Journal: The Journal of Neuroscience

Article Title: N-Cadherin Mediates Axon-Aligned Process Growth and Cell–Cell Interaction in Rat Schwann Cells

doi: 10.1523/JNEUROSCI.22-10-04066.2002

Figure Lengend Snippet: SC adhesion and network formation are perturbed when N-cadherin function is blocked

Article Snippet: Positive controls were protein lysates of epidermoid carcinoma cell line A 431 (E-cadherin), rat brain (N-cadherin, R-cadherin), mouse neonate (M-cadherin) (all from BD Transduction), and Madin-Darby canine kidney (MDCK) epithelial cells (K-cadherin).

Techniques:

Journal: The Journal of Neuroscience

Article Title: N-Cadherin Mediates Axon-Aligned Process Growth and Cell–Cell Interaction in Rat Schwann Cells

doi: 10.1523/JNEUROSCI.22-10-04066.2002

Figure Lengend Snippet: N-cadherin is present in developing peripheral nerves and SCs cultured in the presence of mitogens. A, N-cadherin expression was analyzed in embryonic and adult peripheral nerves. N-cadherin is detected as a strong band at 130 kDa in embryonic limb (E15), but a much weaker signal is seen in adult sciatic nerve (ASN). B, N-cadherin protein amounts increase after culturing adult sciatic nerve-derived SCs in the presence of mitogens. Dissociated SCs from adult sciatic nerve explants (P1) were cultured for 3 d in the absence (P1-m) or presence (P1+m) of mitogens (forskolin, pituitary extract, and heregulin β1). The Western blots show weak N-cadherin immunoreactivity in SCs in the absence of mitogens (P1-m). Normalized values measured in four different autoradiograms using background-corrected ratios of N-cadherin divided by β-actin show a mean 1.3-fold increase in N-cadherin protein amounts after mitogen treatment (P1+m; p < 0.05; Tukey–Kramer, multiple comparisons) compared with SCs cultured without mitogens (P1-m). C, Cell density regulates N-cadherin protein amount in cultured rat SCs. Equal amounts of protein (15 μg per lane) from purified SCs of the same sciatic nerve preparation and passage number (P3) but cultured at various cell densities (1.8 × 106 SCs/100 mm2, subconfluent; 3.7 × 106 SCs/100 mm2, pattern forming; 6.4 × 106 SCs/100 mm2, confluent; and 10.5 × 106 SCs/100 mm2, over-confluent) in the presence of mitogens were analyzed by Western blotting. Strong N-cadherin signals are detected in samples of 1.8, 3.7, and 6.4 × 106SCs/100 mm2, but a weak signal is seen in cells grown at a density of 10.5 × 106 cells/100 mm2. Desitometric analysis shows that N-cadherin protein amount drops more then threefold in over-confluent versus confluent or subconfluent SC cultures.

Article Snippet: Positive controls were protein lysates of epidermoid carcinoma cell line A 431 (E-cadherin), rat brain (N-cadherin, R-cadherin), mouse neonate (M-cadherin) (all from BD Transduction), and Madin-Darby canine kidney (MDCK) epithelial cells (K-cadherin).

Techniques: Cell Culture, Expressing, Derivative Assay, Western Blot, Purification

Journal: The Journal of Neuroscience

Article Title: N-Cadherin Mediates Axon-Aligned Process Growth and Cell–Cell Interaction in Rat Schwann Cells

