure Lengend Snippet: TGF-β1–induced actin reorganization and membrane ruffling in RBL-2H3 cells. (A) RBL-2H3 cells were serum-starved for 12 h and treated with 25 ng/ml TGF-β1 for 5, 15, 30, and 60 min. FcεRI activation was induced by addition of the cross-linking antibody BC4. (B) Monoclonal mouse anti-TGF-β1-3 neutralizing antibody was added to RBL-2H3 cells at a concentration of 25 μg/ml 1 h before stimulation with 25 ng/ml TGF-β1 for 15 min. Filamentous actin was visualized by TRITC-labeled phalloidin. Bar, 20 μm.

    Article Snippet: Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin was from Sigma Chemical.

    Techniques: Activation Assay, Concentration Assay, Labeling

    HGF blocks TGF-β1-induced morphological transformation, F-actin, and vimentin reorganization in renal interstitial fibroblast NRK-49F cells. NRK-49F cells were incubated without (control) ( A , E , I ) or with 2 ng/ml of TGF-β1 ( B , F , J ), 40 ng/ml of HGF ( C , G , K ), or both ( D , H , L ) for 2 days. A to D: TGF-β1 induced morphological transformation of NRK-49F cells into a myofibroblastic appearance ( B ). HGF blocked this transformation ( D ). E to L: Representative micrographs of tetramethyl-rhodamine isothiocyanate-conjugated phalloidin staining ( E–H ) and vimentin staining ( I–L ) showing F-actin and vimentin reorganization in NRK-49F cells induced by TGF-β1 ( F , J ). HGF primarily abolished TGF-β1-induced actin and vimentin reorganization ( H , L ). Scale bar, 20 μm.

    Journal: The American Journal of Pathology

    Article Title: Hepatocyte Growth Factor Suppresses Renal Interstitial Myofibroblast Activation and Intercepts Smad Signal Transduction

    doi:

    Figure Lengend Snippet: HGF blocks TGF-β1-induced morphological transformation, F-actin, and vimentin reorganization in renal interstitial fibroblast NRK-49F cells. NRK-49F cells were incubated without (control) ( A , E , I ) or with 2 ng/ml of TGF-β1 ( B , F , J ), 40 ng/ml of HGF ( C , G , K ), or both ( D , H , L ) for 2 days. A to D: TGF-β1 induced morphological transformation of NRK-49F cells into a myofibroblastic appearance ( B ). HGF blocked this transformation ( D ). E to L: Representative micrographs of tetramethyl-rhodamine isothiocyanate-conjugated phalloidin staining ( E–H ) and vimentin staining ( I–L ) showing F-actin and vimentin reorganization in NRK-49F cells induced by TGF-β1 ( F , J ). HGF primarily abolished TGF-β1-induced actin and vimentin reorganization ( H , L ). Scale bar, 20 μm.

    Article Snippet: For visualizing F-actin, cells were stained with tetramethyl-rhodamine isothiocyanate-conjugated phalloidin (Sigma).

    Techniques: Transformation Assay, Incubation, Staining

    Effects of Hic-5 overexpression on cell spreading. (A) NIH 3T3 cells transfected with pCG- pax (HA-tagged paxillin, panels 1 and 2) or pCG-LD1m hic-5 (HA-tagged LD1mHic-5, panels 3 and 4) were allowed to spread on fibronectin-coated coverslips for 30 min, fixed with 3.7% formalin, and then immunostained with anti-HA antibody (panels 1 and 3) or stained with TRITC-conjugated phalloidin (panels 2 and 4). Arrows indicate the transfected cells. (B) NIH 3T3 cells were transfected with vector (●), expression vector of paxillin (▴), or Hic-5 (■). After 48 h, cell spreading was quantified by allowing the cells to spread on fibronectin-coated coverslips for the indicated times. Cells were stained with anti-HA antibody, and the percentages of spread cells among transfected cells stained with anti-HA antibody at each time point were calculated. Values represent the means of at least three independent experiments ± the standard deviation (SD). (C) NIH 3T3 cells transfected with pEGFP-N3 (GFP), pCG- pax (paxillin), pCG-h hic-5 (hHic-5), pCG-LD1m hic-5 (LD1mHic-5), and pCG-m hic-5 (mHic-5) were allowed to spread on fibronectin-coated coverslips for 30 min. The percentages of the spread cells were determined. Each bar represents the mean of at least three independent experiments ± the SD. (D) Equal amounts of total cell lysates from cells used in the spreading assay were subjected to SDS-PAGE and immunoblotted with anti-HA antibody. Lane 1, primary MEFs; lanes 2 to 6, NIH 3T3 cells transfected with vector (lane 2) or the expression vectors of paxillin (lane 3), hHic-5 (lane 4), LD1mHic-5 (lane 5), or mHic-5 (lane 6).

    Journal: Molecular and Cellular Biology

    Article Title: Hic-5-Reduced Cell Spreading on Fibronectin: Competitive Effects between Paxillin and Hic-5 through Interaction with Focal Adhesion Kinase

    doi: 10.1128/MCB.21.16.5332-5345.2001

    Figure Lengend Snippet: Effects of Hic-5 overexpression on cell spreading. (A) NIH 3T3 cells transfected with pCG- pax (HA-tagged paxillin, panels 1 and 2) or pCG-LD1m hic-5 (HA-tagged LD1mHic-5, panels 3 and 4) were allowed to spread on fibronectin-coated coverslips for 30 min, fixed with 3.7% formalin, and then immunostained with anti-HA antibody (panels 1 and 3) or stained with TRITC-conjugated phalloidin (panels 2 and 4). Arrows indicate the transfected cells. (B) NIH 3T3 cells were transfected with vector (●), expression vector of paxillin (▴), or Hic-5 (■). After 48 h, cell spreading was quantified by allowing the cells to spread on fibronectin-coated coverslips for the indicated times. Cells were stained with anti-HA antibody, and the percentages of spread cells among transfected cells stained with anti-HA antibody at each time point were calculated. Values represent the means of at least three independent experiments ± the standard deviation (SD). (C) NIH 3T3 cells transfected with pEGFP-N3 (GFP), pCG- pax (paxillin), pCG-h hic-5 (hHic-5), pCG-LD1m hic-5 (LD1mHic-5), and pCG-m hic-5 (mHic-5) were allowed to spread on fibronectin-coated coverslips for 30 min. The percentages of the spread cells were determined. Each bar represents the mean of at least three independent experiments ± the SD. (D) Equal amounts of total cell lysates from cells used in the spreading assay were subjected to SDS-PAGE and immunoblotted with anti-HA antibody. Lane 1, primary MEFs; lanes 2 to 6, NIH 3T3 cells transfected with vector (lane 2) or the expression vectors of paxillin (lane 3), hHic-5 (lane 4), LD1mHic-5 (lane 5), or mHic-5 (lane 6).

    Article Snippet: Cells were blocked with 3% BSA in PBS containing 0.1% Tween 20, treated with anti-HA antibody (12CA5) at 1:300 or with anti-Myc antibody (9E10) at 1:300 for 1 h, and then incubated with tetramethyl rhodamine isocyanate (TRITC)-conjugated phalloidin (Sigma) at 1:300 and with FITC-conjugated anti-mouse IgG (Dako) at 1:300 for 1 h. After being washed with PBS containing 0.1% Tween 20, the cells were mounted and visualized under a low-magnification fluorescence microscope.

    Techniques: Hydrophobic Interaction Chromatography, Over Expression, Transfection, Staining, Plasmid Preparation, Expressing, Standard Deviation, SDS Page

    Effect of overexpression of FRMD7 on the actin cytoskeleton in NIH3T3 cells. NIH3T3 cells transfected with mouse FRMD7-pEGFP-n1 (green) or the empty pEGFP-n1 vector (green) (Mock) were cultured for 24 hours and then cultured for 16 hours without FBS. F-actin was stained with TRITC-conjugated rhodamine-phalloidin (red). NIH3T3 cells transfected with the mouse full-length FRMD7 displayed some lamellipodia and ruffles, consistent with Rac1 activation. Normal NIH3T3 cells served as the control (N). Scale bars: 20 µm.

    Journal: PLoS ONE

    Article Title: FERM Domain Containing Protein 7 Interacts with the Rho GDP Dissociation Inhibitor and Specifically Activates Rac1 Signaling

    doi: 10.1371/journal.pone.0073108

    Figure Lengend Snippet: Effect of overexpression of FRMD7 on the actin cytoskeleton in NIH3T3 cells. NIH3T3 cells transfected with mouse FRMD7-pEGFP-n1 (green) or the empty pEGFP-n1 vector (green) (Mock) were cultured for 24 hours and then cultured for 16 hours without FBS. F-actin was stained with TRITC-conjugated rhodamine-phalloidin (red). NIH3T3 cells transfected with the mouse full-length FRMD7 displayed some lamellipodia and ruffles, consistent with Rac1 activation. Normal NIH3T3 cells served as the control (N). Scale bars: 20 µm.

    Article Snippet: F-actin was stained using TRITC-conjugated rhodamine–phalloidin (77481, Sigma Aldrich, St Louis, MO, USA) fluorescein diluted in PBS and bovine serum albumin for 45 min at room temperature.

    Techniques: Over Expression, Transfection, Plasmid Preparation, Cell Culture, Staining, Activation Assay

    Immunofluorescence microscopy to examine phospho-Cav1 and β-actin localization during C. neoformans infection . The HBMEC monolayers were incubated with 10 6 C. neoformans strain B-4500FO2 for 0 min, 5 min and 60 min. In parallel, untreated HBMEC were used as a negative control, indicated as 0 min in the figure. β-Actin was stained with phalloidin-rhodamine conjugate (red) (second column) and the phosphor-Cav1 was stained with anti-phospho-Cav1 (Tyr-14) antibody and second antibody-FITC conjugate (green). Nuclei were stained with DAPI (blue). Bar: 15 μm. A similar study was performed in the bottom panel, in which tubulin was stained with anti-tubulin antibodies and rhodamine conjugate (second column) at the 60 min incubation time point.

    Journal: Journal of Biomedical Science

    Article Title: Lipid raft/caveolae signaling is required for Cryptococcus neoformans invasion into human brain microvascular endothelial cells

    doi: 10.1186/1423-0127-19-19

    Figure Lengend Snippet: Immunofluorescence microscopy to examine phospho-Cav1 and β-actin localization during C. neoformans infection . The HBMEC monolayers were incubated with 10 6 C. neoformans strain B-4500FO2 for 0 min, 5 min and 60 min. In parallel, untreated HBMEC were used as a negative control, indicated as 0 min in the figure. β-Actin was stained with phalloidin-rhodamine conjugate (red) (second column) and the phosphor-Cav1 was stained with anti-phospho-Cav1 (Tyr-14) antibody and second antibody-FITC conjugate (green). Nuclei were stained with DAPI (blue). Bar: 15 μm. A similar study was performed in the bottom panel, in which tubulin was stained with anti-tubulin antibodies and rhodamine conjugate (second column) at the 60 min incubation time point.

    Article Snippet: Co. (#C1655, 1~2 μg/mL); anti-Cav1(tyr-14) was purchased from Cell Signaling Technology (#3251); actin filaments were stained with phalloidin-rhodamine conjugate (Sigma Chem Co. cat #P1951), and anti-tubulin antibody was obtained from Sigma Chem Co., (#T5168).

