Journal: PLoS ONE

Article Title: Mesenchymal Stromal Cell Secretome Up-Regulates 47 kDa CXCR4 Expression, and Induce Invasiveness in Neuroblastoma Cell Lines

doi: 10.1371/journal.pone.0120069

Figure Lengend Snippet: Antibodies used in western blot studies.

Article Snippet: After blocking with blocking buffer for 30 minutes at room temperature, the membranes were incubated overnight at 4°C with human CXCL12 (Miltenyi Biotec, Cat No #130–093–997) in TBS-T.

Techniques: Western Blot, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Mesenchymal Stromal Cell Secretome Up-Regulates 47 kDa CXCR4 Expression, and Induce Invasiveness in Neuroblastoma Cell Lines

doi: 10.1371/journal.pone.0120069

Figure Lengend Snippet: a. The membrane proteins from 3 highly invasive neuroblastoma cell lines, MNB-OZ, NB 19 and SK-N-SH, and 2 less invasive cell lines, IMR 32 and SJ-N-KP were extracted, electrophoresed and blotted on to PVDF membrane. Protein detection was carried out using an anti-CXCR4 polyclonal antibody. Shown in figure is the representative result from 5 experiments. b. CXCR4 immune-precipiate from 3 highly invasive (NB 19, MNB-OZ & SK-N-SH) and 3 less invasive (SJ-N-KP, IMR 32 & NB Mass) cell lines were electrophoresed and blotted on to PVDF membrane. The membrane was incubated overnight with human CXCL12 in TBS-T at 4°C. The blot was washed and protein detection was carried out with anti-CXCL12 antibody. Shown in figure is the representative result from 3 experiments.

Article Snippet: After blocking with blocking buffer for 30 minutes at room temperature, the membranes were incubated overnight at 4°C with human CXCL12 (Miltenyi Biotec, Cat No #130–093–997) in TBS-T.

Techniques: Membrane, Incubation

Journal: PLoS ONE

Article Title: Mesenchymal Stromal Cell Secretome Up-Regulates 47 kDa CXCR4 Expression, and Induce Invasiveness in Neuroblastoma Cell Lines

doi: 10.1371/journal.pone.0120069

Figure Lengend Snippet: a. (left panel) CXCR4 immune precipitates from cell lines MNB-OZ, NB 19 and SK-N-SH, serum-starved (control) and pre-treated with mRPMI were subjected to western blot analysis. Protein detection was carried out using anti-CXCR4 polyclonal antibody. Shown in figure is the representative result from 3 experiments. (right panel) CXCR4 immune precipitates from cell lines MNB-OZ, NB 19 and SK-N-SH, serum-starved (control) and pre-treated with mRPMI were electrophoresed and blotted on to PVDF membrane. The membrane was incubated with human CXCL12 overnight at 4°C. Protein detection was carried out using anti-CXCL12 antibody. Shown in figure is the representative result from 3 experiments. b. The blots were quantified using imageJ. Plotted are the mean relative expression of CXCR4 from 3 experiments. (* p < 0.05) c. Plotted are the mean relative CXCR4 bound CXCL12 from 3 experiments. (* p < 0.05). d. Culture supernatants from cells serum-starved (control) and pre-treated with mRPMI were harvested and subjected to gelatin zymography. The gels were scanned and the images were analyzed using imageJ. The relative secretion of MMP-9 was calculated from 3 experiments (*p < 0.05). e. Total cell lysate from serum-starved and mRPMI treated SK-N-SH cells were subjected to western blot analysis, and protein detection was carried out using anti-integrin α3 or anti-integrin β1 antibodies. Anti-actin antibody was used for loading control.

Article Snippet: After blocking with blocking buffer for 30 minutes at room temperature, the membranes were incubated overnight at 4°C with human CXCL12 (Miltenyi Biotec, Cat No #130–093–997) in TBS-T.

Techniques: Control, Western Blot, Membrane, Incubation, Expressing, Zymography

Journal: PLoS ONE

Article Title: Mesenchymal Stromal Cell Secretome Up-Regulates 47 kDa CXCR4 Expression, and Induce Invasiveness in Neuroblastoma Cell Lines

doi: 10.1371/journal.pone.0120069

Figure Lengend Snippet: a. Neuroblastoma cells serum-starved (control) and pre-treated with CXCL12, were assayed for invasive potential using transwell invasion assays. Plotted is the mean number of cells migrating across the mesh from 5 experiments. b. Invasion index of neuroblastoma cell lines serum-starved (control) and pretreated with CXCL12. Plotted is the invasion index calculated from 5 experiments. c. Neuroblastoma cells serum-starved (control) and pre-treated with mRPMI, were assayed for invasive potential using transwell invasion assays. Plotted is the mean number of cells migrating across the mesh from 5 experiments. (*p<0.05) d. Invasion index of neuroblastoma cell lines serum-starved (control) and pretreated with mRPMI. Plotted is the invasion index calculated from 5 experiments. (*p<0.05)

Article Snippet: After blocking with blocking buffer for 30 minutes at room temperature, the membranes were incubated overnight at 4°C with human CXCL12 (Miltenyi Biotec, Cat No #130–093–997) in TBS-T.

