Journal: The Journal of Cell Biology

Article Title: The stromal cell–derived factor-1α/CXCR4 ligand–receptor axis is critical for progenitor survival and migration in the pancreas

doi: 10.1083/jcb.200304153

Figure Lengend Snippet: SDF-1α and CXCR4 colocalize in the islets of NOD and IFNγNOD pancreas. Panel A depicts a confocal image of NOD islet stained with SDF-1α (red) and CXCR4 (green) antibodies. Panel B shows SDF-1α and CXCR4 staining in an area of ductal proliferation and islet formation in the IFNγNOD pancreas. The central region of B which comprises the islet mass is magnified in C. Note that several islet cells exhibit SDF-1α (red) expression; others exhibit CXCR4 (green) expression, whereas the majority of cells exhibit double staining (yellow). A second region of ductal proliferation is shown in D. A prominent ductal region shown in the square is magnified in E. Most of the ductal cells in this region stain for CXCR4 only, with several of the cells exhibiting colocalization of CXCR4 and SDF-1α. Panel F shows nuclear staining (blue) of the region in E, confirming its ductal morphology (d, duct; i, islet). Bars, 25 μm.

Article Snippet: Paraffin embedded tissue was cut into 4-μm sections and stained using rabbit polyclonal anti–SDF-1α antibody against mouse SDF-1α (Cell Sciences), goat polyclonal anti-CXCR4 antibody raised against the NH 2 -terminal extracellular domain of mouse CXCR4 receptor (Capralogics Inc.), or guinea pig antibody against insulin (DakoCytomation).

Techniques: Staining, Expressing, Double Staining

Journal: The Journal of Cell Biology

Article Title: The stromal cell–derived factor-1α/CXCR4 ligand–receptor axis is critical for progenitor survival and migration in the pancreas

doi: 10.1083/jcb.200304153

Figure Lengend Snippet: SDF-1a and CXCR4 expression in embryonic NOD pancreas. Panel A illustrates SDF-1α expression by DAB staining in primitive islet structures in the fetal pancreas. Ductal areas are clear of SDF-1α staining. Panel B depicts CXCR4 expression in primitive islets and also in some ductal cells (d, duct; i, islet). (C) Representative double immunofluorescent images of SDF-1α (green) and insulin (red) reveal extensive colocalization (yellow) in the E18 pancreas with a population of cells expressing SDF-1α alone. (D) Insulin (red) and CXCR4 (green) immunofluorescent staining demonstrating that some cells display coexpression of CXCR4 and insulin (yellow), with a significant number of cells staining only for CXCR4. (E) Double immunofluorescent staining of CXCR4 (green) and SDF1-α (red) demonstrates that contiguous cells in the primitive islet clusters can express the ligand, the receptor, or both (yellow). (F) A ductal region surrounded by developing islet clusters magnified from E. Arrows point to duct cells. Bars, 25 μm.

Article Snippet: Paraffin embedded tissue was cut into 4-μm sections and stained using rabbit polyclonal anti–SDF-1α antibody against mouse SDF-1α (Cell Sciences), goat polyclonal anti-CXCR4 antibody raised against the NH 2 -terminal extracellular domain of mouse CXCR4 receptor (Capralogics Inc.), or guinea pig antibody against insulin (DakoCytomation).

Techniques: Expressing, Staining

Journal: The Journal of Cell Biology

Article Title: The stromal cell–derived factor-1α/CXCR4 ligand–receptor axis is critical for progenitor survival and migration in the pancreas

doi: 10.1083/jcb.200304153

Figure Lengend Snippet: CXCR4 neutralizing antibody treatment diminishes BrdU incorporation in the regenerating pancreatic duct cells of the IFNγNOD mouse. The ratio of BrdU-positive ductal cells to the total number of ductal cells is expressed as a mean percentage ± SEM (P < 0.005).