doi: 10.1523/JNEUROSCI.22-10-04066.2002

Figure Lengend Snippet: N-cadherin is localized at regions of SC and SC–axon contacts. A, B, Confocal images show SCs cultured 6 hr (A) and 24 hr (B) on ammoniated collagen that were immunostained for N-cadherin (red) and S100 (green). An intense band of N-cadherin is seen at intercellular junctions between SCs (A,arrow). Strong N-cadherin signals are detected in process tips of SCs (A, arrowheads; scale bar, 25 μm) and at cell–cell contact areas (B, arrowhead; scale bar, 50 μm). C, N-cadherin localization is shown in SC–DRG cocultures 24 hr after seeding SCs onto DRG neurons. Confocal images show vitally stained SCs (Cell Tracker,green), immunostained axons (anti-neurofilament M, Cy5; color coded in blue), and N-cadherin (red/pink/yellow). N-cadherin is accumulated at sites of contact with axons (arrows) and at filopodia and lamellipodia of SC processes (arrowheads). D, N-cadherin (red) is shown in an SC (green) associated with a small axon bundle (blue) after 24 hr of coculturing with DRG neurons. Out of a series of five optical sections, one confocal section of 0.4 μm is shown (XY,center). Orthogonal sectioning through the stack of all five images (depth 1.6 μm) is shown in horizontal (XZ) and vertical (YZ) planes. In all three planes, intense accumulation of N-cadherin (red) between SC process (green) and axons (blue,arrowheads) is seen in all areas of contact. Scale bar, 10 μm. E, SCs are in alignment with axons after 4 d of coculturing with DRG neurons. Confocal images show triple labeling using Cell Tracker to stain SCs (green) and anti-neurofilament M antibody to label axons (blue) and anti-N-cadherin (red). N-cadherin immunofluorescence is weaker and more evenly distributed than after 24 hr. This image was recorded using higher detector voltage compared with Cto increase signal intensity for N-cadherin. Scale bar, 20 μm.

Article Snippet: Positive controls were protein lysates of epidermoid carcinoma cell line A 431 (E-cadherin), rat brain (N-cadherin, R-cadherin), mouse neonate (M-cadherin) (all from BD Transduction), and Madin-Darby canine kidney (MDCK) epithelial cells (K-cadherin).

Techniques: Cell Culture, Staining, Labeling, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: N-Cadherin Mediates Axon-Aligned Process Growth and Cell–Cell Interaction in Rat Schwann Cells

doi: 10.1523/JNEUROSCI.22-10-04066.2002

Figure Lengend Snippet: Calcium is required for SC–SC and SC–axon interaction. The contribution of cadherins to SC contact behavior is shown by the effect of lowering calcium in SC–SC cultures (A, B) and SC–DRG cocultures (C–F). A, Vitally labeled SCs (green) cultured for 24 hr in normal calcium (N2 medium, 1% FBS) are lined up in arrays.Inset in A shows SCs in normal calcium with intense N-cadherin-positive cell–cell contacts (red, arrows). B, The SC network is perturbed in low calcium (N2 medium, 1% FBS, 0.22 mm Ca2+), and SCs were not interacting with each other. B, Inset, SC processes failed to contact other SCs, and N-cadherin is shown more evenly distributed on the cell surface (red/yellow). Scale bar, 20 μm.C–F, SCs (Cell Tracker,green) were plated onto axons of DRG neurons (anti-neurofilament M, red) in either normal calcium levels (1.15 mm Ca2+; C,E) or low calcium levels (0.15 mmCa2+; D, F) medium. C, E, SCs are observed in alignment with axons at 4 hr (C) and 24 hr (E) after plating. C,Inset, Confocal image shows an SC adjoining a small axon bundle with its processes aligned. D, F, By comparison, SCs in low calcium medium fail to align with axons at either 4 hr (D) or 24 hr (F) of coculturing. D,Inset, One SC (green) not aligning with axons but crossing the axon bundle (red).

Article Snippet: Positive controls were protein lysates of epidermoid carcinoma cell line A 431 (E-cadherin), rat brain (N-cadherin, R-cadherin), mouse neonate (M-cadherin) (all from BD Transduction), and Madin-Darby canine kidney (MDCK) epithelial cells (K-cadherin).