    Techniques: Immunofluorescence, Microscopy, Infection, Incubation, Negative Control, Staining

    Pten deletion in excitatory cortical neurons results in increased soma size. a Brain sections obtained from P0 wild type (WT) mice in the NEX- Pten colony were processed for immunofluorescence using Cre antibodies. A low magnification confocal image shows widespread expression in the cerebral cortex (CX) and hippocampus (HC), but not in the midbrain (MB) or cerebellum (CB). b Western blot analysis of the forebrain and cerebellum of newborn wild type (WT), heterozygous (Het), and homozygous (Hom) NEX- Pten littermates. The blot, which is representative of data obtained from 3 sets of littermates, was probed with Pten antibodies and then reprobed with actin antibodies to ensure equal protein loading. Pten expression is progressively reduced in the forebrain of heterozygous and homozygous mutants. c, d Pten immunohistochemistry staining (brown) of brain sections obtained from WT and Hom NEX- Pten littermates. Sections were counterstained with cresyl violet. Brightfield images of the upper cortical layers show that the majority of cells are Pten-positive in wild type and Pten-negative in homozygous mutants. Pten-negative cells appeared enlarged, and some protrude in the marginal zone near the pial surface. e, f Confocal images of representative dissociated cortical neurons cultured for 15 DIV and triple-labeled with rhodamine-phalloidin (red), Pten antibodies (green) and DAPI (blue). g Soma size comparison between Pten-positive ( n = 35) and Pten-negative cultured neurons ( n =22). The difference is statistically significant ( p

    Journal: Developmental neuroscience

    Article Title: Development and characterization of NEX-Pten, a novel forebrain excitatory neuron-specific knockout mouse

    doi: 10.1159/000337229

    Figure Lengend Snippet: Pten deletion in excitatory cortical neurons results in increased soma size. a Brain sections obtained from P0 wild type (WT) mice in the NEX- Pten colony were processed for immunofluorescence using Cre antibodies. A low magnification confocal image shows widespread expression in the cerebral cortex (CX) and hippocampus (HC), but not in the midbrain (MB) or cerebellum (CB). b Western blot analysis of the forebrain and cerebellum of newborn wild type (WT), heterozygous (Het), and homozygous (Hom) NEX- Pten littermates. The blot, which is representative of data obtained from 3 sets of littermates, was probed with Pten antibodies and then reprobed with actin antibodies to ensure equal protein loading. Pten expression is progressively reduced in the forebrain of heterozygous and homozygous mutants. c, d Pten immunohistochemistry staining (brown) of brain sections obtained from WT and Hom NEX- Pten littermates. Sections were counterstained with cresyl violet. Brightfield images of the upper cortical layers show that the majority of cells are Pten-positive in wild type and Pten-negative in homozygous mutants. Pten-negative cells appeared enlarged, and some protrude in the marginal zone near the pial surface. e, f Confocal images of representative dissociated cortical neurons cultured for 15 DIV and triple-labeled with rhodamine-phalloidin (red), Pten antibodies (green) and DAPI (blue). g Soma size comparison between Pten-positive ( n = 35) and Pten-negative cultured neurons ( n =22). The difference is statistically significant ( p

    Article Snippet: In order to label actin, rhodamine phalloidin conjugate (10 µl; Invitrogen) was added to the blocking solution for 15 min followed by 3 PBS washes.

    Techniques: Mouse Assay, Immunofluorescence, Expressing, Western Blot, Immunohistochemistry, Staining, Cell Culture, Labeling

    Effects of Klotho on Ang II-induced migration of VSMC. ( A ) Wound healing assay. Conflunet VSMCs were scapes wounded and allowed to migrate for 24 h. ( B ) Transwell chemotactic assay. Klotho inhibited Ang II-induced VSMCs miagration. ( C ) Cell morphology and actin filaments analysis by laser confocal microscopy. VSMCs were incubated with Alexa 546-conjugated rhodamine phalloidin (red) and the nuclei were stained with DAPI (blue). * p

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Klotho Inhibits Proliferation and Migration of Angiotensin II-Induced Vascular Smooth Muscle Cells (VSMCs) by Modulating NF-κB p65, Akt, and Extracellular Signal Regulated Kinase (ERK) Signaling Activities

    doi: 10.12659/MSM.908038

    Figure Lengend Snippet: Effects of Klotho on Ang II-induced migration of VSMC. ( A ) Wound healing assay. Conflunet VSMCs were scapes wounded and allowed to migrate for 24 h. ( B ) Transwell chemotactic assay. Klotho inhibited Ang II-induced VSMCs miagration. ( C ) Cell morphology and actin filaments analysis by laser confocal microscopy. VSMCs were incubated with Alexa 546-conjugated rhodamine phalloidin (red) and the nuclei were stained with DAPI (blue). * p

    Article Snippet: The cells were washed with PBS and then incubated in the dark with Alexa 546-conjugated rhodamine phalloidin (5 U/mL, 1: 100, Invitrogen, Carlsbad, CA, USA) for 30 min. Then, the nuclei were stained with DAPI (Sigma) for 3 min in the dark.

    Techniques: Migration, Wound Healing Assay, Chemotaxis Assay, Confocal Microscopy, Incubation, Staining

    The F-actin bundling activity of DdVASP is required for filopodium formation in vivo . ( a ) 3D reconstructions of tetramethylrhodamine B isothiocyanate–phalloidin-labeled knockout and reconstituted cells as indicated. (Scale bar: 5 μm.)

    Journal:

    Article Title: The bundling activity of vasodilator-stimulated phosphoprotein is required for filopodium formation

    doi: 10.1073/pnas.0511243103

    Figure Lengend Snippet: The F-actin bundling activity of DdVASP is required for filopodium formation in vivo . ( a ) 3D reconstructions of tetramethylrhodamine B isothiocyanate–phalloidin-labeled knockout and reconstituted cells as indicated. (Scale bar: 5 μm.)

    Article Snippet: F-actin was labeled with tetramethylrhodamine B isothiocyanate-conjugated phalloidin (Sigma), and microscopic analysis of F-actin structures was performed as described previously ( ).

    Techniques: Activity Assay, In Vivo, Labeling, Knock-Out

    PKA activation inhibits EphA2-dependent cell retraction. (A) Representative images of phalloidin-labeled PC3 cells stimulated for 12 min with 0.5 μg/ml control Fc or ephrin-A1 Fc or pretreated for 40 min with 20 μM forskolin or 200 μM of the PKA agonist 6-Benz-cAMP before ephrin-A1 Fc stimulation. Scale bar, 50 μm. (B) Histogram showing average cell areas ± SE under the different conditions (693 cells/condition from three experiments in each of which 77 cells/well from three wells were counted). **** p

    Journal: Molecular Biology of the Cell

    Article Title: Protein kinase A can block EphA2 receptor–mediated cell repulsion by increasing EphA2 S897 phosphorylation

    doi: 10.1091/mbc.E16-01-0048

    Figure Lengend Snippet: PKA activation inhibits EphA2-dependent cell retraction. (A) Representative images of phalloidin-labeled PC3 cells stimulated for 12 min with 0.5 μg/ml control Fc or ephrin-A1 Fc or pretreated for 40 min with 20 μM forskolin or 200 μM of the PKA agonist 6-Benz-cAMP before ephrin-A1 Fc stimulation. Scale bar, 50 μm. (B) Histogram showing average cell areas ± SE under the different conditions (693 cells/condition from three experiments in each of which 77 cells/well from three wells were counted). **** p

    Article Snippet: The cells were then fixed for 15 min in 4% formaldehyde in PBS, permeabilized for 3 min in 0.5% Triton X-100 in Tris-buffered saline, and stained with rhodamine-conjugated phalloidin to visualize F-actin.

    Techniques: Activation Assay, Labeling

    Inhibition of EphA2-dependent cell retraction by representative hit compounds. (A, B) “No compound” refers to cells treated with Fc control or ephrin-A1 Fc and DMSO. Other cells were pretreated for 40 min with the indicated compounds and then for 10 min with Fc or ephrin-A1 Fc in the presence of the compounds. Forskolin was used at 20 μM and dibutyryl cyclical AMP (dbcAMP) at 0.5 mM. The other compounds were used at 5 μM. NECA, 5′-( N -ethylcarboxamido) adenosine. Cells were stained for F-actin with fluorescent phalloidin (red), and nuclei were labeled with DAPI (blue). The inactive compound (O100, oxotremorine methiodide) did not inhibit retraction and rounding of the cells induced by ephrin-A1 Fc, similar to the control. All other compounds shown inhibited retraction, and the cells remained flat. (C) Dose–response curve for forskolin-mediated inhibition of cell retraction analyzed by automated image analysis in the 384-well format. Averages and SEs from quadruplicate measurements from two experiments carried out on different days.

    Journal: Molecular Biology of the Cell

    Article Title: Protein kinase A can block EphA2 receptor–mediated cell repulsion by increasing EphA2 S897 phosphorylation

    doi: 10.1091/mbc.E16-01-0048

    Figure Lengend Snippet: Inhibition of EphA2-dependent cell retraction by representative hit compounds. (A, B) “No compound” refers to cells treated with Fc control or ephrin-A1 Fc and DMSO. Other cells were pretreated for 40 min with the indicated compounds and then for 10 min with Fc or ephrin-A1 Fc in the presence of the compounds. Forskolin was used at 20 μM and dibutyryl cyclical AMP (dbcAMP) at 0.5 mM. The other compounds were used at 5 μM. NECA, 5′-( N -ethylcarboxamido) adenosine. Cells were stained for F-actin with fluorescent phalloidin (red), and nuclei were labeled with DAPI (blue). The inactive compound (O100, oxotremorine methiodide) did not inhibit retraction and rounding of the cells induced by ephrin-A1 Fc, similar to the control. All other compounds shown inhibited retraction, and the cells remained flat. (C) Dose–response curve for forskolin-mediated inhibition of cell retraction analyzed by automated image analysis in the 384-well format. Averages and SEs from quadruplicate measurements from two experiments carried out on different days.

    Article Snippet: The cells were then fixed for 15 min in 4% formaldehyde in PBS, permeabilized for 3 min in 0.5% Triton X-100 in Tris-buffered saline, and stained with rhodamine-conjugated phalloidin to visualize F-actin.

    Techniques: Inhibition, Staining, Labeling

    Inhibition of EphA2-dependent cell retraction by cAMP requires S897 phosphorylation. (A) Representative images of phalloidin-labeled, EphA2-knockdown PC3 cell populations infected with pLVX-IRES-Neo lentiviral vector (Vector) or lentivirus encoding WT EphA2 (WT) or the indicated EphA2 mutants. The cells, pretreated or not for 40 min with 20 μM forskolin, were stimulated for 12 min with 0.5 μg/ml control Fc or ephrin-A1 Fc. (B) Histogram showing average cell areas ± SE for the different conditions (160 cells/condition from an experiment in which 80 cells/well from two wells were measured). **** p

    Journal: Molecular Biology of the Cell

    Article Title: Protein kinase A can block EphA2 receptor–mediated cell repulsion by increasing EphA2 S897 phosphorylation

    doi: 10.1091/mbc.E16-01-0048

    Figure Lengend Snippet: Inhibition of EphA2-dependent cell retraction by cAMP requires S897 phosphorylation. (A) Representative images of phalloidin-labeled, EphA2-knockdown PC3 cell populations infected with pLVX-IRES-Neo lentiviral vector (Vector) or lentivirus encoding WT EphA2 (WT) or the indicated EphA2 mutants. The cells, pretreated or not for 40 min with 20 μM forskolin, were stimulated for 12 min with 0.5 μg/ml control Fc or ephrin-A1 Fc. (B) Histogram showing average cell areas ± SE for the different conditions (160 cells/condition from an experiment in which 80 cells/well from two wells were measured). **** p

    Article Snippet: The cells were then fixed for 15 min in 4% formaldehyde in PBS, permeabilized for 3 min in 0.5% Triton X-100 in Tris-buffered saline, and stained with rhodamine-conjugated phalloidin to visualize F-actin.

