Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Motility of glioblastoma cells is driven by netrin-1 induced gain of stemness

doi: 10.1186/s13046-016-0482-0

Figure Lengend Snippet: Netrin-1 is associated with poor patient prognosis in gliomas. The association of NTN1 with the survival of glioma patients was assessed using Kaplan-Meier survival analysis. NTN1 association to ( a ) glioma specific survival (hazard ratio [HR] = 1.73, 95% confidence interval [95% CI] = 1.11 to 2.71; p = 0.015) and ( b ) to recurrence-free survival were analyzed (HR = 1.62, 95% CI = 1.04 to 2.53; p < 0.001). c The localization of NTN1 in low grade gliomas was investigated by immunofluorescence staining of paraffin embedded tumor tissue. Scale bars represent 50 μm. d The localization of NTN1 in GBM tissue was analyzed similarly as in ( c ). Representative images of two tumors are presented. Scale bars represent 100 μm

Article Snippet: Either DMEM/F12 as such or supplemented with 50 ng/ml of recombinant NTN1 (R&D Systems) was added to the lower chamber.

Techniques: Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Motility of glioblastoma cells is driven by netrin-1 induced gain of stemness

doi: 10.1186/s13046-016-0482-0

Figure Lengend Snippet: Netrin-1 co-exists with glioblastoma stem like cells in tumor stroma and infiltrative sprouts. a Colocalization of NTN1 and Jagged1 was analyzed by immunofluorescence staining of human GBM tissue. Representative area of the full image is surrounded with white box and presented as enlargement in lower panel. Scale bars represent 50 μm in full image and 20 μm in the enlargement. Merged image with all channels and separate channels of the enlargement are presented. NTN1 is marked with green , Jagged1 with red and nuclei with blue . b Colocalization of NTN1 and Notch2 was visualized by immunofluorescence staining of human GBM tissue similarly as in ( a ). NTN1 is marked with green , Notch2 with red and nuclei with blue . c Colocalization of NTN1 and Nestin was analyzed by immunofluorescence staining of human GBM tissue similarly as described in ( a ). NTN1 is marked with red , nestin with green and nuclei with blue . c Colocalization of NTN1 and CD133 was studied by immunofluorescence staining of human GBM tissue similarly as described in ( a ). NTN1 is marked with green , CD133 with red and nuclei with blue . e and f To study invasive front of human GBM tumor, tissue pieces were grown in 3D Matrigel ex vivo. The location of NTN1 and nestin ( e ) or NTN1 and Notch2 ( f ) was analyzed with immunofluorescence microscopy. The border of tumor tissue and invasive cells is implicated with dashed line . Enlargement of the boxed area is presented. In the enlargement white arrow points the direction of the migration. Green arrows point to NTN1 positive cells and red arrows Nestin positive cells in ( e ) and Notch2 positive cells in ( f )

Article Snippet: Either DMEM/F12 as such or supplemented with 50 ng/ml of recombinant NTN1 (R&D Systems) was added to the lower chamber.

Techniques: Immunofluorescence, Staining, Ex Vivo, Microscopy, Migration

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Motility of glioblastoma cells is driven by netrin-1 induced gain of stemness

doi: 10.1186/s13046-016-0482-0

Figure Lengend Snippet: Netrin-1 promotes glioblastoma growth in vivo. a–d U87MG or ( e–i ) U373MG cells expressing firefly luciferase and full-length NTN1 (NTN1FH) or its central domain (NTN1(II)FH) were intracranial implanted into nude mice. Five mice per cell-line was xenografted. a, e and f The growth of the tumors was followed with bioluminescent imaging. Photons emitted by the tumor were recorded and quantitated. The photons emitted by the U373MG xenografts were quantitated separately from the head area of the mice ( e ) and together from the head and spine ( f ). Growth curves represent the average photons/second emitted by the tumors in each group. Error bars represent the standard error of the mean. * p value is <0,05. b-d and f-h Representative image of mice in each xenograft group at the experiment end point are presented. The color bars indicate the intensity of photons emitted by the tumor. The color bar scales are set on equal levels within the groups of U87MG xenografts ( b-d ) and within the groups of U373MG xenografts ( f-h )

Article Snippet: Either DMEM/F12 as such or supplemented with 50 ng/ml of recombinant NTN1 (R&D Systems) was added to the lower chamber.