Article Snippet: Paraffin embedded tissue was cut into 4-μm sections and stained using rabbit polyclonal anti–SDF-1α antibody against mouse SDF-1α (Cell Sciences), goat polyclonal anti-CXCR4 antibody raised against the NH 2 -terminal extracellular domain of mouse CXCR4 receptor (Capralogics Inc.), or guinea pig antibody against insulin (DakoCytomation).

Techniques: BrdU Incorporation Assay

Journal: The Journal of Cell Biology

Article Title: The stromal cell–derived factor-1α/CXCR4 ligand–receptor axis is critical for progenitor survival and migration in the pancreas

doi: 10.1083/jcb.200304153

Figure Lengend Snippet: CXCR4 neutralization increases apoptosis in the pancreatic duct cells of the IFNγNOD mouse. Sections from the pancreas of two mice each from the CXCR4 antibody–treated and control groups were assessed for apoptosis using the TUNEL method. The number of apoptotic nuclei was quantitated as a percentage of the total number of duct cells in the pancreas. Values are expressed as mean ± SEM (P < 0.003).

Article Snippet: Paraffin embedded tissue was cut into 4-μm sections and stained using rabbit polyclonal anti–SDF-1α antibody against mouse SDF-1α (Cell Sciences), goat polyclonal anti-CXCR4 antibody raised against the NH 2 -terminal extracellular domain of mouse CXCR4 receptor (Capralogics Inc.), or guinea pig antibody against insulin (DakoCytomation).

Techniques: Neutralization, Control, TUNEL Assay

Journal: The Journal of Cell Biology

Article Title: The stromal cell–derived factor-1α/CXCR4 ligand–receptor axis is critical for progenitor survival and migration in the pancreas

doi: 10.1083/jcb.200304153

Figure Lengend Snippet: CXCR4 neutralization causes reduced numbers of PDX1-positive progenitor cells within the ductal cell population in the IFNγNOD pancreas . Sections from the pancreas of four mice each from the CXCR4 antibody treatment and control groups were assessed for PDX1 expression. Values are expressed as mean ± SEM (P < 0.03).

Article Snippet: Paraffin embedded tissue was cut into 4-μm sections and stained using rabbit polyclonal anti–SDF-1α antibody against mouse SDF-1α (Cell Sciences), goat polyclonal anti-CXCR4 antibody raised against the NH 2 -terminal extracellular domain of mouse CXCR4 receptor (Capralogics Inc.), or guinea pig antibody against insulin (DakoCytomation).

Techniques: Neutralization, Control, Expressing

Journal: The Journal of Cell Biology

Article Title: The stromal cell–derived factor-1α/CXCR4 ligand–receptor axis is critical for progenitor survival and migration in the pancreas

doi: 10.1083/jcb.200304153

Figure Lengend Snippet: Disruption of the CXCR4 SDF-1α axis shifts the distribution of the duct cell population into smaller ducts. Hematoxylin stained sections from BOUIN's fixed pancreas from CXCR4 neutralizing antibody ( n = 7) or rabbit IgG ( n = 7) were scored for ductal cells. Values are expressed as mean ± SEM (P < 0.001).

Article Snippet: Paraffin embedded tissue was cut into 4-μm sections and stained using rabbit polyclonal anti–SDF-1α antibody against mouse SDF-1α (Cell Sciences), goat polyclonal anti-CXCR4 antibody raised against the NH 2 -terminal extracellular domain of mouse CXCR4 receptor (Capralogics Inc.), or guinea pig antibody against insulin (DakoCytomation).