    Techniques: Inhibition, Labeling, Infection, Plasmid Preparation

    The in vitro assessments of FGM biocompatibility. Notes: ( A ) Cell metabolic activity measured by the Alamar Blue assay. ( B ) Vital cell density determined by a DAPI assay. ( C ) Immunofluorescence images of MSCs seeded on the control and PDLLA nanofiber-coated cell culture dishes after 1 day. F-actin was labeled by rhodamine-conjugated phalloidin (red), and nuclei was labeled by DAPI (blue). Magnification: 400×. Scale bar: 50 μm (* P

    Journal: International Journal of Nanomedicine

    Article Title: PDGF-metronidazole-encapsulated nanofibrous functional layers on collagen membrane promote alveolar ridge regeneration

    doi: 10.2147/IJN.S137342

    Figure Lengend Snippet: The in vitro assessments of FGM biocompatibility. Notes: ( A ) Cell metabolic activity measured by the Alamar Blue assay. ( B ) Vital cell density determined by a DAPI assay. ( C ) Immunofluorescence images of MSCs seeded on the control and PDLLA nanofiber-coated cell culture dishes after 1 day. F-actin was labeled by rhodamine-conjugated phalloidin (red), and nuclei was labeled by DAPI (blue). Magnification: 400×. Scale bar: 50 μm (* P

    Article Snippet: The viability of the cells was assessed by 4,6-diamidino-2-phenylindole (DAPI) assay (Sigma-Aldrich, St Louis, MO, USA) for vital cell nucleus, and by rhodamine-conjugated phalloidin (Santa Cruz Biotech Inc., Dallas, TX, USA) to label filament-actin (F-actin) for cell morphology.

    Techniques: In Vitro, Activity Assay, Alamar Blue Assay, Immunofluorescence, Cell Culture, Labeling

    ( A ) Portion of a kelch neo stage 10 egg chamber stained with rhodamine-conjugated phalloidin and examined by confocal microscopy. A number of striated actin bundles can be seen ( arrowheads ). Of interest here are the distorted regions characterized by angular intersections of relatively straight bundles ( arrows ). Similar distortions can be seen in wild-type egg chambers, especially where the actin bundles contact the nuclear envelope (e.g., Fig. 2 b ). This is a 3-μm optical section. ( B ) Thin-section through the portion of the nurse cell cytoplasm from a kelch neo stage 11 egg follicle. Of interest is that the cable appears broken. This occurs at the point of overlap of two modules. Bars: ( A ) 5 μm; ( B ) 0.1 μm.

    Journal: The Journal of Cell Biology

    Article Title: Actin Filament Cables in Drosophila Nurse Cells Are Composed of Modules That Slide Passively Past One Another during Dumping

    doi:

    Figure Lengend Snippet: ( A ) Portion of a kelch neo stage 10 egg chamber stained with rhodamine-conjugated phalloidin and examined by confocal microscopy. A number of striated actin bundles can be seen ( arrowheads ). Of interest here are the distorted regions characterized by angular intersections of relatively straight bundles ( arrows ). Similar distortions can be seen in wild-type egg chambers, especially where the actin bundles contact the nuclear envelope (e.g., Fig. 2 b ). This is a 3-μm optical section. ( B ) Thin-section through the portion of the nurse cell cytoplasm from a kelch neo stage 11 egg follicle. Of interest is that the cable appears broken. This occurs at the point of overlap of two modules. Bars: ( A ) 5 μm; ( B ) 0.1 μm.

    Article Snippet: The follicles were then stained in PBS containing 4% paraformaldehyde, 0.1% Triton X-100, and 1 μM phalloidin conjugated to rhodamine ( Sigma Chemical Co. , St. Louis, MO).

    Techniques: Staining, Confocal Microscopy

    Portions of wild-type stage 10B egg chambers stained with rhodamine-conjugated phalloidin and examined by confocal microscopy. ( a ) Cortical actin staining outlines 7 of the 15 nurse cells ( left ) and the anterior portion of the oocyte ( right ). Two ring canals ( rc ) that connect the oocyte to the nurse cells are also indicated ( arrows ). The nurse cells contain prominent arrays of actin bundles extending from the plasma membrane toward the nucleus (dark regions within the nurse cells). These cables are located on plasma membranes adjacent to the oocyte ( 1 ), adjacent to other nurse cells with a common ring canal ( 2 ), adjacent to other nurse cells without a ring canal connection ( 3 ), and adjacent to follicle cells surrounding the egg chamber ( 4 ). The densities of these cables along the nurse cell plasma membrane are indicated in Table I . This is a 14-μm optical section. ( b ) The actin cables originate from the plasma membrane and extend toward and outline the nucleus (dark region). The striated actin cables exhibit gaps at low ( leftward arrows ), medium ( upward arrowheads ), and high ( downward arrowheads ) frequencies. In addition, the F-actin staining intensity in the gaps between the striations is often well above background. Also, the fluorescent intensity of adjacent modules in the same cable can be quite different ( leftward arrows ) and probably reflects the presence of overlapping actin bundles. The oocyte (not shown) of this egg chamber is connected to this nurse cell. This is a 6-μm optical section. Bars: ( a ) = 100 μm; ( b ) 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Actin Filament Cables in Drosophila Nurse Cells Are Composed of Modules That Slide Passively Past One Another during Dumping

    doi:

    Figure Lengend Snippet: Portions of wild-type stage 10B egg chambers stained with rhodamine-conjugated phalloidin and examined by confocal microscopy. ( a ) Cortical actin staining outlines 7 of the 15 nurse cells ( left ) and the anterior portion of the oocyte ( right ). Two ring canals ( rc ) that connect the oocyte to the nurse cells are also indicated ( arrows ). The nurse cells contain prominent arrays of actin bundles extending from the plasma membrane toward the nucleus (dark regions within the nurse cells). These cables are located on plasma membranes adjacent to the oocyte ( 1 ), adjacent to other nurse cells with a common ring canal ( 2 ), adjacent to other nurse cells without a ring canal connection ( 3 ), and adjacent to follicle cells surrounding the egg chamber ( 4 ). The densities of these cables along the nurse cell plasma membrane are indicated in Table I . This is a 14-μm optical section. ( b ) The actin cables originate from the plasma membrane and extend toward and outline the nucleus (dark region). The striated actin cables exhibit gaps at low ( leftward arrows ), medium ( upward arrowheads ), and high ( downward arrowheads ) frequencies. In addition, the F-actin staining intensity in the gaps between the striations is often well above background. Also, the fluorescent intensity of adjacent modules in the same cable can be quite different ( leftward arrows ) and probably reflects the presence of overlapping actin bundles. The oocyte (not shown) of this egg chamber is connected to this nurse cell. This is a 6-μm optical section. Bars: ( a ) = 100 μm; ( b ) 10 μm.

    Article Snippet: The follicles were then stained in PBS containing 4% paraformaldehyde, 0.1% Triton X-100, and 1 μM phalloidin conjugated to rhodamine ( Sigma Chemical Co. , St. Louis, MO).

    Techniques: Staining, Confocal Microscopy

    Portions of singed X2 stage 10 egg chambers stained with rhodamine-conjugated phalloidin and examined by confocal microscopy. ( a ) The cortical actin and two ring canals between three nurse cells are shown. There is a wide variety of ring canal morphologies in sn X2 egg chambers (unpublished observations). The ring canals shown here look nearly wild type in appearance. ( b ) The cortical actin and striated actin cables between two nurse cells are shown. For comparison, a wild-type nurse cell at approximately the same stage is shown in Fig. 2 b. These are 6- ( a ) and 7-μm ( b ) optical sections. Bars: ( a ) 10 μm; ( b ) 5 μm.

    Journal: The Journal of Cell Biology

    Article Title: Actin Filament Cables in Drosophila Nurse Cells Are Composed of Modules That Slide Passively Past One Another during Dumping

    doi:

    Figure Lengend Snippet: Portions of singed X2 stage 10 egg chambers stained with rhodamine-conjugated phalloidin and examined by confocal microscopy. ( a ) The cortical actin and two ring canals between three nurse cells are shown. There is a wide variety of ring canal morphologies in sn X2 egg chambers (unpublished observations). The ring canals shown here look nearly wild type in appearance. ( b ) The cortical actin and striated actin cables between two nurse cells are shown. For comparison, a wild-type nurse cell at approximately the same stage is shown in Fig. 2 b. These are 6- ( a ) and 7-μm ( b ) optical sections. Bars: ( a ) 10 μm; ( b ) 5 μm.

    Article Snippet: The follicles were then stained in PBS containing 4% paraformaldehyde, 0.1% Triton X-100, and 1 μM phalloidin conjugated to rhodamine ( Sigma Chemical Co. , St. Louis, MO).

    Techniques: Staining, Confocal Microscopy

    Portions of wild-type egg chambers stained with rhodamine-conjugated phalloidin and examined by confocal microscopy. Regions containing many overlapping bundles are shown. ( a ) Stage 10B. Gaps emphasizing the striations ( arrows ). ( b ) Stage 11. Gaps emphasizing the striations ( arrows ) are more frequent per unit length than in earlier stages. In addition, F-actin staining intensity in the gaps is well above background. ( c ) Stage 12. Here many actin bundles have been compressed and have coalesced to form high density and relatively striation-free bundles. Bars, 5 μm.

    Journal: The Journal of Cell Biology

    Article Title: Actin Filament Cables in Drosophila Nurse Cells Are Composed of Modules That Slide Passively Past One Another during Dumping

    doi:

    Figure Lengend Snippet: Portions of wild-type egg chambers stained with rhodamine-conjugated phalloidin and examined by confocal microscopy. Regions containing many overlapping bundles are shown. ( a ) Stage 10B. Gaps emphasizing the striations ( arrows ). ( b ) Stage 11. Gaps emphasizing the striations ( arrows ) are more frequent per unit length than in earlier stages. In addition, F-actin staining intensity in the gaps is well above background. ( c ) Stage 12. Here many actin bundles have been compressed and have coalesced to form high density and relatively striation-free bundles. Bars, 5 μm.

    Article Snippet: The follicles were then stained in PBS containing 4% paraformaldehyde, 0.1% Triton X-100, and 1 μM phalloidin conjugated to rhodamine ( Sigma Chemical Co. , St. Louis, MO).

    Techniques: Staining, Confocal Microscopy

    The actin cytoskeleton in eye imaginal discs is regulated by Kette and PTP61F. All transgenic constructs were driven by GMR-GAL4 and maintained at 25°C. (A) The phenotype of adult compound eyes was examined by SEM. Ectopic expression of Kette Myr resulted in a rough-eye phenotype. The roughness was enhanced upon RNAi-mediated knockdown of endogenous PTP61F in combination with Kette Myr ( i2 - 5; Kette Myr ). In contrast, forced expression of PTP61Fm rescued the Kette Myr -induced eye defect ( m2 - 2; Kette Myr ). (B) All images were prepared from the 40-h pupal eye imaginal discs stained with rhodamine-phalloidin (top row and second row from top) and anti-ELAV antibody (third row from top). F-actin organization is shown at both apical (top row) and basal (second row from top) levels, whereas the neuronal pattern of photoreceptor cells is shown at the basal level (third row from top). Merged images of F-actin and photoreceptor cells at the basal level are shown in the bottom row. The staining of F-actin (images 1 and 5) and photoreceptor cells (images 9 and 13) in the WT pupal eye discs reveals the organized pattern of ommatidia. This pattern became disorganized in response to ectopic expression of Kette Myr (images 3, 7, 11, and 15). In GMR-Gal4 -driven i2 - 5; Kette Myr flies, severe defects in the F-actin organization (images 4, 8, and 16) and disturbed localization of photoreceptor cells (images 12 and 16) were observed.