Techniques: In Vivo, Expressing, Luciferase, Imaging

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Motility of glioblastoma cells is driven by netrin-1 induced gain of stemness

doi: 10.1186/s13046-016-0482-0

Figure Lengend Snippet: Netrin-1 increases the invasiveness of GBM stem-like cells in vivo. a–c The tumor growth in xenograft models was evaluated by immunohistochemistry with human specific nestin antibody. Representative tile images of tissue sections of each U87MG xenograft group are presented. The area covered with tumor is surrounded with dashed line . d To evaluate the effect of NTN1FH or NTN1(II)FH on GBM cell invasiveness xenograft tissue section were stained with human specific Lamin A/C antibody and the invasive colonies were counted. The average number of invasive colonies in each group is presented. Error bars represent +/− standard error of the mean. * p value is <0,05. e–g Immunofluorescence microscopy images of the tumor border show the infiltrative growth of nestin positive GBM cells in each U87MG xenograft groups. Scale bars represent 100 μm. h–i The invasive tumor sprouts of U87MG-NTN1FH tumors were further characterized with immunofluorescence stainings of the tissue sections. Dashed line borders the tumor stroma and the white arrow points the direction of invasion. In ( h ) red arrows point NTN1 positive cells and green arrows Nestin positive cells. In ( i ) green arrows point NTN1 positive cells and red arrows the Notch2 positive cells. Scale bars represent 50 μm

Article Snippet: Either DMEM/F12 as such or supplemented with 50 ng/ml of recombinant NTN1 (R&D Systems) was added to the lower chamber.

Techniques: In Vivo, Immunohistochemistry, Staining, Immunofluorescence, Microscopy

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Motility of glioblastoma cells is driven by netrin-1 induced gain of stemness

doi: 10.1186/s13046-016-0482-0

Figure Lengend Snippet: Netrin-1 promotes the maintenance and motility of U251MG stem-like cells. a Either wild-type or NTN1 overexpressing U251MG cells were cultured under condition that favor neural stem cell proliferation for 3 weeks. The formed neurospheres were calculated. The number of the neurospheres is presented as relative to the wildtype U251MG. Error bars represent SD. * = p < 0,05. b The formed U251MG neurospheres were collected and the expression of stem cell markers was evaluated with quantitative real time PCR. The mRNA expression is normalized to the expression of GAPDH. The results are expressed as relative to the mRNA expression levels of U251MG cells cultured under standard conditions. Error bars represent the standard deviation. c The expression of the markers was also confirmed with immunofluorescence microscopy. Scale bars represent 50 μm. d U251MG neurospheres were dissociated and plated into Matrigel coated Boyden chambers. Cells were allowed to invade for 8 h and then quantitated. Number of invaded cells are presented as relative to the control group. Error bars represent SEM and * = p < 0,05. e Neurospheres were embedded into 3D Matrigel matrix and monitored for 24 h. The area of the spheroids was measured at the starting and ending point of the experiment. The graphs represent the relative change in the area of the spheroid. Error bars represent SEM and * = p < 0,05. f Images represent the endpoint of the 3D Matrigel invasion assays. The invasion of the control spheroids is presented also on Additional file 4: Video 1 and the invasion of the rNTN1 treated spheroids in Additional file 5: Video2. Scale bars represent 200 μm

Article Snippet: Either DMEM/F12 as such or supplemented with 50 ng/ml of recombinant NTN1 (R&D Systems) was added to the lower chamber.