Techniques: Disruption, Staining

Journal: The Journal of Biological Chemistry

Article Title: The Chlamydia Effector TarP Mimics the Mammalian Leucine-Aspartic Acid Motif of Paxillin to Subvert the Focal Adhesion Kinase during Invasion *

doi: 10.1074/jbc.M114.604876

Figure Lengend Snippet: Focal adhesion components associate with TarP and its conserved LD-like motif. A , ClustalW sequence alignment showing the putative actin binding domains ( ABD ; open box ) from Tarp orthologs with the conserved residues of the LD motif highlighted in yellow . The red asterisk indicates the recently identified F-actin binding domains (FAB1). The consensus sequence of the LD domain within TarP was aligned with the paxillin LD domain to highlight homology. Species analyzed include C. caviae (GPIC), Chlamydophila abortus ( CAb ), Chlamydophila felis ( CFe ), Chlamydia muridarum ( MoPn ), C. trachomatis serovars A, L2, and D. B , the TarP-LD-like motif was ectopically expressed in Cos7 cells for either 24 or 8 h and tested for the ability to co-recruit pY397-FAK and actin ( white arrowheads ). When transient expression was limited to 8 h rather than 24 h, the TarP-LD specifically localized to FAK-rich focal adhesion structures and to various points along actin stress fibers. Large arrowheads in the 24-h panel indicate the co-recruitment of pY397-FAK and actin to TarP-LD aggregates, whereas in the 8-h panel, they indicate the localization of TarP-LD to focal adhesion structures. Small arrowheads indicate the localization of TarP-LD along the F-actin. Scale bars , 10 μm.

Article Snippet: Coverslips were washed 3 times in PBS and fixed with 4% paraformaldehyde for 15 min. Extracellular EBs were labeled with a mouse IgG anti- C. trachomatis LPS 1:750 (Abcam) primary and goat anti-mouse IgG Alexa Fluor 488-conjugated secondary antibody (Invitrogen).

Techniques: Sequencing, Binding Assay, Expressing

Journal: Journal of cell science

Article Title: The chemokine SDF-1 differentially regulates axonal elongation and branching in hippocampal neurons.

doi: 10.1242/jcs.01694

Figure Lengend Snippet: Fig. 1. Western blot analysis of CXCR4 in cultured hippocampal neurons. Cultured neurons and rat brain membrane homogenates were immunoblotted with anti-CXCR4 antibody. The antibody revealed a 45 kDa band in both cultured neurons and rat brain samples, corresponding to the expected molecular mass for CXCR4. Molecular weight markers are indicated on the right.

Article Snippet: The membranes were incubated overnight in polyclonal goat anti-CXCR4 antibody (1:2000; Santa Cruz Biotechnology) diluted in TBST containing 8% dehydrated milk, washed with TBST, incubated for 1 hour in HRP-conjugated secondary goat anti-mouse antibody (1:20,000; Jackson ImmunoResearch) and visualized using chemiluminescent substrate (Amersham) and exposure to X-ray film.

Techniques: Western Blot, Cell Culture, Membrane, Molecular Weight

Journal: Journal of cell science

Article Title: The chemokine SDF-1 differentially regulates axonal elongation and branching in hippocampal neurons.

doi: 10.1242/jcs.01694

Figure Lengend Snippet: Fig. 2. CXCR4 is distributed in a non-uniform manner both in the somatodendritic and axonal domains, but is preferentially associated with axons. Hippocampal neurons at day 10 in culture were fixed and immunostained for CXCR4 (A1,B1) and either the dendritic marker MAP2 (A2) or the synaptic marker SV2 (B2). CXCR4 immunoreactivity was detected within neuronal cell bodies and a subset of nerve cell processes identified as MAP2-positive and -negative processes, indicating a somatodendritic and axonal localization of CXCR4 (A1-A3). The extent of axonal CXCR4 immunostaining was however more prominent than the dendritic labelling. CXCR4 immunoreactivity was not uniformly distributed but was often observed in large clusters along the cell body and processes (arrows in insets corresponding to higher magnification of boxed regions in A3). Double labelling with SV2 (B3) shows that these CXCR4 clusters did not overlap with the synaptic marker, indicating that CXCR4 clusters were localized at nonsynaptic sites. Bar, 20 µm.

