Figure 5

DVL-enhanced proliferation does not operate through β-CATENIN. (A) Representative images of proliferating C25 myoblasts after stimulation with 5 mM LiCl for 24 h showing incorporated EdU (red), with all nuclei counterstained with DAPI (blue). Scale bar represents 100 µm. Quantification was performed for 4 biological replicates, and significant difference was assessed using a student’s T-test. Three asterisks indicates p < 0.001. (B) Quantification of western blot for total β-CATENIN and non-phosphorylated active β-CATENIN in nuclear fractions of proliferating C25 myoblasts stimulated with WNT ligands WNT7a or WNT3a for 24 h. A representative blot is shown that includes cytoplasmic housekeeper VINCULIN, nuclear housekeeper TBP, total β-CATENIN and active β-CATENIN. Band intensity was normalised to the nuclear housekeeper TBP and changes are shown as fold change to unstimulated control. Labels indicate protein sizes. N = 3 independent biological replicates, significant difference between treatment and Ctrl was assessed using a One-Way ANOVA, with an asterisk denoting p < 0.05. (C) Representative images of proliferating C25 myoblasts stimulated with WNT ligands WNT7a or WNT3a for 24 h, with quantification of the percentage of EdU containing nuclei. N = 3, no significant differences were found using a One-Way ANOVA with Dunnett’s post-hoc test, comparing each group to the control. Scale bar equal 100 µm. (D) Western blot for non-phosphorylated active β-CATENIN in proliferating control C25, and C25 DVL1⁺ or DVL3⁺ myoblasts constitutively overexpressing DVL1 or DVL3. Band intensities were quantified and normalised to TBP, expression is shown as fold change to control (Ctrl). N = 3 independent biological replicates, no statistical differences observed between the overexpressing samples and the controls, assessed with a One-Way ANOVA with Dunnett’s post-hoc test, comparing each group to the control.