Figure 5. Analysis of the olfactory neuroepithelium (ON) cell’s
cytoskeletal morphology, adhesion and migration. (A) Cells were
incubated for 1 hour or 5 days under proliferative conditions, after
which they were analysed by immunofluorescences (IF) or in western blots
(WB). (B, C) Representative immunofluorescence images of
cultured cells derived from a control subject and a cannabis user,
stained with anti- β-III-Tubulin (red) and DAPI (blue). The area and
perimeter of cells were quantified by defining the stained area with
ImageJ (dotted lines) (D) Quantification of cell area.(E) Quantification of the percentage of cells with specific
roundness value intervals. (F, G ) Quantification of β-actin
(β-A) by western blots (r.u. = relative units) normalized to the total
protein content (TP). (H, I) Representative bright field images
of cells 1 hour after plating. (J) Quantification of the number
of attached cells 1 hour after plating. (K, L ) Representative
immunofluorescence images of cells in culture derived from a control
subject and a cannabis user, stained for vinculin (green) and with DAPI
(blue). (M) The number of vinculin points of adhesion (VPA)
were quantified. (N) Representative bright field images at 0,
24 and 48 hours after scratching the cell monolayer. (O)Quantification of the percentage of closure in the wound area at 24 and
48 hours, relative to the baseline (t: 0h). (P) Quantification
of the number of cells that migrated into the scratched area 24 h after
scratching. Scale bar = 50 μm. * p < 0.05; ** p <
0.001; *** p < 0.001 (Student’s t-test , n=5).