Article Snippet: The membranes were incubated overnight in polyclonal goat anti-CXCR4 antibody (1:2000; Santa Cruz Biotechnology) diluted in TBST containing 8% dehydrated milk, washed with TBST, incubated for 1 hour in HRP-conjugated secondary goat anti-mouse antibody (1:20,000; Jackson ImmunoResearch) and visualized using chemiluminescent substrate (Amersham) and exposure to X-ray film.

Techniques: Marker, Immunostaining

Journal: Journal of cell science

Article Title: The chemokine SDF-1 differentially regulates axonal elongation and branching in hippocampal neurons.

doi: 10.1242/jcs.01694

Figure Lengend Snippet: Fig. 3. The somatodendritic and axonal CXCR4 receptors are functionally coupled to intracellular Ca2+ mobilization. (A) Phase contrast and pseudocolour images of a field containing dendrites and axons. Neurons were loaded with Fura-2 AM and exposed to 20 nM SDF-1. Pseudocolour images were taken 5 seconds before SDF-1 stimulation (–5 sec), 7 seconds and 40 seconds after SDF-1 stimulation. Most processes displayed SDF-1-induced [Ca2+]i increase. The colour calibration bar shows pseudocolour mapping of the ratio of fluorescence emission at 340 and 380 nm. (B) Higher magnification of the boxed regions shown in A show a dendrite and an axon based on morphological features analysed by phase-contrast microscopy. (C) Traces of variations of [Ca2+]i recorded in the boxed regions depicted in A. A rise in [Ca2+]i levels was observed with a similar time course for dendrites and axons, 3 seconds after SDF-1 exposure as symbolized by asterisks, and recovered to near basal levels within 1 minute. Bar, 10 µm.

Article Snippet: The membranes were incubated overnight in polyclonal goat anti-CXCR4 antibody (1:2000; Santa Cruz Biotechnology) diluted in TBST containing 8% dehydrated milk, washed with TBST, incubated for 1 hour in HRP-conjugated secondary goat anti-mouse antibody (1:20,000; Jackson ImmunoResearch) and visualized using chemiluminescent substrate (Amersham) and exposure to X-ray film.

Techniques: Microscopy

Journal: Journal of cell science

Article Title: The chemokine SDF-1 differentially regulates axonal elongation and branching in hippocampal neurons.

doi: 10.1242/jcs.01694

Figure Lengend Snippet: Fig. 4. Developmental distribution of CXCR4 in hippocampal neurons from day 2 to day 7. Hippocampal cultures were fixed at day 2 (A1,A2), day 4 (B1-B4) and day 7 (C1,C2) and immunostained for endogenous CXCR4 at all stages and for MAP2 at day 4 (B3,B4) or were transfected at the time of plating with CXCR4-GFP (D1,D2) or the parent vector GFP (E1,E2). At day 2 and day 4, the tips of the axon and the other neurites visualized in the phase-contrast images (A1,B1) were frequently enriched in CXCR4 immunoreactivity as seen in the paired immunolabelled images (A2,B2). This CXCR4 accumulation at the leading edge of processes was observed in the absence (A1-A2, arrows) and in the presence of contacts with other neurons (B1-B4, arrows). At day 4, the tips of two processes originating from the same cell body were highly immunopositive for CXCR4 at the site of contact with neighbouring neurons (B1-B4, arrows). At day 7 (C1,C2), CXCR4 immunoreactivity was no longer enriched at growing tips, but was rather detected along a subset of processes. At day 2, in neurons transfected with CXCR4-GFP cDNA the fluorescence was preferentially concentrated at the tips of processes (D1,D2), whereas in neurons transfected with the parent GFP vector the fluorescence was diffusely distributed throughout the cell (E1,E2). Bar, 20 µm.