Techniques: Control

Journal: Journal of Clinical Investigation

Article Title: Implantation of olfactory ensheathing cells promotes neuroplasticity in murine models of stroke

doi: 10.1172/jci34363

Figure Lengend Snippet: Figure 1 Phenotypic characterization of hOECs/ONFs. (A) The appearance of hOECs/ONFs by phase-contrast microscopy was either spindle shaped (arrow) or astrocyte-like (arrowhead). (B) Immunofluorescence colocalization analysis of hOECs/ONFs showed coexpression of p75 and GFAP, p75 and FN, p75 and S100, and GFAP and S100. (C) The proteins identified in our study as being produced by hOECs/ONFs including SDF-1α and its receptor, CXCR4, were coexpressed with p75 and GFAP in a double immunofluorescence study in hOECs/ONFs. Scale bars: 50 μm.

Article Snippet: In immunofluorescence colocalization of hOEC/ONF cells and cell-specific marker studies, each coronal section was treated with cell-specific antibodies: vWF (for endothelial cells; 1:200; Dako), GFAP (for astrocytes; 1:300), Neu-N (for neuronal nuclei; 1:50), nestin (for neuronal dendrites; 1:50), and MAP-2 (for neuronal dendrites; 1:300), all from Millipore; SDF-1α (1:200) and CXCR4 (1:200) from Torrey Pines Biolabs; and PrPC (1:300; M20; Santa Cruz Biotechnology Inc.).

Techniques: Microscopy, Immunofluorescence, Produced

Journal: Journal of Clinical Investigation

Article Title: Implantation of olfactory ensheathing cells promotes neuroplasticity in murine models of stroke

doi: 10.1172/jci34363

Figure Lengend Snippet: Figure 2 hOECs/ONFs upregulated the synthesis of soluble factors via the specific signaling pathway. (A–C) Under conditions of OGD, significantly increased expression of SDF-1α and CXCR4 was found by ELISA and Western blot analyses, respectively. (D and E) Expression of sig- nal transduction proteins (p-Akt and p-ERK1/2) increased significantly by 1 hour after treatment. (F) The upregulation of SDF-1α could be blocked by specific inhibitors of p-Akt and p-ERK1/2 (LY294002 and PD98059). Data are expressed as mean ± SEM. *P < 0.05 and **P < 0.01 versus control.

Article Snippet: In immunofluorescence colocalization of hOEC/ONF cells and cell-specific marker studies, each coronal section was treated with cell-specific antibodies: vWF (for endothelial cells; 1:200; Dako), GFAP (for astrocytes; 1:300), Neu-N (for neuronal nuclei; 1:50), nestin (for neuronal dendrites; 1:50), and MAP-2 (for neuronal dendrites; 1:300), all from Millipore; SDF-1α (1:200) and CXCR4 (1:200) from Torrey Pines Biolabs; and PrPC (1:300; M20; Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Transduction, Control

Journal: Journal of Clinical Investigation

Article Title: Implantation of olfactory ensheathing cells promotes neuroplasticity in murine models of stroke

doi: 10.1172/jci34363

Figure Lengend Snippet: Figure 6 Biological mechanism of neuro- plastic effects on the ischemic brain after intracerebral transplantation of hOECs/ONFs. (A) In a representa- tive brain section of a GFP-chime- ric mouse treated with or without hOECs/ONFs (white arrow indicates the injection site), GFP+ cells are seen dispersed over the periphery of the transplanted hOECs/ONFs and were significantly increased in quantity in the hOEC/ONF-treated mice in comparison with controls. In FISH analysis (white arrow, 2 red spots), hOECs/ONFs were shown to be of human origin (inset square in left panel). (B) IHC of hOEC/ONF treatment in the BrdU-labeled mice. Many BrdU+nestin+ cells were dis- tributed around the transplanted hOECs/ONFs. (C) Interestingly, 1 cell with 2 nuclei (cell fusion) was found in the implanted hOECs/ ONFs (white arrows, blue nucleus) and GFP+ cells (white arrowheads, red nucleus). The nucleic dye TOTO-3 (red) was used to define the outline of all nuclei in the sec- tion. (D) In a colocalization study (3D image) some bis-benzimide– labeled cells and some GFP+ cells colocalized with MAP-2+, vWF+, and GFAP+ cells in the penumbra of hOEC/ONF-treated ischemic rat brains. (E) SDF-1α–immunoreac- tive cells colocalized with a few bis- benzimide–labeled hOECs/ONFs and GFP+ cells. Data are expressed as mean ± SEM. *P < 0.05 versus control. Scale bars: 50 μm.

Article Snippet: In immunofluorescence colocalization of hOEC/ONF cells and cell-specific marker studies, each coronal section was treated with cell-specific antibodies: vWF (for endothelial cells; 1:200; Dako), GFAP (for astrocytes; 1:300), Neu-N (for neuronal nuclei; 1:50), nestin (for neuronal dendrites; 1:50), and MAP-2 (for neuronal dendrites; 1:300), all from Millipore; SDF-1α (1:200) and CXCR4 (1:200) from Torrey Pines Biolabs; and PrPC (1:300; M20; Santa Cruz Biotechnology Inc.).

Techniques: Transplantation Assay, Injection, Comparison, Labeling, Control