Fig. 5: L1CAM knockdown inhibits sphere formation in OVCAR8 cells.

a Western blot analysis of L1CAM expression in OVCAR8 and isogenic OVCAR8-L1^low cells (passage 10). b Phase contrast images representing OVCAR8 and OVCAR8-L1^low cells grown for 6 days under 2D conditions. c Phase contrast images representing OVCAR8 and OVCAR8-L1^low cells grown for 1, 2 and 6 days grown under 3D conditions and for 6 days. d Colony formation assay was quantified for colony size in different OVCAR8 cell populations e Cells were measured for compaction (area change). f Relative invasion of OVCAR8 and OVCAR8-L1^low cells. g OVCAR8 and OVCAR8-L1^low cells analyzed by bright field microscopy (lower row) and for 3D colonies with crystal violet (upper row) after culturing under 3D conditions in serum free media. (h, left panel) Fluorescent images representing OVCAR8 and OVCAR8-L1^low stained for calcein (green) and ethidium bromide (red). (g, right panel) Dot plot showing the ratio between living and dead cells, assessed by measuring the ratio between calcein (green) and ethidium bromide (red) intensity in OVCAR8 and OVCAR8-L1^low cells after culturing for 7 days under 3D and serum free conditions. i, j Rescue experiment: Re-expression of L1CAM analyzed by Western blot (i) in OVCAR8-L1^low and its effect on sphere formation. j Dot plot representing number of colonies after cells were transferred from 3D to 2D cell culture. k Effects of L1CAM antibody treatment (anti-L1- 9.3) on sphere size after 48 h. b, c, g, h, j Scalebar represents 200 µm. Three independent experiments were performed (d, e, f, j). P-values were calculated with an unpaired two-sided t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001. k ANOVA and Dunnett multiple comparison tests were used to calculate significance.