    Journal: Molecular and Cellular Biology

    Article Title: Organization of F-Actin via Concerted Regulation of Kette by PTP61F and dAbl ▿Organization of F-Actin via Concerted Regulation of Kette by PTP61F and dAbl ▿ †

    doi: 10.1128/MCB.00229-09

    Figure Lengend Snippet: The actin cytoskeleton in eye imaginal discs is regulated by Kette and PTP61F. All transgenic constructs were driven by GMR-GAL4 and maintained at 25°C. (A) The phenotype of adult compound eyes was examined by SEM. Ectopic expression of Kette Myr resulted in a rough-eye phenotype. The roughness was enhanced upon RNAi-mediated knockdown of endogenous PTP61F in combination with Kette Myr ( i2 - 5; Kette Myr ). In contrast, forced expression of PTP61Fm rescued the Kette Myr -induced eye defect ( m2 - 2; Kette Myr ). (B) All images were prepared from the 40-h pupal eye imaginal discs stained with rhodamine-phalloidin (top row and second row from top) and anti-ELAV antibody (third row from top). F-actin organization is shown at both apical (top row) and basal (second row from top) levels, whereas the neuronal pattern of photoreceptor cells is shown at the basal level (third row from top). Merged images of F-actin and photoreceptor cells at the basal level are shown in the bottom row. The staining of F-actin (images 1 and 5) and photoreceptor cells (images 9 and 13) in the WT pupal eye discs reveals the organized pattern of ommatidia. This pattern became disorganized in response to ectopic expression of Kette Myr (images 3, 7, 11, and 15). In GMR-Gal4 -driven i2 - 5; Kette Myr flies, severe defects in the F-actin organization (images 4, 8, and 16) and disturbed localization of photoreceptor cells (images 12 and 16) were observed.

    Article Snippet: For F-actin staining, discs were reacted with tetramethyl rhodamine isocyanate-conjugated phalloidin (Sigma).

    Techniques: Transgenic Assay, Construct, Expressing, Staining

    JARID2 significantly promotes invasion and metastasis of HCC cells in vitro and in vivo ( A ) Knockdown of JARID2 expression inhibited HCCLM3 ( A1 ) and MHCC97-H ( A2 ) cells migration, whereas overexpressed JARID2 in HepG2 cells promoted cell migration ( A3 ) in wound-healing assays. ( B ) The transwell assays showed that knockdown of JARID2 expression inhibited HCCLM3 ( B1 ) and MHCC97-H ( B2 ) cells invasion, but JARID2 overexpression enhanced HepG2 cells ( B3 ) invasion. ( C ) Immunofluorescence assays of cytoskeleton of HCCLM3 control and HCCLM3 shJARID2-1 ( C1 ), MHCC97-H control and MHCC97-H shJARID2-1 ( C2 ), HepG2 Vector and HepG2 JARID2 cells ( C3 ). F-actin filaments were visualized in cells using rhodamine-phalloidin. ( D ) The HCC metastatic mouse model was constructed by using HCCLM3 and MHCC97-H cells transfected with shJARID2-1 or control vector, and HepG2 cells transfected with JARID2 or control vector as described in the Materials and Methods. The size of liver tumors in each group was calculated and compared in HCCLM3 ( D1 ), MHCC97-H ( D2 ) and HepG2 ( D3 ) cells. Scale bar, 1 cm. ( E ) Representative pictures for intrahepatic ( E1 ) and lung metastasis ( E2 ). ( F ) The number of metastatic nodules per liver or lung was calculated and compared between HCCLM3 control and HCCLM3 shJARID2-1 ( F1 ), MHCC97-H control and MHCC97-H shJARID2-1 ( F2 ), HepG2 Vector and HepG2 JARID2 cells group ( F3 , F4 ). * P

    Journal: Oncotarget

    Article Title: JARID2 promotes invasion and metastasis of hepatocellular carcinoma by facilitating epithelial-mesenchymal transition through PTEN/AKT signaling

    doi: 10.18632/oncotarget.9733

    Figure Lengend Snippet: JARID2 significantly promotes invasion and metastasis of HCC cells in vitro and in vivo ( A ) Knockdown of JARID2 expression inhibited HCCLM3 ( A1 ) and MHCC97-H ( A2 ) cells migration, whereas overexpressed JARID2 in HepG2 cells promoted cell migration ( A3 ) in wound-healing assays. ( B ) The transwell assays showed that knockdown of JARID2 expression inhibited HCCLM3 ( B1 ) and MHCC97-H ( B2 ) cells invasion, but JARID2 overexpression enhanced HepG2 cells ( B3 ) invasion. ( C ) Immunofluorescence assays of cytoskeleton of HCCLM3 control and HCCLM3 shJARID2-1 ( C1 ), MHCC97-H control and MHCC97-H shJARID2-1 ( C2 ), HepG2 Vector and HepG2 JARID2 cells ( C3 ). F-actin filaments were visualized in cells using rhodamine-phalloidin. ( D ) The HCC metastatic mouse model was constructed by using HCCLM3 and MHCC97-H cells transfected with shJARID2-1 or control vector, and HepG2 cells transfected with JARID2 or control vector as described in the Materials and Methods. The size of liver tumors in each group was calculated and compared in HCCLM3 ( D1 ), MHCC97-H ( D2 ) and HepG2 ( D3 ) cells. Scale bar, 1 cm. ( E ) Representative pictures for intrahepatic ( E1 ) and lung metastasis ( E2 ). ( F ) The number of metastatic nodules per liver or lung was calculated and compared between HCCLM3 control and HCCLM3 shJARID2-1 ( F1 ), MHCC97-H control and MHCC97-H shJARID2-1 ( F2 ), HepG2 Vector and HepG2 JARID2 cells group ( F3 , F4 ). * P

    Article Snippet: Rhodamine-conjugated phalloidin, DAPI and fluorescence labeled secondary antibody were obtained from Beyotime Institute of Biotechnology (Shanghai, China).

    Techniques: In Vitro, In Vivo, Expressing, Migration, Over Expression, Immunofluorescence, Plasmid Preparation, Construct, Transfection

    Organization of the serotonin-like immunoreactive nervous system in a 24-day-old larva. Micrographs of a live animal (A), Z-projections (B-I) of larvae after mono- and double staining with antibodies against 5-HT (serotonin) (green), as well as staining with phalloidin (grey), and Hoechst (violet), and 3-D reconstructions (J-K). ( A ) General view of a live larva showing the apical plate (ap), the border of the preoral coelom (bc1), vestibulum (v), esophagus (eso), stomach (st), midgut (mg), proctodaeum (pr), the border of the trunk coelom (bc3), tentacle (t), telotroch (tt), and primordium of the metasomal sac (pms). Lateral view; apical is to the top; ventral is to the right. ( B ) Nervous system with all major elements: apical organ (ao), median neurite bundle (men), frontal organ (fo), oral nerve ring (or), tentacular neurite bundle (tn) with dorsal commissure (dc), and nerve ring of the telotroch (nt). Lateral view; apical is to the top, ventral to the right. ( C ) Dorsal view of branched proximal ends of the tentacular neurite bundle (br) and apical organ, which is composed of monopolar (mo) and bipolar or multipolar perikarya. The latter forms the larger group on the left (lgn), which rises above the neuropil (np). Anterior is to the upper right. ( D ) Overview of the musculature and serotonin-like immunoreactive nervous system showing apical organ (ao), tentacular neurite bundle (tn) which branches (br) on the dorsal side, thin trunk neurites (trn), and nerve ring of the telotroch (nt). Ventral view; apical is to the top; preoral lobe bends backward. ( E ) Dorso-lateral view of the preoral lobe showing apical organ (ao), median neurite bundle (men), and branches (br) of the tentacular neurite bundle (tn). Anterior is to the right. ( F ) Left portion of the apical organ showing monopolar perikarya (mo) and the left group of bipolar or multipolar perikarya (lgn), which gives rise to the left branch of the tentacular neurite bundle (ltn) and median neurite bundle (mn) of the preoral lobe. Lateral view; apical is to the top; anterior of the preoral lobe is to the right. ( G ) Details of a monopolar perikaryon; lateral view showing perikaryon (mo) and basal projection (bp) that passes from the median part of the cell to the neuropil (np). ( H ) Lateral view of the preoral lobe showing anterior part of the apical organ (ao), median neurite bundle (mn), and frontal organ (fo), which is composed of several weakly stained perikarya. Anterior of the preoral lobe is to the right. ( I ) Dorsal commissure (dc) between two dorso-lateral branches of left (ltn) and right (rtn) branches of the tentacular neurite bundle. Dorsal view; apical is to the top. ( J ) Posterior view of the apical organ that is composed of two groups of bipolar or multipolar perikarya (light blue), which give rise to two dorso-lateral branches of the tentacular neurite bundle (light pink) and median neurite bundle (magenta). Monopolar perikarya form the upper large neuropil (golden). Anterior is to the right; left is to the bottom. ( K ) Top view of the apical organ showing large neuropil and monopolar perikarya (golden), neuropil and bipolar or multipolar perikarya (light blue), tentacular neurite bundle (light pink), median neurite bundle (magenta), and oral nerve ring (pale blue).

    Journal: BMC Evolutionary Biology

    Article Title: Development of the nervous system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) reveals both deuterostome- and trochozoan-like features

    doi: 10.1186/1471-2148-12-121

    Figure Lengend Snippet: Organization of the serotonin-like immunoreactive nervous system in a 24-day-old larva. Micrographs of a live animal (A), Z-projections (B-I) of larvae after mono- and double staining with antibodies against 5-HT (serotonin) (green), as well as staining with phalloidin (grey), and Hoechst (violet), and 3-D reconstructions (J-K). ( A ) General view of a live larva showing the apical plate (ap), the border of the preoral coelom (bc1), vestibulum (v), esophagus (eso), stomach (st), midgut (mg), proctodaeum (pr), the border of the trunk coelom (bc3), tentacle (t), telotroch (tt), and primordium of the metasomal sac (pms). Lateral view; apical is to the top; ventral is to the right. ( B ) Nervous system with all major elements: apical organ (ao), median neurite bundle (men), frontal organ (fo), oral nerve ring (or), tentacular neurite bundle (tn) with dorsal commissure (dc), and nerve ring of the telotroch (nt). Lateral view; apical is to the top, ventral to the right. ( C ) Dorsal view of branched proximal ends of the tentacular neurite bundle (br) and apical organ, which is composed of monopolar (mo) and bipolar or multipolar perikarya. The latter forms the larger group on the left (lgn), which rises above the neuropil (np). Anterior is to the upper right. ( D ) Overview of the musculature and serotonin-like immunoreactive nervous system showing apical organ (ao), tentacular neurite bundle (tn) which branches (br) on the dorsal side, thin trunk neurites (trn), and nerve ring of the telotroch (nt). Ventral view; apical is to the top; preoral lobe bends backward. ( E ) Dorso-lateral view of the preoral lobe showing apical organ (ao), median neurite bundle (men), and branches (br) of the tentacular neurite bundle (tn). Anterior is to the right. ( F ) Left portion of the apical organ showing monopolar perikarya (mo) and the left group of bipolar or multipolar perikarya (lgn), which gives rise to the left branch of the tentacular neurite bundle (ltn) and median neurite bundle (mn) of the preoral lobe. Lateral view; apical is to the top; anterior of the preoral lobe is to the right. ( G ) Details of a monopolar perikaryon; lateral view showing perikaryon (mo) and basal projection (bp) that passes from the median part of the cell to the neuropil (np). ( H ) Lateral view of the preoral lobe showing anterior part of the apical organ (ao), median neurite bundle (mn), and frontal organ (fo), which is composed of several weakly stained perikarya. Anterior of the preoral lobe is to the right. ( I ) Dorsal commissure (dc) between two dorso-lateral branches of left (ltn) and right (rtn) branches of the tentacular neurite bundle. Dorsal view; apical is to the top. ( J ) Posterior view of the apical organ that is composed of two groups of bipolar or multipolar perikarya (light blue), which give rise to two dorso-lateral branches of the tentacular neurite bundle (light pink) and median neurite bundle (magenta). Monopolar perikarya form the upper large neuropil (golden). Anterior is to the right; left is to the bottom. ( K ) Top view of the apical organ showing large neuropil and monopolar perikarya (golden), neuropil and bipolar or multipolar perikarya (light blue), tentacular neurite bundle (light pink), median neurite bundle (magenta), and oral nerve ring (pale blue).