Article Snippet: The membranes were incubated overnight in polyclonal goat anti-CXCR4 antibody (1:2000; Santa Cruz Biotechnology) diluted in TBST containing 8% dehydrated milk, washed with TBST, incubated for 1 hour in HRP-conjugated secondary goat anti-mouse antibody (1:20,000; Jackson ImmunoResearch) and visualized using chemiluminescent substrate (Amersham) and exposure to X-ray film.

Techniques: Transfection, Plasmid Preparation

Journal: Journal of cell science

Article Title: The chemokine SDF-1 differentially regulates axonal elongation and branching in hippocampal neurons.

doi: 10.1242/jcs.01694

Figure Lengend Snippet: Fig. 5. SDF-1 regulates axonal patterning without influencing the other neurites. Hippocampal neurons were treated at day 1 for 16 hours with (B) or without (A) 50 nM SDF-1 and were fixed at day 2 for analysis by phase-contrast microscopy. Quantification of the length of the axon and the other neurites in the presence and absence of the glial feeder layer (C), the growth cone area (D), the soma area (E), the number of processes emerging from the cell body (F), the number of primary branch points per 100 µm of axon and other neurites (G) and the number of growth cones per process (H) was carried out. The analysis was performed on control cells, cells treated with 50 nM SDF-1, cells treated with 50 nM SDF-1 in the presence of 1 µM bicyclam, a CXCR4 antagonist, and cells treated with bicyclam alone. Data are presented as the mean±s.e.m. of two to three independent experiments. **P<0.01, ***P<0.001 (Student’s t-test) compared to corresponding measurements in control cells. Bar, 12 µm.

Article Snippet: The membranes were incubated overnight in polyclonal goat anti-CXCR4 antibody (1:2000; Santa Cruz Biotechnology) diluted in TBST containing 8% dehydrated milk, washed with TBST, incubated for 1 hour in HRP-conjugated secondary goat anti-mouse antibody (1:20,000; Jackson ImmunoResearch) and visualized using chemiluminescent substrate (Amersham) and exposure to X-ray film.

Techniques: Microscopy, Control

Journal: Journal of cell science

Article Title: The chemokine SDF-1 differentially regulates axonal elongation and branching in hippocampal neurons.

doi: 10.1242/jcs.01694

Figure Lengend Snippet: Fig. 6. SDF-1 secreted from transfected COS cells acts as an extracellular signal regulating axonal patterning of CXCR4-GFP- expressing neurons. (A,B) COS cells transfected with CXCR4-GFP were incubated with or without 100 nM SDF-1 for 2 hours. In unstimulated cells, CXCR4-GFP was diffusely distributed throughout the cells (A) whereas SDF-1 stimulation induced the formation of CXCR4-GFP-containing intracellular clusters (B). (C) COS cells were separately transfected with either CXCR4-GFP (green) or SDF-1 (red, identified with an anti-SDF-1 antibody), and were subsequently co-cultured. CXCR4-GFP distribution exhibits a typical internalization pattern only if an SDF-1-expressing cell was located in the proximity of the former. By contrast, in cells distant from any SDF-1-transfected cells, CXCR4-GFP was distributed diffusely over the cell. (D,E) Hippocampal neurons were transfected at the time of plating with CXCR4-GFP. COS cells previously transfected with SDF-1 were added to the neuron culture at day 1 and the co-culture was fixed at day 2. CXCR4-GFP-expressing neurons in contact with SDF-1-expressing COS cells showed a reduced axonal length (E1,E2) compared to neurons distant from any SDF-1-transfected cells (D1,D2). Bar, 20 µm.

Article Snippet: The membranes were incubated overnight in polyclonal goat anti-CXCR4 antibody (1:2000; Santa Cruz Biotechnology) diluted in TBST containing 8% dehydrated milk, washed with TBST, incubated for 1 hour in HRP-conjugated secondary goat anti-mouse antibody (1:20,000; Jackson ImmunoResearch) and visualized using chemiluminescent substrate (Amersham) and exposure to X-ray film.

Techniques: Transfection, Expressing, Incubation, Cell Culture, Co-Culture Assay