Techniques: Labeling, Cell Culture

Journal: The Journal of Neuroscience

Article Title: N-Cadherin Mediates Axon-Aligned Process Growth and Cell–Cell Interaction in Rat Schwann Cells

doi: 10.1523/JNEUROSCI.22-10-04066.2002

Figure Lengend Snippet: Removal of full-size cadherins from the SC surface prevents SC contact formation and alignment to axons. A, The Western blots show the effect of trypsin digestion on N-cadherin in the presence (TCa2+) and absence (TE) of calcium ions. Cultured rat SCs were treated for 10 min in TE (lanes 1–3) or TCa2+(lanes 4–6), and protein samples were harvested immediately after digestion (lanes 1,4), as well as after 4 hr (lanes 2, 5) and 24 hr (lanes 3,6) of culturing. Samples were probed for N-cadherin and β-actin. The 130 kDa N-cadherin band was almost absent in the TE-treated SCs (lane 1) and then recovered to ∼50% within 4 hr (lane 2) and almost fully within 24 hr (lane 3). TE-treated SCs show an N-cadherin fragment of ∼66 kDa (lane 1–3); this band disappeared within 24 hr. In the TCa2+-treated SCs, the 130 kDa N-cadherin band was detected with unreduced intensity (lanes 4-6), even immediately after trypsin digestion (lane 4), and the 66 kDa fragment was not detectable.B, C, The interaction of SCs treated for 10 min in TE (B) or TCa2+(C) are shown after 4 hr of culturing.B, TE-treated SCs fail to form N-cadherin-positive contacts. B, Inset, A vitally labeled, TE-treated SC exhibits weak and discontinuous surface N-cadherin and an intense aggregate of N-cadherin in the cytoplasm (yellow). C, Cadherin-protected SCs (TCa2+) formed numerous cell–cell N-cadherin-positive contacts. C, Inset, Three SCs (green) show bands of N-cadherin staining (red) at cell surfaces, particularly at cell–cell contact sites. D, E, The interaction of TE- and TCa2+-treated SCs (Cell Tracker, green) with axons (N-cadherin immunofluorescence, red) after 4 hr plating onto DRG neurons is shown. D, The confocal image shows a TE-treated SC not aligning with the axon bundles. E, A pair of cadherin-protected (TCa2+) SCs display all processes in alignment with axons. Scale bar, 20 μm.

Article Snippet: Positive controls were protein lysates of epidermoid carcinoma cell line A 431 (E-cadherin), rat brain (N-cadherin, R-cadherin), mouse neonate (M-cadherin) (all from BD Transduction), and Madin-Darby canine kidney (MDCK) epithelial cells (K-cadherin).

Techniques: Western Blot, Cell Culture, Labeling, Staining, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: N-Cadherin Mediates Axon-Aligned Process Growth and Cell–Cell Interaction in Rat Schwann Cells

doi: 10.1523/JNEUROSCI.22-10-04066.2002

Figure Lengend Snippet: N-cadherin is the most abundant cadherin expressed in cultured rat SCs and DRG neurons. The Western blots were performed using protein lysates from cultured rat DRG neurons (N) and purified rat SCs, cultured in mitogens (S). They were compared with positive control samples (+) for the various cadherins. These controls were lysates of epidermoid carcinoma cell line A 431 (E-cad), rat brain (N-cad, R-cad), mouse neonate (M-cad), and MDCK cells (K-cad). Positive controls gave strong signals for all tested cadherins. The autoradiogram shows no signals for R-cadherin, E-cadherin, and M-cadherin; however, strong N-cadherin signals were detected in cultured SCs and DRG neurons. Faint bands were detected for K-cadherin in cultured SCs and DRG neurons.

Article Snippet: Positive controls were protein lysates of epidermoid carcinoma cell line A 431 (E-cadherin), rat brain (N-cadherin, R-cadherin), mouse neonate (M-cadherin) (all from BD Transduction), and Madin-Darby canine kidney (MDCK) epithelial cells (K-cadherin).