    Article Snippet: Then, the specimens were washed in PBT/BSA and incubated in a mixture of rhodamine-conjugated phalloidin (1:50) (Fisher Scientific, Pittsburgh, PA, USA) and Hoechst (1:1000) (Fisher Scientific, Pittsburgh, PA, USA) for 1 h at RT in the dark.

    Techniques: Double Staining, Staining

    Organization of the FMRFamide-like immunoreactive nervous system in 5-day-old (A-E) and 6-day-old (F-H) larvae. Z-projections of larvae after double and triple staining with antibodies against FMRFamide (cyan), as well as staining with phalloidin (blue), and Hoechst (violet). Apical is to the top in all cases except for E, where it is to the upper right. ( A ) Overview showing apical organ (ao), marginal neurite bundle (mn), tentacular neurite bundle (tn), and midgut perikarya (mgp); dorsal view. ( B ) Oral field and basal part of the preoral lobe of a larva with oral nerve ring (or), marginal neurite bundle (mn), ventral neurite bundles (vnb), upper ventro-lateral perikarya (uvl), ventro-lateral neurites (vln), and lower ventro-lateral perikarya (lvl). Ventral view; the image is composed of the most ventral optical sections. ( C ) Lateral part of the apical organ with monopolar perikarya (mo), their basal processes (bp), neuropil (np), and bipolar or multipolar perikaryon (arrowheads). ( D ) Dorsal part of the apical organ with main bipolar or multipolar perikarya (mpe) and two groups of additional bipolar or multipolar perikarya (ape) that connect to the neuropil (np) via a prominent neurite bundle (double arrows). ( E ) Dorsal side of a larva with thin tentacular neurite bundle (tn) that arises from main bipolar or multipolar perikarya (mpe) and then forms two branches. Midgut perikarya (mgp) are shown. The micrograph contains the most dorsal optical sections only. ( F ) Overview of the musculature and FMRFamide-like immunoreactive nervous system showing apical organ (ao), perikarya of the preoral lobe (plp), marginal neurite bundle (mn), oral ring (or), ventral neurite bundles (vnb) with commissures (arrowheads), ventro-lateral neurites (vln), upper ventro-lateral perikarya (uvl), lower ventro-lateral perikarya (lvl), tentacular neurite bundle (tn), and midgut perikarya (mgp). Ventral view. ( G ) Part of the preoral lobe with marginal neurite bundle (mn) and perikaryon (pe) with neurites (arrowheads). ( H ) Ventral neurite bundleswith paired perikarya (pe) and commissures (arrowheads). Ventral view; four most ventral optical sections were used only to produce this image.

    Journal: BMC Evolutionary Biology

    Article Title: Development of the nervous system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) reveals both deuterostome- and trochozoan-like features

    doi: 10.1186/1471-2148-12-121

    Figure Lengend Snippet: Organization of the FMRFamide-like immunoreactive nervous system in 5-day-old (A-E) and 6-day-old (F-H) larvae. Z-projections of larvae after double and triple staining with antibodies against FMRFamide (cyan), as well as staining with phalloidin (blue), and Hoechst (violet). Apical is to the top in all cases except for E, where it is to the upper right. ( A ) Overview showing apical organ (ao), marginal neurite bundle (mn), tentacular neurite bundle (tn), and midgut perikarya (mgp); dorsal view. ( B ) Oral field and basal part of the preoral lobe of a larva with oral nerve ring (or), marginal neurite bundle (mn), ventral neurite bundles (vnb), upper ventro-lateral perikarya (uvl), ventro-lateral neurites (vln), and lower ventro-lateral perikarya (lvl). Ventral view; the image is composed of the most ventral optical sections. ( C ) Lateral part of the apical organ with monopolar perikarya (mo), their basal processes (bp), neuropil (np), and bipolar or multipolar perikaryon (arrowheads). ( D ) Dorsal part of the apical organ with main bipolar or multipolar perikarya (mpe) and two groups of additional bipolar or multipolar perikarya (ape) that connect to the neuropil (np) via a prominent neurite bundle (double arrows). ( E ) Dorsal side of a larva with thin tentacular neurite bundle (tn) that arises from main bipolar or multipolar perikarya (mpe) and then forms two branches. Midgut perikarya (mgp) are shown. The micrograph contains the most dorsal optical sections only. ( F ) Overview of the musculature and FMRFamide-like immunoreactive nervous system showing apical organ (ao), perikarya of the preoral lobe (plp), marginal neurite bundle (mn), oral ring (or), ventral neurite bundles (vnb) with commissures (arrowheads), ventro-lateral neurites (vln), upper ventro-lateral perikarya (uvl), lower ventro-lateral perikarya (lvl), tentacular neurite bundle (tn), and midgut perikarya (mgp). Ventral view. ( G ) Part of the preoral lobe with marginal neurite bundle (mn) and perikaryon (pe) with neurites (arrowheads). ( H ) Ventral neurite bundleswith paired perikarya (pe) and commissures (arrowheads). Ventral view; four most ventral optical sections were used only to produce this image.

    Article Snippet: Then, the specimens were washed in PBT/BSA and incubated in a mixture of rhodamine-conjugated phalloidin (1:50) (Fisher Scientific, Pittsburgh, PA, USA) and Hoechst (1:1000) (Fisher Scientific, Pittsburgh, PA, USA) for 1 h at RT in the dark.

    Techniques: Staining, Plasmid Purification

    The organization of the serotonin-like immunoreactive nervous system in a 13-day-old larva. Micrographs of a live animal (A), Z-projections (B-K) of larvae after mono- and double staining with antibodies against 5-HT (serotonin) (green), as well as staining with phalloidin (grey), and Hoechst (violet), and 3-D reconstruction (L). Apical is to the top in all aspects. ( A ) Lateral view of live larva showing stomach (st), midgut (mg), proctodaeum (pr), and primordia of tentacles (tp). ( B ) Overview of the muscle system; ventral view; preoral lobe bends backward. General view showing preoral lobe (pl), mouth (m), and primordia of tentacles (tp). ( C ) The same larva; overview of the serotonin-likeimmunoreactive system; ventral view showing weakly stained marginal nerves, apical organ (ao), branches (br) of the dorsal portion of the tentacular neurite bundle, oral ring (or), and ventral portion of the tentacular neurite bundle (tn). (D-E) Details of the apical organ; anterior view. ( D ) General view of the apical organ, which is composed of monopolar (arrows) and bipolar or multipolar (arrowheads) perikarya. ( E ) Monopolar perikaryon with varicosities (arrowheads) on the basal process. ( F ) Overview of the musculature and the serotonin-like immunoreactive nervous system showing apical organ (ao), oral ring (or), tentacular neurite bundle (tn), and tentacles (t). Lateral view; ventral is to the right. ( G ) Organization of the ventral portion of the tentacular neurite bundle (tn) which forms a loop in each tentacle. ( H ) Ventral view of the apical plate with apical organ and rows of sensory cells (arrowheads). ( I ) The micrograph comprises the three ventral-most optical sections of the confocal stack, i.e., the region of the mutual position of monopolar perikarya and sensory cells (arrowheads). ( J ) Frontal view of the center of the apical organ showing monopolar perikarya (mo), their basal processes (bp) with varicosities (arrowheads) and neuropil (nmn), and bipolar or multipolar perikarya and neuropil (nmu). ( K ) Organization of bipolar or multipolar perikarya (arrowheads); ventral view. ( L ) Three-dimensional reconstruction of the apical organ. Light blue indicates multipolar or bipolar perikarya, which form a horseshoe-shaped row under the neuropil of the monopolar perikarya (golden); ventral view.

    Journal: BMC Evolutionary Biology

    Article Title: Development of the nervous system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) reveals both deuterostome- and trochozoan-like features

    doi: 10.1186/1471-2148-12-121

    Figure Lengend Snippet: The organization of the serotonin-like immunoreactive nervous system in a 13-day-old larva. Micrographs of a live animal (A), Z-projections (B-K) of larvae after mono- and double staining with antibodies against 5-HT (serotonin) (green), as well as staining with phalloidin (grey), and Hoechst (violet), and 3-D reconstruction (L). Apical is to the top in all aspects. ( A ) Lateral view of live larva showing stomach (st), midgut (mg), proctodaeum (pr), and primordia of tentacles (tp). ( B ) Overview of the muscle system; ventral view; preoral lobe bends backward. General view showing preoral lobe (pl), mouth (m), and primordia of tentacles (tp). ( C ) The same larva; overview of the serotonin-likeimmunoreactive system; ventral view showing weakly stained marginal nerves, apical organ (ao), branches (br) of the dorsal portion of the tentacular neurite bundle, oral ring (or), and ventral portion of the tentacular neurite bundle (tn). (D-E) Details of the apical organ; anterior view. ( D ) General view of the apical organ, which is composed of monopolar (arrows) and bipolar or multipolar (arrowheads) perikarya. ( E ) Monopolar perikaryon with varicosities (arrowheads) on the basal process. ( F ) Overview of the musculature and the serotonin-like immunoreactive nervous system showing apical organ (ao), oral ring (or), tentacular neurite bundle (tn), and tentacles (t). Lateral view; ventral is to the right. ( G ) Organization of the ventral portion of the tentacular neurite bundle (tn) which forms a loop in each tentacle. ( H ) Ventral view of the apical plate with apical organ and rows of sensory cells (arrowheads). ( I ) The micrograph comprises the three ventral-most optical sections of the confocal stack, i.e., the region of the mutual position of monopolar perikarya and sensory cells (arrowheads). ( J ) Frontal view of the center of the apical organ showing monopolar perikarya (mo), their basal processes (bp) with varicosities (arrowheads) and neuropil (nmn), and bipolar or multipolar perikarya and neuropil (nmu). ( K ) Organization of bipolar or multipolar perikarya (arrowheads); ventral view. ( L ) Three-dimensional reconstruction of the apical organ. Light blue indicates multipolar or bipolar perikarya, which form a horseshoe-shaped row under the neuropil of the monopolar perikarya (golden); ventral view.

