Fig 1: Cells adhered to N-cadherin surfaces. A) Cells equally adhere to positive control (Matrigel®) (n = 6) and N-cad 5 (n = 3) and N-cad 3 (n = 5) N-cadherin substrates. Significant differences were found between the negative control (protein A) (n = 5) and the positive control, N-cad 5, and N-cad 3. N-cadherin substrates N-cad 2 (n = 3) or N-cad 1 (n = 6) did not differ. B) Regardless of the number of cells that adhered for N-Cad 3 or Matrigel, they had the same doubling time.
Fig 2: YAP translocation to the nucleus was analyzed (A) and showed positive control (Matrigel®) (1.82 ± 0.183) and N-cad 1(1.57 ± 0.262) did not differ. N-cad 2 (2.14 ± 0.0945) was significantly higher than N-cad 1; N-cad 3 showed significantly more translocation compared to all groups (2.72 ± 0.217). (B) The percentage of cells with YAP colocalized with the actin rings, control (0.0% ± 0.0%), N-cad 1 (4.16 ± 3.81), N-cad 2 (26.8 ± 12.4), N-cad 3 (62.2 ± 4.59). Lines represent mean +/- standard deviation. (C) Representative images of hNSCs taken at 63X, with nuclei (blue), YAP (green) and F-actin (red). Scale bar 10 µm.
Fig 3: Cell morphology of hNSCs on N-cadherin surfaces is affected by cadherin concentration. A. Representative images of a single cell on the cadherin surface at 63X; scale bar is 10 μm in each image. Nuclei (blue), β-catenin (green), N-cadherin (magenta) and F-actin (red). For positive control, N-cad 1, N-cad 2, N-cad 3, and N-cad 5. Scale bar 10 μm. B. Average cell area (μm2). C. Average nucleus area (μm2). D. Average number of cells polls per cell.
Fig 4: A. Schematic of 4 step process for recombinant cadherin tethering on 2D glass. B. FTIR analysis following each subsequent surface fabrication step. C. Quantification of eluted N-cadherin from surfaces via ELISA. Significantly less N-cadherin was eluted from N-Cad 1 surfaces (3.5 fmol) compared to N-Cad 5 (17 fmol) substrates.
Fig 5: Cadherin concentration alters adherens junction morphology. Actin analysis showed (A) percent of cells with an F-actin ring. (B) Average area of the actin ring. β-catenin analysis showed the (C) average number of adherens points per cell. (D) Location of β-catenin staining showed percentage of cells with β-catenin staining at the edges of the cells. E. Percent of cells with β-catenin staining at the actin ring. All lines represent mean +/− standard deviation. (F) Image taken with a Zeiss LSM 880 with adjusted pinhole of a single cell on the N-cad 3 cadherin surface at 63X. (G) Nuclei (blue) (H) β-catenin (green) (I) N-cadherin (magenta) and (J) F-actin (red) are shown separately. Scale bar is 10 μm.
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