Techniques: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Immunofluorescence, Microscopy, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Motility of glioblastoma cells is driven by netrin-1 induced gain of stemness

doi: 10.1186/s13046-016-0482-0

Figure Lengend Snippet: Netrin-1 promotes the motility of the primary glioblastoma stem-like cells. a Primary human GBM stem-like cells, GBM9, were mixed with GFP positive U251MG cells, that were either wildtype (U251), full length NTN1 producing (U251-NTN1FH) or NTN1 central domain producing (NTN1(II)FH). The spheroids were implanted into 3D Matrigel and their growth was monitored with Cell-IQ imaging system for 24 h. Gray arrows mark sprouts that are led by GBM9 cells. Green arrows mark sprouts that are led by GFP-positive U251MG cells. The invasion of the spheroids is presented also on Additional file 6: Video3, Additional file 7: Video4 and Additional file 8: Video5. b The number of sprouts led by the GFP-negative (GFP-) primary cells or GFP-positive (GFP+) U251MG cells were quantitated. The distribution as percentage is presented. Error bars represent SD. * = p < 0,05. c After 24 h 3D Matrigel invasion spheroids consisting of GBM9 cells mixed with U251-NTN1FH or U251-NTN1(II)FH were fixed and used for immunofluorescence staining against Nestin and HA. HA marks either full-length NTN1 (NTN1FH) or the central fragment of NTN1 (NTN1(II)FH)

Article Snippet: Either DMEM/F12 as such or supplemented with 50 ng/ml of recombinant NTN1 (R&D Systems) was added to the lower chamber.

Techniques: Imaging, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Motility of glioblastoma cells is driven by netrin-1 induced gain of stemness

doi: 10.1186/s13046-016-0482-0

Figure Lengend Snippet: Invasive primary GBM cells increase Notch2 activation upon NTN1FH expression. a After 24 h 3D Matrigel invasion spheroids consisting of GBM10 cells mixed with control U251, U251-NTN1FH or U251-NTN1(II)FH were fixed and used for immunofluorescence staining against cleaved Notch2 and HA. HA marks either full-length NTN1 (NTN1FH) or the central fragment of NTN1 (NTN1(II)FH). b Similarly as in ( a ) the invaded spheroids consisting of GM10 cells mixed with GFP positive U251MG cells were stained against cleaved Notch2. c Model of NTN1 induced invasion of GBM cells

Article Snippet: Either DMEM/F12 as such or supplemented with 50 ng/ml of recombinant NTN1 (R&D Systems) was added to the lower chamber.

Techniques: Activation Assay, Expressing, Control, Immunofluorescence, Staining

Journal: The Journal of Neuroscience

Article Title: Somatotopy of Mouse Spinothalamic Innervation and the Localization of a Noxious Stimulus Requires Deleted in Colorectal Carcinoma Expression by Phox2a Neurons