    Article Snippet: Then, the specimens were washed in PBT/BSA and incubated in a mixture of rhodamine-conjugated phalloidin (1:50) (Fisher Scientific, Pittsburgh, PA, USA) and Hoechst (1:1000) (Fisher Scientific, Pittsburgh, PA, USA) for 1 h at RT in the dark.

    Techniques: Double Staining, Staining

    Organization of the FMRFamide-like immunoreactive nervous system in the late gastrula (A, B), preactinotrocha (C), and young larva (D-K). Z-projections of larvae after double and triple staining with antibodies against FMRFamide (cyan), as well as staining with phalloidin (blue), and Hoechst (violet). Apical is to the top in all cases except C, where it is to the upper left. ( A ) Overview showing apical organ, which is represented by a horseshoe-shaped neuropil (np), archenteron (ar), and precursor of the proctodaeum (ppr). Dorso-lateral view. ( B ) Median region of the anterior body part showing apical plate (ap) with neuropil (np), mesodermal cells (mc), and mouth (m); ventral view. ( C ) Overview of a preactinotrocha with large apical neuropil (np) and marginal neurite bundle (mn) running along edge of the preoral lobe (pl). Lateral view; ventral side is to the right. ( D ) Overview of nuclei and FMRFamide-like immunoreactive nervous system showing marginal neurite bundle (mn), neuropil (np), first perikarya (n) of the apical organ, oral nerve ring (or), first neurites of the ventral neurite bundle (vnb), and tentacular ridge (tr). Ventral view. ( E ) Overview of the musculature and FMRFamide-like immunoreactive nervous system showing marginal neurite bundle (mn), perikarya (n) of the apical organ, the tentacular neurite bundle (tn), oral ring (or), lower ventro-lateral perikarya (lvl) of the oral field, perikarya associated with the ventral neurite bundle (vnb), and perikarya associated with the midgut (mgp). Ventral view. ( F ) Lateral view of a young larva with apical organ (ao), marginal neurite bundle (mn), thin tentacular neurite bundle (tn), upper ventro-lateral perikaryon (uvl), lower ventro-lateral perikaryon (lvl), perikarya of the midgut (mgp), and perikarya of the ventral neurite bundles (vnb). Lateral view; ventral side is to the right. ( G ) Monopolar perikaryon of the apical organ with bright apical cell part (apt), nucleus (nu), and basal process (bp) projecting to the neuropil (np). ( H ) Ventral body wall of a larva with perikarya (pe) and neurites (ne) of the ventral neurite bundles. ( I ) Bipolar perikaryon of the apical organ with two processes: anterior process (apr), which runs into the neuropil (np), and posterior process (pth), which is the precursor of one stem of the tentacular neurite bundle. Lateral view; anterior is to the right. ( J ) Ventro-lateral body wall with large upper ventro-lateral perikaryon (uvl) and the ventro-lateral neurite (pvln). Lateral view; ventral is to the right. ( K ) Posterior body part with midgut (mg), proctodaeum (pr), and perikarya of the midgut, which contain the non-stained nuclei (nu) and form basal processes (bp). Lateral view; ventral is to the right.

    Journal: BMC Evolutionary Biology

    Article Title: Development of the nervous system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) reveals both deuterostome- and trochozoan-like features

    doi: 10.1186/1471-2148-12-121

    Figure Lengend Snippet: Organization of the FMRFamide-like immunoreactive nervous system in the late gastrula (A, B), preactinotrocha (C), and young larva (D-K). Z-projections of larvae after double and triple staining with antibodies against FMRFamide (cyan), as well as staining with phalloidin (blue), and Hoechst (violet). Apical is to the top in all cases except C, where it is to the upper left. ( A ) Overview showing apical organ, which is represented by a horseshoe-shaped neuropil (np), archenteron (ar), and precursor of the proctodaeum (ppr). Dorso-lateral view. ( B ) Median region of the anterior body part showing apical plate (ap) with neuropil (np), mesodermal cells (mc), and mouth (m); ventral view. ( C ) Overview of a preactinotrocha with large apical neuropil (np) and marginal neurite bundle (mn) running along edge of the preoral lobe (pl). Lateral view; ventral side is to the right. ( D ) Overview of nuclei and FMRFamide-like immunoreactive nervous system showing marginal neurite bundle (mn), neuropil (np), first perikarya (n) of the apical organ, oral nerve ring (or), first neurites of the ventral neurite bundle (vnb), and tentacular ridge (tr). Ventral view. ( E ) Overview of the musculature and FMRFamide-like immunoreactive nervous system showing marginal neurite bundle (mn), perikarya (n) of the apical organ, the tentacular neurite bundle (tn), oral ring (or), lower ventro-lateral perikarya (lvl) of the oral field, perikarya associated with the ventral neurite bundle (vnb), and perikarya associated with the midgut (mgp). Ventral view. ( F ) Lateral view of a young larva with apical organ (ao), marginal neurite bundle (mn), thin tentacular neurite bundle (tn), upper ventro-lateral perikaryon (uvl), lower ventro-lateral perikaryon (lvl), perikarya of the midgut (mgp), and perikarya of the ventral neurite bundles (vnb). Lateral view; ventral side is to the right. ( G ) Monopolar perikaryon of the apical organ with bright apical cell part (apt), nucleus (nu), and basal process (bp) projecting to the neuropil (np). ( H ) Ventral body wall of a larva with perikarya (pe) and neurites (ne) of the ventral neurite bundles. ( I ) Bipolar perikaryon of the apical organ with two processes: anterior process (apr), which runs into the neuropil (np), and posterior process (pth), which is the precursor of one stem of the tentacular neurite bundle. Lateral view; anterior is to the right. ( J ) Ventro-lateral body wall with large upper ventro-lateral perikaryon (uvl) and the ventro-lateral neurite (pvln). Lateral view; ventral is to the right. ( K ) Posterior body part with midgut (mg), proctodaeum (pr), and perikarya of the midgut, which contain the non-stained nuclei (nu) and form basal processes (bp). Lateral view; ventral is to the right.

    Article Snippet: Then, the specimens were washed in PBT/BSA and incubated in a mixture of rhodamine-conjugated phalloidin (1:50) (Fisher Scientific, Pittsburgh, PA, USA) and Hoechst (1:1000) (Fisher Scientific, Pittsburgh, PA, USA) for 1 h at RT in the dark.

    Techniques: Staining

    Organization of the FMRFamide-like immunoreactive nervous system in 13-day-old (A, A-insert) and 24-day-old (B-F) larvae, respectively. Z-projections of larvae after mono- and double staining with antibodies against FMRFamide (cyan), as well as staining with phalloidin (blue). Apical is to the top in all cases except for A, where it is to the upper right. ( A ) Overview of the musculature and FMRFamide-like immunoreactive nervous system showing apical organ (ao), marginal neurite bundle (mn), oral ring (or), ventral neurite bundles (paired perikarya are shown by arrowheads), upper ventro-lateral perikarya (uvl), ventro-lateral neurites (vln), lower ventro-lateral perikarya (lvl), tentacular neurite bundle (tn), and neurites of the telotroch (nt). Ventral view. (A-insert) Apical organ with two groups of monopolar perikarya (mo) and bipolar or multipolar perikarya (bn) that give rise to the tentacular neurite bundle (tn). Dorsal view. ( B ) Overview showing apical organ (ao), marginal neurite bundle (mn), oral ring (or), upper branch of the tentacular neurite bundle (ubt), lower branch of the tentacular neurite bundle (lbt), midgut perikarya (mgp), and neurites of the telotroch (nt). Dorsal view. ( C ). Ventral view of the apical organ with neuropil (np), monopolar perikarya (mo), and bipolar or multipolar perikarya (bn). ( D ) Ventral view of the preoral lobe showing apical organ (ao), marginal neurite bundles (mn), and median neurite bundle (men) with concentration of perikarya (cp). ( E ). Ventral view of the oral field with prominent oral nerve ring (or), ventral neurite bundles (vnc; paired perikarya are shown by arrowheads), upper ventro-lateral perikarya (uvl), and ventro-lateral neurites (vln). ( F ) Part of the proctodaeum (pr) and midgut (mg) with perikarya (mgp); dorsal view.

    Journal: BMC Evolutionary Biology

    Article Title: Development of the nervous system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) reveals both deuterostome- and trochozoan-like features

    doi: 10.1186/1471-2148-12-121

    Figure Lengend Snippet: Organization of the FMRFamide-like immunoreactive nervous system in 13-day-old (A, A-insert) and 24-day-old (B-F) larvae, respectively. Z-projections of larvae after mono- and double staining with antibodies against FMRFamide (cyan), as well as staining with phalloidin (blue). Apical is to the top in all cases except for A, where it is to the upper right. ( A ) Overview of the musculature and FMRFamide-like immunoreactive nervous system showing apical organ (ao), marginal neurite bundle (mn), oral ring (or), ventral neurite bundles (paired perikarya are shown by arrowheads), upper ventro-lateral perikarya (uvl), ventro-lateral neurites (vln), lower ventro-lateral perikarya (lvl), tentacular neurite bundle (tn), and neurites of the telotroch (nt). Ventral view. (A-insert) Apical organ with two groups of monopolar perikarya (mo) and bipolar or multipolar perikarya (bn) that give rise to the tentacular neurite bundle (tn). Dorsal view. ( B ) Overview showing apical organ (ao), marginal neurite bundle (mn), oral ring (or), upper branch of the tentacular neurite bundle (ubt), lower branch of the tentacular neurite bundle (lbt), midgut perikarya (mgp), and neurites of the telotroch (nt). Dorsal view. ( C ). Ventral view of the apical organ with neuropil (np), monopolar perikarya (mo), and bipolar or multipolar perikarya (bn). ( D ) Ventral view of the preoral lobe showing apical organ (ao), marginal neurite bundles (mn), and median neurite bundle (men) with concentration of perikarya (cp). ( E ). Ventral view of the oral field with prominent oral nerve ring (or), ventral neurite bundles (vnc; paired perikarya are shown by arrowheads), upper ventro-lateral perikarya (uvl), and ventro-lateral neurites (vln). ( F ) Part of the proctodaeum (pr) and midgut (mg) with perikarya (mgp); dorsal view.

    Article Snippet: Then, the specimens were washed in PBT/BSA and incubated in a mixture of rhodamine-conjugated phalloidin (1:50) (Fisher Scientific, Pittsburgh, PA, USA) and Hoechst (1:1000) (Fisher Scientific, Pittsburgh, PA, USA) for 1 h at RT in the dark.

