human ggf2  (BPS Bioscience)

Journal: Oncotarget

Article Title: Neuroblastoma in dialog with its stroma: NTRK1 is a regulator of cellular cross-talk with Schwann cells

doi:

Figure Lengend Snippet: (A) Graphs display NRG1 , FGF5 , PDGFA and CXCL12 expression in SY5Y-NTRK1 (green circles), SY5Y-NTRK2 (red circles) and SY5Y-vec (black circles) cells. The R2 platform was used to extract data from previously obtained microarray analyses . (B) Bars represent expression of NRG1 , FGF5 , PDGFA and CXCL12 measured using real-time RT-PCR and normalized to the geometric mean of GAPDH, UBC and HPRT expression in SY5Y-NTRK1 (green), SY5Y-NTRK2 (red) and control SY5Y-vec (black) cells. *p<0.05, ***p<0.0001 (C) NRG1 expression was analyzed in whole-cell lysates from SY5Y-NTRK1, SY5Y-NTRK2 and SY5Y-vec cells and in medium conditioned by the respective cell lines (CM). In whole-cell lysates β-actin served as the loading control. (D) NTRK1 (green squares) and NRG1 (blue dots) expression were re-analyzed using the R2 platform in previously generated exon array data from a representative cohort of 101 primary neuoblastomas . p=4×10 −12 , r=0.622.

Article Snippet: Briefly, 3 500 cells were seeded into 96-well plates, and changes in cellular indices (CI) as a surrogate marker for cell proliferation were detected after addition of conditioned media, 12.5μg/ml recombinant NRG1 with or without forskolin (Merck), a polyclonal antibody specific for human NRG1 (4μg/ml, cat.# AF2015, R&D Systems) or an isotype control for the anti-NRG1 antibody (8μg/ml, cat.# 02-6202, Invitrogen).

Techniques: Expressing, Microarray, Quantitative RT-PCR, Control, Generated

Journal: Oncotarget

Article Title: Neuroblastoma in dialog with its stroma: NTRK1 is a regulator of cellular cross-talk with Schwann cells

doi:

Figure Lengend Snippet: (A) Dual immunofluorescence staining using anti-rat NGFR primary antibody followed by goat anti-mouse IgG-FITC secondary antibody and DAPI counterstaining in Schwann cells in short-term culture from P3 rats (scale bar 50μm). (B) Bars represent the fractions of NGFR-positive and -negative cells from the total population of Schwann cells before and after positive selection using anti-rat NGFR primary antibody and rat anti-mouse IgG1 microbeads. (C) Growth curves of Schwann cells over a period of 80h as detected by electric cell-substrate impedance sensing in real-time following addition of medium conditioned by SY5Y-NTRK1 cells (NTRK1 CM), recombinant NRG1 + forskolin (NRG1 + forskolin), recombinant NRG1 (NRG1) or medium without additives (no additives). (D) Growth curves of Schwann cells over a period of 80h as detected by electric cell-substrate impedance sensing in real-time following addition of medium conditioned by SY5Y-NTRK1 cells (NTRK1 CM), medium conditioned by SY5Y-NTRK1 cells supplemented with anti-NRG1 isotype control (NTRK1 CM + isotype ctrl) or anti-NRG1 antibody (NTRK1 CM + anti-NRG1) or medium conditioned by SY5Y-vec cells (vec CM). For all growth curves in (C) and (D), cell index was normalized each at a base-time of 24h following plating of Schwann cells using an RTCA software-based algorithm. *p<0.05, **p<0.01, ***p<0.0001 (E) Schwann cells invading the membranes in a Boyden chamber assay 9 days after equal quantities of Schwann cells were plated in the upper chambers. Lower chambers contained SY5Y-NTRK1 cells cultured without additives (NTRK1), with anti-NRG1 antibody (NTRK1 + anti-NRG1) or with isotype control (NTRK1 + isotype ctrl) or SY5Y-vec cells (vec). Representative images are shown. Schwann cell nuclei are DAPI-stained. (F) Statistical analysis of Boyden chamber assays at conditions described in (E). Bars represent the percentage of Schwann cells that migrated through the membrane in relation to the number of cells initially seeded onto the upper membrane surface. ***p<0.0001, *p<0.05.