Techniques: Cell Culture, Western Blot, Purification, Positive Control

Journal: The Journal of Neuroscience

Article Title: N-Cadherin Mediates Axon-Aligned Process Growth and Cell–Cell Interaction in Rat Schwann Cells

doi: 10.1523/JNEUROSCI.22-10-04066.2002

Figure Lengend Snippet: N-cadherin-blocking antibodies perturb SC network formation. A–D, L7 IgG perturbs adhesion of NC+L cells using a 24 hr aggregation assay. A, Control (ctrl) culture of Cell Tracker-labeled NC+L cells (green), kept in N2 medium plus 1% FBS, show “cobblestone”-like arrangement and N-cadherin-positive bands between cells (red).B, Cultures in low calcium (low Ca2+) exhibit spherically shaped, single L-cells and only rare N-cadherin-positive cell–cell contacts.C, In the presence of control antibody (ctrl ab; L4 IgG 1:50 in N2), cell clusters with N-cadherin-positive junctions are shown similar to those of controls (A). D, Cultures treated with N-cadherin-binding antibody (N-cad ab; L7 IgG, 1:50 in N2) display single cells and less frequent N-cadherin-positive cell–cell contacts. E, F, Vitally stained SCs (green) cultured for 24 hr in the presence of control antibody (ctrl ab; rabbit IgG 1:50 in N2) form arrays of cells (E) and display N-cadherin-positive bands (E′, red,arrows). SCs treated with N-cadherin-binding antibodies (N-cad ab; L7 IgG 1:50 in N2 medium) fail to form arrays (F), and N-cadherin-containing contacts between SCs are rarely seen (F′). Scale bar, 20 μm.G, Plotted are the averages of the adhesion factor obtained from four SC cultures under each condition. The adhesion factor was determined by dividing the number of N-cadherin-positive cell–cell contacts by the number of SCs found in groups (contacting neighboring SCs). Control cultures were SCs in normal calcium medium (N2) as well as SCs in low calcium medium (−Ca2+). Treated cultures were incubated with N-cadherin-binding antibodies (N-cad ab; L7 IgG) and nonbinding control antibody (ctrl; L4 IgG). The adhesion factor is significantly lower under low calcium conditions and in L7 IgG-treated SC cultures (p < 0.01; Tukey–Kramer multiple comparisons).

Article Snippet: Positive controls were protein lysates of epidermoid carcinoma cell line A 431 (E-cadherin), rat brain (N-cadherin, R-cadherin), mouse neonate (M-cadherin) (all from BD Transduction), and Madin-Darby canine kidney (MDCK) epithelial cells (K-cadherin).

Techniques: Blocking Assay, Labeling, Binding Assay, Staining, Cell Culture, Incubation

Journal: The Journal of Neuroscience

Article Title: N-Cadherin Mediates Axon-Aligned Process Growth and Cell–Cell Interaction in Rat Schwann Cells

doi: 10.1523/JNEUROSCI.22-10-04066.2002

Figure Lengend Snippet: N-cadherin function-blocking agents decrease the number of SCs aligning to axons. A, B, Cell Tracker-labeled SCs (green) were cocultured with DRG neurons in the presence of 0.5 mg/ml of the cyclic pentapeptides HGV (0.5 mg/ml) or HAV (0.5 mg/ml). After 4 hr, cultures were fixed and axons were stained for neurofilament M (red). A, Spindle-shaped SCs are seen with their extensions aligned to axons in cultures treated with the control peptide (HGV). B, In the presence of blocking peptide HAV, SCs display multiple processes that failed to align with axons. Scale bar, 20 μm. C,D, SCs (Cell Tracker, green) were cocultured with DRG neurons for 24 hr in defined medium (ctrl; N2) or the presence of N-cadherin function-blocking guinea pig antibody (Ncad ab; gp1260 IgG, 1:100). C, In control cultures (N2), most SCs show processes well aligned to axon bundles. Both N-cadherin-blocking antibodies (L7 IgG and gp1260) were used to treat SC–DRG cocultures.D, A coculture treated with N-cadherin-blocking antibody (here gp1260 IgG) shows SCs that failed to associate with axons (arrows) as well as SCs with short or no processes (∗) and some SCs with axon-aligned processes (arrowheads). Scale bar, 20 μm. E, Plotted are average percentages of SCs aligned with axons. Error bars represent SDs from three cultures of each condition. Significantly fewer SCs align to axons in HAV as well as N-cadherin-blocking antibody (here data from L7 IgG)-treated cocultures compared with untreated (N2) and controls treated (average of HGV, rabbit IgG, and L4 IgG combined;p < 0.01, Tukey–Kramer multiple comparisons). There was no significant difference between HAV and low Ca2+ (Tukey–Kramer, multiple comparisons).