    Techniques: Double Staining, Staining, Concentration Assay

    The organization of the serotonin-like immunoreactive nervous system in the mid-gastrula (A-D), late gastrula I (E-F), late gastrula II (G-J), and preactinotrocha (K-O). Photographs of live animals (A, E, K) and Z-projections of embryos after mono-, double, and triple staining for 5-HT (serotonin) (green), phalloidin (grey), and Hoechst (violet). Apical is to the top on all micrographs except for B and C, where it is to the upper right. ( A ) Mid-gastrula gross anatomy showing the apical plate (ap), the archenteron (ar), the apical tuft (at), the blastocoel (bl), and the mesodermal cells (mc); lateral view, ventral side is to the right. ( B ) Overview of a mid-gastrula stage with apical organ (ao). ( C ) The same embryo with one perikaryon (n). Image created from selected optical sections from the mid region of the specimen. (C-insert 1) Detail of a perikaryon with short basal process (bp). (C-insert 2) Detail of the nucleus of the perikaryon, which has an upper thin protrusion (arrowhead). ( D ) Anterior portion of a mid-gastrula with apical organ, which consists of four perikarya. Some perikarya have a special area (as) devoid of signal under the wide part of the cell containing the nucleus. Ventral view showing mesodermal cells (mc) and mouth (m). ( E ) Ventral view of live late gastrula I showing apical plate, mouth, and archenteron. ( F ) Serotonin-like immunoreactive nervous system in late gastrula I: the apical organ is formed by 6–7 perikarya. Ventral view showing mesodermal cells, mouth, and archenteron. Image created from selected optical sections from the mid region of the specimen. ( G ) Optical sagittal section through a late gastrula II, which has a large apical organ, the precursor of the preoral lobe (ppl) with spacious blastocoels (bl) and apical plate, esophagus (eso), stomach (st), and the precursor of the proctodaeum (ppr). (H-J) Detail of perikarya of a late gastrula II. ( H ) Two perikarya with two separated apical parts (apt) and one basal process (arrowheads). ( I ) Two perikarya with special areas (as), which are located under the nuclei and lack staining. ( J ) Small neuropil (np) and perikaryon with long basal neurite (bp). ( K ) Lateral view (ventral side on the right) of a live preactinotrocha with complete digestive tract, which consists of esophagus, stomach, midgut (mg), proctodaeum (pr), and small preoral coelom (c1) under the apical plate and blastocoel (bl) in the preoral lobe. ( L ) Overview of a preactinotrocha, dorso-lateral view showing apical organ, preoral lobe, and tentacular ridge. (M-O) Details of the organization of parikarya of the apical organ. Arrowhead indicates an individual perikaryon, which has a nucleus with apical protrusion. ( M ) General view of perikarya. ( N ) Body shape of perikarya with constricted area (double arrow) between the apical and basal parts. ( O ) Nuclei of perikarya; arrowhead indicates apical protrusion of one nucleus.

    Journal: BMC Evolutionary Biology

    Article Title: Development of the nervous system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) reveals both deuterostome- and trochozoan-like features

    doi: 10.1186/1471-2148-12-121

    Figure Lengend Snippet: The organization of the serotonin-like immunoreactive nervous system in the mid-gastrula (A-D), late gastrula I (E-F), late gastrula II (G-J), and preactinotrocha (K-O). Photographs of live animals (A, E, K) and Z-projections of embryos after mono-, double, and triple staining for 5-HT (serotonin) (green), phalloidin (grey), and Hoechst (violet). Apical is to the top on all micrographs except for B and C, where it is to the upper right. ( A ) Mid-gastrula gross anatomy showing the apical plate (ap), the archenteron (ar), the apical tuft (at), the blastocoel (bl), and the mesodermal cells (mc); lateral view, ventral side is to the right. ( B ) Overview of a mid-gastrula stage with apical organ (ao). ( C ) The same embryo with one perikaryon (n). Image created from selected optical sections from the mid region of the specimen. (C-insert 1) Detail of a perikaryon with short basal process (bp). (C-insert 2) Detail of the nucleus of the perikaryon, which has an upper thin protrusion (arrowhead). ( D ) Anterior portion of a mid-gastrula with apical organ, which consists of four perikarya. Some perikarya have a special area (as) devoid of signal under the wide part of the cell containing the nucleus. Ventral view showing mesodermal cells (mc) and mouth (m). ( E ) Ventral view of live late gastrula I showing apical plate, mouth, and archenteron. ( F ) Serotonin-like immunoreactive nervous system in late gastrula I: the apical organ is formed by 6–7 perikarya. Ventral view showing mesodermal cells, mouth, and archenteron. Image created from selected optical sections from the mid region of the specimen. ( G ) Optical sagittal section through a late gastrula II, which has a large apical organ, the precursor of the preoral lobe (ppl) with spacious blastocoels (bl) and apical plate, esophagus (eso), stomach (st), and the precursor of the proctodaeum (ppr). (H-J) Detail of perikarya of a late gastrula II. ( H ) Two perikarya with two separated apical parts (apt) and one basal process (arrowheads). ( I ) Two perikarya with special areas (as), which are located under the nuclei and lack staining. ( J ) Small neuropil (np) and perikaryon with long basal neurite (bp). ( K ) Lateral view (ventral side on the right) of a live preactinotrocha with complete digestive tract, which consists of esophagus, stomach, midgut (mg), proctodaeum (pr), and small preoral coelom (c1) under the apical plate and blastocoel (bl) in the preoral lobe. ( L ) Overview of a preactinotrocha, dorso-lateral view showing apical organ, preoral lobe, and tentacular ridge. (M-O) Details of the organization of parikarya of the apical organ. Arrowhead indicates an individual perikaryon, which has a nucleus with apical protrusion. ( M ) General view of perikarya. ( N ) Body shape of perikarya with constricted area (double arrow) between the apical and basal parts. ( O ) Nuclei of perikarya; arrowhead indicates apical protrusion of one nucleus.

    Article Snippet: Then, the specimens were washed in PBT/BSA and incubated in a mixture of rhodamine-conjugated phalloidin (1:50) (Fisher Scientific, Pittsburgh, PA, USA) and Hoechst (1:1000) (Fisher Scientific, Pittsburgh, PA, USA) for 1 h at RT in the dark.

    Techniques: Staining

    Three-dimensional reconstructions of the serotonin-like immunoreactive nervous system in consecutive stages of development. Color: yellow – apical organ; pink – tentacular neurite bundle; light green – telotroch neurites; dark green – nerve net around the proctodaeum; cyan – ventral nerve cord; pale blue – oral nerve ring; dark blue – marginal neurites of the preoral lobe; brown – trunk neurites; grey/white – muscle system and all actin-containing structures (parietal cytoplasm, microvilliy, etc.). ( A ) Mid-gastrula, dorsal view, apical is to the top. The mouth (m) and archenteron (ar) are visible. ( B ) Dorsal view of the apical organ which consists of four or five perikarya and their neurites. ( C ) Two adjacent perikarya which have two apical parts (apt) and one basal process (bp). ( D ) Lateral view of a preactinotrocha (ventral side to the right) showing apical plate (ap), preoral lobe, and tentacular ridge (tr). ( E ) Dorsal view of the apical organ in a preactinotrocha showing perikarya (prk), neuropil (np), and constricted areas (ca) between the apical and basal parts of some perikarya. ( F ) Dorsal view of the nervous system in a young actinotrocha, which has a large apical organ and two thin dorso-lateral branches of the tentacular neurite bundles. The branches bifurcate (bf) at the terminal end. ( G ) Dorso-lateral view of a 6-day-old actinotrocha (preoral lobe bends backward) showing midgut (mg), proctodaeum (pr), pyloric sphincter (ps), and tentacular ridge (tr). The two dorsal branches of the tentacular neurite bundle form close contact (cc) on the dorsal side. ( H ) The same larva, anterior view. The tentacular ridge contains latero-frontal cells, which have long thick microvilli strongly stained by phalloidin. The mouth is marked by thick muscles; the preoral lobe (pl) bends backward. ( I ) The same larva, lateral view of the nervous system. ( J ) The same larva; the image includes only the ventral part of the nervous system of the oral field. The paired ventral neurite bundle contains paired perikarya (pn) and thin commissures (arrowheads). ( K ) Ventro-lateral view of the nervous system in a 24-day-old larva. The tentacular neurite bundle is very thick and forms several loops in each tentacle. The two dorsal branches of the tentacular neurite bundle interconnect via a dorsal commissure (dc). ( L ) The same larva, ventro-lateral top view. Ring-shaped neurites around the mouth and the telotroch are visible.

    Journal: BMC Evolutionary Biology

    Article Title: Development of the nervous system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) reveals both deuterostome- and trochozoan-like features

    doi: 10.1186/1471-2148-12-121

    Figure Lengend Snippet: Three-dimensional reconstructions of the serotonin-like immunoreactive nervous system in consecutive stages of development. Color: yellow – apical organ; pink – tentacular neurite bundle; light green – telotroch neurites; dark green – nerve net around the proctodaeum; cyan – ventral nerve cord; pale blue – oral nerve ring; dark blue – marginal neurites of the preoral lobe; brown – trunk neurites; grey/white – muscle system and all actin-containing structures (parietal cytoplasm, microvilliy, etc.). ( A ) Mid-gastrula, dorsal view, apical is to the top. The mouth (m) and archenteron (ar) are visible. ( B ) Dorsal view of the apical organ which consists of four or five perikarya and their neurites. ( C ) Two adjacent perikarya which have two apical parts (apt) and one basal process (bp). ( D ) Lateral view of a preactinotrocha (ventral side to the right) showing apical plate (ap), preoral lobe, and tentacular ridge (tr). ( E ) Dorsal view of the apical organ in a preactinotrocha showing perikarya (prk), neuropil (np), and constricted areas (ca) between the apical and basal parts of some perikarya. ( F ) Dorsal view of the nervous system in a young actinotrocha, which has a large apical organ and two thin dorso-lateral branches of the tentacular neurite bundles. The branches bifurcate (bf) at the terminal end. ( G ) Dorso-lateral view of a 6-day-old actinotrocha (preoral lobe bends backward) showing midgut (mg), proctodaeum (pr), pyloric sphincter (ps), and tentacular ridge (tr). The two dorsal branches of the tentacular neurite bundle form close contact (cc) on the dorsal side. ( H ) The same larva, anterior view. The tentacular ridge contains latero-frontal cells, which have long thick microvilli strongly stained by phalloidin. The mouth is marked by thick muscles; the preoral lobe (pl) bends backward. ( I ) The same larva, lateral view of the nervous system. ( J ) The same larva; the image includes only the ventral part of the nervous system of the oral field. The paired ventral neurite bundle contains paired perikarya (pn) and thin commissures (arrowheads). ( K ) Ventro-lateral view of the nervous system in a 24-day-old larva. The tentacular neurite bundle is very thick and forms several loops in each tentacle. The two dorsal branches of the tentacular neurite bundle interconnect via a dorsal commissure (dc). ( L ) The same larva, ventro-lateral top view. Ring-shaped neurites around the mouth and the telotroch are visible.

    Article Snippet: Then, the specimens were washed in PBT/BSA and incubated in a mixture of rhodamine-conjugated phalloidin (1:50) (Fisher Scientific, Pittsburgh, PA, USA) and Hoechst (1:1000) (Fisher Scientific, Pittsburgh, PA, USA) for 1 h at RT in the dark.