Article Snippet: Briefly, 3 500 cells were seeded into 96-well plates, and changes in cellular indices (CI) as a surrogate marker for cell proliferation were detected after addition of conditioned media, 12.5μg/ml recombinant NRG1 with or without forskolin (Merck), a polyclonal antibody specific for human NRG1 (4μg/ml, cat.# AF2015, R&D Systems) or an isotype control for the anti-NRG1 antibody (8μg/ml, cat.# 02-6202, Invitrogen).

Techniques: Immunofluorescence, Staining, Selection, Electric Cell-substrate Impedance Sensing, Recombinant, Control, Software, Boyden Chamber Assay, Cell Culture, Membrane

Journal: Oncotarget

Article Title: Neuroblastoma in dialog with its stroma: NTRK1 is a regulator of cellular cross-talk with Schwann cells

doi:

Figure Lengend Snippet: (A) Bars represent NGF concentrations (measured by ELISA) in a priori NGF-free medium conditioned by Schwann cells cultured without additives (no additives), with recombinant NRG1 (+recNRG1) or with medium conditioned by SY5Y-NTRK1 (+NTRK1 CM) or by SY5Y-vec (+vec CM) cells. NGF was not detected in medium conditioned by SY5Y-NTRK1 or SY5Y-vec cells. ***p<0.0001 (B) Detection of phosphorylated and total NTRK1 receptor expression (p-NTRK1 and NTRK1, respectively) by western blotting of whole-cell lysates of SY5Y cells with tetracycline-conditional expression of NTRK1 (SY5Y-TR-NTRK1) or control cells not expressing NTRK1 (SY5Y-TR). Prior to protein extraction, cells were exposed for 48h to tetracycline and Schwann cell-conditioned media (Tet + SC CM) or to nothing (no additives) or tetracycline alone (Tet) as negative controls, or to tetracycline and recombinant NGF (Tet + NGF) to induce and activate NTRK1 as a positive control. Experiments were repeated using control cells as negative controls. Actin expression was used as a loading control. (C-D) Differentiation of SY5Y-TR-NTRK1 cells after 5 days of tetracycline-induced NTRK1 expression in combination with stimulation by media conditioned by Schwann cells (Tet + SC CM) was assessed by measuring expression of the specific differentiation marker, GAP43 , using real-time RT-PCR (C) and assessing phenotypic signs of differentiation in cultures stained with rhodamine-labeled phalloidin (red) and counterstained with DAPI (blue) to visualize the differentiation marker F-actin and nuclei, respectively (D). Cells were exposed to nothing (no additives) or tetracycline (Tet) alone as negative controls or to tetracycline and recombinant NGF in combination (Tet + NGF) to activate NTRK1 as a positive control.

Article Snippet: Briefly, 3 500 cells were seeded into 96-well plates, and changes in cellular indices (CI) as a surrogate marker for cell proliferation were detected after addition of conditioned media, 12.5μg/ml recombinant NRG1 with or without forskolin (Merck), a polyclonal antibody specific for human NRG1 (4μg/ml, cat.# AF2015, R&D Systems) or an isotype control for the anti-NRG1 antibody (8μg/ml, cat.# 02-6202, Invitrogen).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant, Expressing, Western Blot, Control, Protein Extraction, Positive Control, Marker, Quantitative RT-PCR, Staining, Labeling

Journal: Oncotarget

Article Title: Neuroblastoma in dialog with its stroma: NTRK1 is a regulator of cellular cross-talk with Schwann cells

doi:

Figure Lengend Snippet: Expression of NTRK1 on neuroblastic cells induces production of NRG1, which attracts Schwann cells. Infiltrating Schwann cells, in turn, secrete the specific ligand of NTRK1, NGF. Neuroblastic cells in this scenario (right) are triggered to differentiate by the activation of the NTRK1 receptor via NGF binding. In the absence of NTRK1 expression (left), neuroblastic cells do not differentiate and continue to proliferate, fueling the phenotype of progressive disease.

Article Snippet: Briefly, 3 500 cells were seeded into 96-well plates, and changes in cellular indices (CI) as a surrogate marker for cell proliferation were detected after addition of conditioned media, 12.5μg/ml recombinant NRG1 with or without forskolin (Merck), a polyclonal antibody specific for human NRG1 (4μg/ml, cat.# AF2015, R&D Systems) or an isotype control for the anti-NRG1 antibody (8μg/ml, cat.# 02-6202, Invitrogen).

Techniques: Expressing, Activation Assay, Binding Assay