Necl2 overexpression influences keratinocyte adhesion and motility.
(A) Identification of cell doublets based on Draq5 nuclear area/aspect
ratio (left panel; blue gate) and characterisation of doublets by labelling
with FITC- and RPE-conjugated anti-α6-integrin antibodies (right).
Insert shows representative RPE-RPE (top), RPE-FITC (middle) and FITC-FITC
(bottom) stained cell doublets. (B) Quantification of relative RPE-FITC
labelled EV-EV, Necl2-EV and Necl2-Necl2 doublets. (C-G) Representative
images (C-F) and quantification (G) of Necl2 expression in control (C,E) and
Necl2-transduced (D,F) cells cultured under standard (high; C,D) and
low-calcium (E,F) conditions. (H-L) Representative images (H-K) and
quantification (L) of E-cadherin expression in control (H,J) and
Necl2-transduced (I,K) cells cultured under standard (high; H,I) and
low-calcium (J,K) conditions. (G,L) Quantification of pixel intensity at
cell-cell borders. (M-O) Control (M) and Necl2-transduced (N)
cell motility tracks and maximum migration distances (O) relative to a
starting position (x,y=0,0) at time=0 minutes. Each line in M,N shows
motility of an individual cell. (B,G,L) Error bars represent s.e.m.;
*P<0.05. Scale bars: 10 μm in C-F,H-K.