doi: 10.1523/JNEUROSCI.1164-22.2022

Figure Lengend Snippet: Resources and reagents used

Article Snippet: Reagent or resource Source/reference Identifier Mice (MGI notation) Dcc flox Krimpenfort et al. 2012 MGI: 3665466 Phox2a Cre Roome et al. 2022 RRID: NA Ai14 B6;129S6-Gt (ROSA)26Sortm14(CAG- tdTomato ) Hze/J The Jackson Laboratory Catalog #JAX:007908 RRID: IMSR_JAX:007908 Cdx2 FlpO Tg(CDX2-flpo)#Gld Dr. Martyn Goulding, Salk Institute, San Diego MGI: 5911680 RRID: NA Ai65 B6;129S-Gt ROSA)26Sortm65.1(CAG- dTomato) Hze/J The Jackson Laboratory Catalog #JAX:021875 RRID: IMSR_JAX:021875 Ntn1 Bgeo Skarnes et al., 1995 RRID: NA 129S1/SvImJ The Jackson Laboratory Catalog #JAX:002448 RRID: IMSR_JAX:002448 B6C3F1/J The Jackson Laboratory Catalog #JAX: 100010 RRID: IMSR_JAX:100010 Primary antibodies Goat anti-mouse DCC (1:500) R&D Systems Catalog #AF844 RRID: AB_2089765 Rabbit anti-Phox2a (1:10,000 from frozen stock) Jean-François Brunet (École Normale Supérieure, Paris) ( Tiveron et al., 1996 ) RRID: AB_2315159 Rabbit anti-RFP (red fluorescent protein; 1:1000) Rockland Catalog #600-401-379 RRID: AB_2209751 Goat anti-tdT (red fluorescent protein; 1:300) SICGEN Catalog #AB8181-200 RRID: AB_2722750 Rabbit anti-GFP (1:1000) Life Technologies Catalog #A-11122 RRID: AB_221569 Goat anti-netrin-1 (1:200) R&D Systems Catalog #AF1109, RRID: AB_2298775 Rabbit anti-c- fos (1:500) Cell Signaling Technology Catalog #2250S RRID: AB_2247211 Rabbit anti-Pax6 (1:500) Millipore Catalog #AB2237, RRID: AB_1587367 Rabbit anti-Sox2 (1:200) Abcam Catalog #97959 RRID: AB_2341193 Secondary antibodies Alexa 488 Donkey anti-Rabbit (1:500) Jackson ImmunoResearch Laboratories Catalog #711–545-152 Lot #141848 RRID: AB_2313584 Alexa 488 Donkey anti-Goat (1:500) Jackson ImmunoResearch Laboratories Catalog #705–545-147 Lot #136089 RRID: AB_2336933 Cy3 Donkey anti-Rabbit (1:500) Jackson ImmunoResearch Laboratories Catalog #711–165-152 Lot #138270 RRID: AB_2307443 Cy3 Donkey anti-Goat (1:500) Jackson ImmunoResearch Laboratories Catalog #705–165-147 Lot #134527 RRID: AB_2307351 Cy5 Donkey anti-Rabbit (1:500) Jackson ImmunoResearch Laboratories Catalog #711–175-152 Lot #138336 RRID: AB_2340607 Cy5 Donkey anti-Goat (1:500) Jackson ImmunoResearch Laboratories Catalog #: 705–175-147 Lot#: 134531 RRID: AB_2340415 Chemicals ( E )-Capsaicin Tocris Bioscience Catalog #0462 Lot #7A/218361 RRID: NA 4′,6-diamidino-2-phenylindole (DAPI) Thermo Fisher Scientific Catalog #D1306 RRID: NA Alexa 488-conjugated Choleratoxin B Thermo Fisher Scientific Catalog # C22841 Lot #2038245 RRID: NA X-gal Millipore Sigma Catalog #7240-90-6 RRID: NA MOWIOL Millipore Sigma Catalog #81381 RRID: NA Open in a separate window MGI, Mouse Genome Informatics; NA, Not applicable.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Netrin 1-Mediated Role of the Substantia Nigra Pars Compacta and Ventral Tegmental Area in the Guidance of the Medial Habenular Axons

doi: 10.3389/fcell.2021.682067

Figure Lengend Snippet: Ntn1 and NTN1 in the SNc and VTA. (A,B) Adjacent coronal paraffin section of a unique E12.5 brain. (A) In situ hybridization for Ntn1. (B) IHC against TH. (C,D) Adjacent coronal paraffin section of a E15.5 brain. (C) In situ hybridization for Ntn1 . (D) IHC against TH. (E–G) Midbrain coronal section of a E13.5 brain with fluorescent immunohistochemistry against NTN1 and TH. Both protein and mRNA are localized in the SNc and VTA. (F,G) are the single channels of (E) . Abbreviations: SNc, substantia nigra pars compacta; fr, fasciculus retroflexus; tst, tectospinal tract; VTA, ventral tegmental area. Scale bar: 200 and 100 μm in (E–G) .

Article Snippet: Samples were blocked using PBS-GT, i.e., PBS containing 0.2% gelatin (Prolabo) and 0.5% Triton X-100 (Sigma-Aldrich) O/N at RT, and then incubated in agitation for 7 days at 37°C with αROBO3 (1:300, R&D Systems AF3076), αTH antibody (1:1000, Abcam AB152), and αNTN1 (1:500, R&D Systems MAB1109).

Techniques: Paraffin Section, In Situ Hybridization, Immunohistochemistry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Netrin 1-Mediated Role of the Substantia Nigra Pars Compacta and Ventral Tegmental Area in the Guidance of the Medial Habenular Axons

doi: 10.3389/fcell.2021.682067

Figure Lengend Snippet: Ntn1 mRNA and NTN1 protein distribution in E13.5 wild type and Gli2 –/– mutant. (A,B) Midbrain coronal sections of E13.5 wild-type brains labeled with Ntn1 in situ hybridization (A) and light-sheet fluorescence microscopy images with NTN1 and TH in toto IHC (B) . (C–F) Coronal sections of E13.5 Gli2 –/– brains labeled with Ntn1 in situ hybridization (C) and with NTN1 and TH IHC by the iDISCO protocol (D–F) . Midbrain level in (C,D) and diencephalic level in (E,F) . Abbreviations: A13, A13 dopaminergic population; SNc, substantia nigra pars compacta; VTA, ventral tegmental area. Scale bar: 200 μm.