Article Snippet: Positive controls were protein lysates of epidermoid carcinoma cell line A 431 (E-cadherin), rat brain (N-cadherin, R-cadherin), mouse neonate (M-cadherin) (all from BD Transduction), and Madin-Darby canine kidney (MDCK) epithelial cells (K-cadherin).

Techniques: Blocking Assay, Labeling, Staining

Journal: The Journal of Neuroscience

Article Title: N-Cadherin Mediates Axon-Aligned Process Growth and Cell–Cell Interaction in Rat Schwann Cells

doi: 10.1523/JNEUROSCI.22-10-04066.2002

Figure Lengend Snippet: SC–axon alignment was decreased using N-cadherin blocking agents

Article Snippet: Positive controls were protein lysates of epidermoid carcinoma cell line A 431 (E-cadherin), rat brain (N-cadherin, R-cadherin), mouse neonate (M-cadherin) (all from BD Transduction), and Madin-Darby canine kidney (MDCK) epithelial cells (K-cadherin).

Techniques: Blocking Assay

Journal: Engineering in Life Sciences

Article Title: Low‐frequency mechanical vibration induces apoptosis of A431 epidermoid carcinoma cells

doi: 10.1002/elsc.201900154

Figure Lengend Snippet: Mechanical vibration induces apoptosis but not necrosis of A431 cells. (A, C, and E) Area of apoptotic A431 cells (A A ) normalized to the value for control cells immediately after mechanical vibration (0 h, A) or after 24‐h (B) and 48‐h (C) incubation. (B, D, and F) As described for (A), (C), and (E), except the area of necrotic cells normalized to the control cells (A N ) is shown. Data are presented as the mean ± SD of n = 3. * P < 0.05 by Student's t ‐test

Article Snippet: The human epidermoid carcinoma cell line A431, which is considered a good model to study the biomechanical response of cancer cells to extracellular stimuli , was obtained from RIKEN (Wako, Saitama, Japan).

Techniques: Incubation

Journal: Engineering in Life Sciences

Article Title: Low‐frequency mechanical vibration induces apoptosis of A431 epidermoid carcinoma cells

doi: 10.1002/elsc.201900154

Figure Lengend Snippet: Mechanical vibration increases glucose consumption by A431 cells. Glucose uptake in culture supernatants was measured between 0 h and 24 h (A) or between 24 h and 48 h (B) after mechanical vibration. Data are presented as the mean ± SD of n = 3

Article Snippet: The human epidermoid carcinoma cell line A431, which is considered a good model to study the biomechanical response of cancer cells to extracellular stimuli , was obtained from RIKEN (Wako, Saitama, Japan).

Techniques:

Journal: Engineering in Life Sciences

Article Title: Low‐frequency mechanical vibration induces apoptosis of A431 epidermoid carcinoma cells

doi: 10.1002/elsc.201900154

Figure Lengend Snippet: Mechanical vibration does not increase HMGB1 release from A431 cells. HMGB1 levels in culture supernatants were analyzed by ELISA immediately after 0 h (A), or 24 h (B) and 48 h (C) after vibration. Data are presented as the mean ± SD of n = 3

Article Snippet: The human epidermoid carcinoma cell line A431, which is considered a good model to study the biomechanical response of cancer cells to extracellular stimuli , was obtained from RIKEN (Wako, Saitama, Japan).

Techniques: Enzyme-linked Immunosorbent Assay