    Techniques: Staining

    The organization of the serotonin-like immunoreactive nervous system in a young actinotrocha (A-I) and in a 5-day-old actinotrocha (K-S). Micrographs of live animals (A, K) and Z-projections of larvae with mono- and double staining with antibodies against 5-HT (serotonin) (green),as well as by phalloidin (grey), and Hoechst (violet). ( A ) Lateral view of a live larva (ventral side is to the right, apical is to the top) showing apical plate (ap), esophagus (eso), stomach (st), midgut (mg), proctodaeum (pr), and tentacular ridge (tr). ( B ) Overview of the musculature and serotonin-like immunoreactive nervous system showing apical organ (ao) and tentacular ridge. Lateral view, ventral side is to the right, apical is at the top. (C-H) Details of the apical organ. ( C ) General view of the apical organ with neuropil (np) and perikarya (prk). Lateral view, apical is to the top; anterior is to the right. ( D ) Dorsal view of the apical organ. The thin area between apical and basal parts of the perikaryon is indicated by two arrowheads. ( E ) Perikaryon with pronounced thin area (two arrowheads) between apical (apt) and basal (bp) parts. ( F ) The same perikaryon with nucleus. Wide basal part is indicated by arrowhead. ( G ) The same perikaryon nucleus (arrowheads), which is located under the common row of nuclei. ( H ) Basal portion of the apical organ showing neuropil and bipolar or multipolar (arrowheads) perikarya. ( I ) Young larva with the first serotonin-like immunoreactive neurites that subsequently form the two dorsal branches of the tentacular neurite bundle (tn). Overview of the musculature and serotonin-like immunoreactive nervous system showing apical organ (ao), mouth (m), and preoral lobe (pl). Ventral view, preoral lobe bends backward. ( J ) The same larva; bifurcated (bf) end of left branch of the tentacular neurite bundle. The micrograph is composed of selected optical sections from the dorsal-most region of the specimen. ( K ) Lateral view of a 5-day-old larva showing preoral lobe, stomach, midgut, and proctodaeum. Apical is to the upper right. ( L ) Overview of the musculature and serotonin-like immunoreactive nervous system showing apical organ, anterior (amn) and posterior (pmn) marginal neurite bundles, tentacular neurite bundle (tn), and tentacular ridge (tr). Lateral view; preoral lobe bends backward; apical is to the upper right. Digestive tract with nonspecific staining (numerous white vesicles in the stomach and proctodaeum). (M-S) Details of the apical organ. ( M ) General view of the apical organ showing neuropil (np), bipolar perikarya (bn), and two dorsal branches of the tentacular neurite bundle (tn). Dorso-lateral view; anterior is to the upper right. ( N ) General view of an individual monopolar perikaryon showing cilium (c), apical part (ap), and basal projection (bp), which connects to the neuropil (np). ( O ) Two serotonin-like immunoreactive perikarya, which are adjacent to each other and form a cluster with two apical parts, two nuclei (arrowheads), and one process. ( P ) The same perikarya. ( Q ) The group of bipolar or multipolar perikarya, nuclei (arrowheads) of which are located close to the neuropil. ( R ) Bipolar perikaryon (arrowheads), which is located on the one branch of the tentacular neurite bundle. ( S ) The same perikaryon with nucleus (arrowheads).

    Journal: BMC Evolutionary Biology

    Article Title: Development of the nervous system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) reveals both deuterostome- and trochozoan-like features

    doi: 10.1186/1471-2148-12-121

    Figure Lengend Snippet: The organization of the serotonin-like immunoreactive nervous system in a young actinotrocha (A-I) and in a 5-day-old actinotrocha (K-S). Micrographs of live animals (A, K) and Z-projections of larvae with mono- and double staining with antibodies against 5-HT (serotonin) (green),as well as by phalloidin (grey), and Hoechst (violet). ( A ) Lateral view of a live larva (ventral side is to the right, apical is to the top) showing apical plate (ap), esophagus (eso), stomach (st), midgut (mg), proctodaeum (pr), and tentacular ridge (tr). ( B ) Overview of the musculature and serotonin-like immunoreactive nervous system showing apical organ (ao) and tentacular ridge. Lateral view, ventral side is to the right, apical is at the top. (C-H) Details of the apical organ. ( C ) General view of the apical organ with neuropil (np) and perikarya (prk). Lateral view, apical is to the top; anterior is to the right. ( D ) Dorsal view of the apical organ. The thin area between apical and basal parts of the perikaryon is indicated by two arrowheads. ( E ) Perikaryon with pronounced thin area (two arrowheads) between apical (apt) and basal (bp) parts. ( F ) The same perikaryon with nucleus. Wide basal part is indicated by arrowhead. ( G ) The same perikaryon nucleus (arrowheads), which is located under the common row of nuclei. ( H ) Basal portion of the apical organ showing neuropil and bipolar or multipolar (arrowheads) perikarya. ( I ) Young larva with the first serotonin-like immunoreactive neurites that subsequently form the two dorsal branches of the tentacular neurite bundle (tn). Overview of the musculature and serotonin-like immunoreactive nervous system showing apical organ (ao), mouth (m), and preoral lobe (pl). Ventral view, preoral lobe bends backward. ( J ) The same larva; bifurcated (bf) end of left branch of the tentacular neurite bundle. The micrograph is composed of selected optical sections from the dorsal-most region of the specimen. ( K ) Lateral view of a 5-day-old larva showing preoral lobe, stomach, midgut, and proctodaeum. Apical is to the upper right. ( L ) Overview of the musculature and serotonin-like immunoreactive nervous system showing apical organ, anterior (amn) and posterior (pmn) marginal neurite bundles, tentacular neurite bundle (tn), and tentacular ridge (tr). Lateral view; preoral lobe bends backward; apical is to the upper right. Digestive tract with nonspecific staining (numerous white vesicles in the stomach and proctodaeum). (M-S) Details of the apical organ. ( M ) General view of the apical organ showing neuropil (np), bipolar perikarya (bn), and two dorsal branches of the tentacular neurite bundle (tn). Dorso-lateral view; anterior is to the upper right. ( N ) General view of an individual monopolar perikaryon showing cilium (c), apical part (ap), and basal projection (bp), which connects to the neuropil (np). ( O ) Two serotonin-like immunoreactive perikarya, which are adjacent to each other and form a cluster with two apical parts, two nuclei (arrowheads), and one process. ( P ) The same perikarya. ( Q ) The group of bipolar or multipolar perikarya, nuclei (arrowheads) of which are located close to the neuropil. ( R ) Bipolar perikaryon (arrowheads), which is located on the one branch of the tentacular neurite bundle. ( S ) The same perikaryon with nucleus (arrowheads).

    Article Snippet: Then, the specimens were washed in PBT/BSA and incubated in a mixture of rhodamine-conjugated phalloidin (1:50) (Fisher Scientific, Pittsburgh, PA, USA) and Hoechst (1:1000) (Fisher Scientific, Pittsburgh, PA, USA) for 1 h at RT in the dark.

    Techniques: Double Staining, Staining

    The organization of the serotonin-like immunoreactive nervous system in a 6-day-old larva. Micrographs of live animals ( A ) and Z-projections of larvae after mono- and double staining with antibodies against serotonin (green), as well as with phalloidin (grey), and Hoechst (violet). Apical is to the upper right on all micrographs except A, C, and D, where apical is to the top. (A) Lateral view with ventral to the right. Larva with apical plate (ap), preoral lobe (pl), vestibulum (v), esophagus (eso), stomach (st), midgut (mg), proctodaeum (pr), and tentacular ridge (tr). ( B ) Overview of the musculature and serotonin-like immunoreactive nervous system showing apical organ (ao), anterior marginal neurite bundle (amn), posterior marginal neurite bundle (pmn), tentacular neurite bundle (tn), and ventral neurite bundles (vnb). Ventral view; preoral lobe bends backward. ( C ) Posterior body part. The micrograph comprises selected optical sections from the mid body region of the specimen and shows the nerve net around the proctodaeum (prn) and the pyloric sphincter (ps), midgut (mg), and protonephridia. ( D ) Lateral view of the apical plate (ap) and large neuropile (np) underneath. The anterior pole of the preoral lobe is to the right. ( E ) Top view of the apical organ with dorsal neuropil (np), dorso-lateral branches of the tentacular neurite bundle (tn), and bipolar perikarya (bn). Some portions of the marginal neurites with perikarya (n) are visible. ( F ) Details of the apical organ, anastomosing neurites are indicated by arrowheads. ( G ) Anterior portion of the edge of the preoral lobe with anterior (amn) and posterior (pmn) marginal neurite bundles with a group of perikarya associated with the latter. ( H ) Oral nerve ring with ventro-lateral, weakly stained perikarya (arrowheads); top view. ( I ) Innervation (oral nerve ring – or) and musculature of the mouth (m); ventral view. ( J ) Dorsal portion of the tentacular neurite bundle (tn) with two branches (br) of the right stem. apical organ (ao), bipolar perikarya (arrowheads), and nerve net of the proctodaeum (prn). ( K ) Oral field with the ventral neurite bundles, which pass from the oral ring (or) with oral perikarya (on) to the tentacular neurite bundle (tn), containing several paired perikarya (pn) and commissures (asterisks). The micrograph is composed of the most ventral optical sections only. ( L ) The image comprises only the four most ventral optical sections of the serotonin labeling and all sections of the muscle staining. Ventral view of a larva showing the marginal neurites (mn) of the preoral lobe, the neuropil (np) of the apical organ, the mouth (m), and the ventral neurite bundles (vnb). ( M ) Some paired perikarya of the ventral neurite bundles with nuclei (arrowheads).

    Journal: BMC Evolutionary Biology

    Article Title: Development of the nervous system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) reveals both deuterostome- and trochozoan-like features

    doi: 10.1186/1471-2148-12-121

    Figure Lengend Snippet: The organization of the serotonin-like immunoreactive nervous system in a 6-day-old larva. Micrographs of live animals ( A ) and Z-projections of larvae after mono- and double staining with antibodies against serotonin (green), as well as with phalloidin (grey), and Hoechst (violet). Apical is to the upper right on all micrographs except A, C, and D, where apical is to the top. (A) Lateral view with ventral to the right. Larva with apical plate (ap), preoral lobe (pl), vestibulum (v), esophagus (eso), stomach (st), midgut (mg), proctodaeum (pr), and tentacular ridge (tr). ( B ) Overview of the musculature and serotonin-like immunoreactive nervous system showing apical organ (ao), anterior marginal neurite bundle (amn), posterior marginal neurite bundle (pmn), tentacular neurite bundle (tn), and ventral neurite bundles (vnb). Ventral view; preoral lobe bends backward. ( C ) Posterior body part. The micrograph comprises selected optical sections from the mid body region of the specimen and shows the nerve net around the proctodaeum (prn) and the pyloric sphincter (ps), midgut (mg), and protonephridia. ( D ) Lateral view of the apical plate (ap) and large neuropile (np) underneath. The anterior pole of the preoral lobe is to the right. ( E ) Top view of the apical organ with dorsal neuropil (np), dorso-lateral branches of the tentacular neurite bundle (tn), and bipolar perikarya (bn). Some portions of the marginal neurites with perikarya (n) are visible. ( F ) Details of the apical organ, anastomosing neurites are indicated by arrowheads. ( G ) Anterior portion of the edge of the preoral lobe with anterior (amn) and posterior (pmn) marginal neurite bundles with a group of perikarya associated with the latter. ( H ) Oral nerve ring with ventro-lateral, weakly stained perikarya (arrowheads); top view. ( I ) Innervation (oral nerve ring – or) and musculature of the mouth (m); ventral view. ( J ) Dorsal portion of the tentacular neurite bundle (tn) with two branches (br) of the right stem. apical organ (ao), bipolar perikarya (arrowheads), and nerve net of the proctodaeum (prn). ( K ) Oral field with the ventral neurite bundles, which pass from the oral ring (or) with oral perikarya (on) to the tentacular neurite bundle (tn), containing several paired perikarya (pn) and commissures (asterisks). The micrograph is composed of the most ventral optical sections only. ( L ) The image comprises only the four most ventral optical sections of the serotonin labeling and all sections of the muscle staining. Ventral view of a larva showing the marginal neurites (mn) of the preoral lobe, the neuropil (np) of the apical organ, the mouth (m), and the ventral neurite bundles (vnb). ( M ) Some paired perikarya of the ventral neurite bundles with nuclei (arrowheads).

    Article Snippet: Then, the specimens were washed in PBT/BSA and incubated in a mixture of rhodamine-conjugated phalloidin (1:50) (Fisher Scientific, Pittsburgh, PA, USA) and Hoechst (1:1000) (Fisher Scientific, Pittsburgh, PA, USA) for 1 h at RT in the dark.

    Techniques: Double Staining, Staining, Labeling