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).