Article Snippet: Samples were blocked using PBS-GT, i.e., PBS containing 0.2% gelatin (Prolabo) and 0.5% Triton X-100 (Sigma-Aldrich) O/N at RT, and then incubated in agitation for 7 days at 37°C with αROBO3 (1:300, R&D Systems AF3076), αTH antibody (1:1000, Abcam AB152), and αNTN1 (1:500, R&D Systems MAB1109).

Techniques: Mutagenesis, Labeling, In Situ Hybridization, Fluorescence, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Netrin 1-Mediated Role of the Substantia Nigra Pars Compacta and Ventral Tegmental Area in the Guidance of the Medial Habenular Axons

doi: 10.3389/fcell.2021.682067

Figure Lengend Snippet: Ectopic Ntn1 -expressing cells attract the mHb rostrally in ONTs. (A) Schematic representation of the transfected cells’ location in E12.5 ONTs. Basal and floor plates are delimited by continuous lines. The floor plate is on the left and caudal to the bottom. (B) Characterization of the control experiment with GFP transfected cells labeled against GFP and DCC; the fr axons maintained their normal trajectory. (C) Characterization of the experiment with Ntn1 -expressing cells labeled against GFP and DCC; the mHb axons are redirected toward the cells located in the rostral diencephalon. (D) Quantification indicating the percentage of experiments with a normal or modified trajectory mHb fr. * p < 0.01. Abbreviations: Hb, habenula; Hyp, hypothalamus; Mb, midbrain; p1, prosomere 1 (pretectum); p2, prosomere 2 (thalamus); p3, prosomere 3 (prethalamus); r1, rhombomere 1; fr, fasciculus retroflexus. Scale bar: 100 μm.

Article Snippet: Samples were blocked using PBS-GT, i.e., PBS containing 0.2% gelatin (Prolabo) and 0.5% Triton X-100 (Sigma-Aldrich) O/N at RT, and then incubated in agitation for 7 days at 37°C with αROBO3 (1:300, R&D Systems AF3076), αTH antibody (1:1000, Abcam AB152), and αNTN1 (1:500, R&D Systems MAB1109).

Techniques: Expressing, Transfection, Control, Labeling, Modification

Journal: Frontiers in Cell and Developmental Biology

Article Title: Netrin 1-Mediated Role of the Substantia Nigra Pars Compacta and Ventral Tegmental Area in the Guidance of the Medial Habenular Axons

doi: 10.3389/fcell.2021.682067

Figure Lengend Snippet: The fr phenotype in Ntn1 –/– . Sagittal section of E16.5 wild-type (A) and Ntn1 –/– (B) brains stained with αDCC. The mHb fr in the mutant displayed an abnormal trajectory; a bunch of these fibers followed the normal path until the basal plate. At that point, they bended caudally toward caudal positions. The dotted line in A indicates the section plane in C–F . Coronal sections of a E16.5 wild type (C,D) and Ntn1 –/– (E,F) labeled for TH and DCC. In the mutant, the SNc presented an abnormal distribution, and the mHb fr was defasciculated and occupied a dorsal position. Abbreviations: mHb fr, medial habenular axons of the fasciculus retroflexus; SNc, substantia nigra pars compacta. Scale bar: 200 μm.

Article Snippet: Samples were blocked using PBS-GT, i.e., PBS containing 0.2% gelatin (Prolabo) and 0.5% Triton X-100 (Sigma-Aldrich) O/N at RT, and then incubated in agitation for 7 days at 37°C with αROBO3 (1:300, R&D Systems AF3076), αTH antibody (1:1000, Abcam AB152), and αNTN1 (1:500, R&D Systems MAB1109).

Techniques: Staining, Mutagenesis, Labeling