Fig. 2: Increased adhesion of ETV1 and ETV1/ETV4/ETV5 deficient hPSCs.

A Growth dynamics of KO, tKO, and WT hPSCs over a 24 h culture monitored by live-cell imaging. Left - representative images of cell confluency at 2, 12, and 24 h culture, marked by green mask. Middle-representative images of cell number at 2, 12, and 24 h culture, marked by nuclei marker SiR DNA (red). Right - representative images of cell spreading (quantified as the ratio of confluency to cell number) at 2, 12, and 24 h culture. A green line marks confluency and nuclei are marked by SiR DNA in red. Scale bar = 400 µm. B Quantification of WT (yellow), KO (green), and tKO (maroon) hPSC confluency over a 24 h culture. tKO cells show the highest confluency, a 166% increase compared to WT cells. N = 3 biological replicates. At 24 h, for KO vs. WT and tKO vs. WT, p < 0.0001, for tKO vs. KO, p = 0.0025. C Quantification of cell number over a 24 h culture. KO (green) and tKO (maroon) show an increase in the cell number compared to WT cells (yellow). The increase in cell number was the most pronounced for tKO cells (66% increase in comparison to WT) and by 36% for KO hPSCs. N = 3 biological replicates. At 24 h, for KO vs. WT, p < 0.0001, for tKO vs. KO, p = 0.039; at 18 h, for tKO vs. WT, p = 0.0171. D Quantification of cell spreading (confluency/cell number) over a 24 h culture. KO (green) and tKO (maroon) show an increase in cell spreading compared to WT cells (yellow). N = 3 biological replicates. At 24 h, for KO vs. WT, p = 0.0008, for tKO vs. WT and tKO vs. KO, p < 0.0001. E Confluency quantification at 2 h post-seeding (corresponds to A). The highest increase in cell confluency was noted for tKO followed by KO and KO2 in comparison to WT hPSCs. N = 3 biological replicates. For KO vs. WT, tKO vs. WT, tKO vs KO, p < 0.0001, for KO2 vs. WT, p = 0.0003. F Cell number quantification at 2 h post-seeding (corresponds to A). The highest increase in cell number was noted for tKO followed by KO and KO2 hPSCs. N = 3 biological replicates. For KO vs. WT and tKO vs. WT, p < 0.0001, for KO2 vs. WT, p = 0.006, tKO vs. KO, p = 0.0006. G Quantification of cell spreading (confluency/cell number) at 2 h post-seeding (corresponds to A). The most pronounced increase in cell spreading was noted for tKO hPSCs followed by KO and KO2. N = 3 biological replicates. For KO vs. WT, tKO vs. WT, tKO vs KO, p < 0.0001, for KO2 vs. WT, p = 0.0009. H Representative crystal violet staining images of cells cultured in the absence (top panel) or presence (bottom panel) of ROCK inhibitor (ROCKi) 24 h after hPSC seeding. The same number of WT and KO cells were seeded on different surface coatings, as indicated. Scale bar = 400 μm. I Quantification of crystal violet staining shows an increase in the attachment of KO (green) and tKO (maroon) compared to WT (yellow) cells, on all tested surface coatings in the absence (top panel) and presence (bottom panel) of ROCKi. N = 4 biological replicates. J Physical cytometer analysis of KO, tKO, and WT hPSC spheres demonstrated enhanced density of KO and tKO compared to WT. N = 3 biological replicates. K Representative images of immunofluorescence staining show ETV1 (green) overexpression in hPSCs induced by 24 h doxycycline treatment (Dox+), compared to untreated (Dox-) cells. L Quantification of adhesion protein levels CDH1 (left) and ITGA5 (right) in WT_OE and KO_OE hPSCs after induction of ETV1 overexpression. CDH1, N = 4 biological replicates; ITGA5, N = 3 biological replicates. For plots B–G, I, J, and L, a one-way ANOVA for multiple comparisons was used to determine the p-values shown on the graph. The data are presented as means ± SDs.