Figure 2

Knockdown of DVL1 or DVL3 reduces proliferation in human myoblasts. (A) Gene expression levels of DVL1 (left), DVL2 (middle) and DVL3 (right) in proliferating C25 myoblasts after knockdown of each isoform for 48 h. N = 3 biological replicates. Data is represented as mean ± SD with significant differences calculated using a One-Way ANOVA with Dunett’s post-hoc test, comparing each group with the control (SiCtrl), where an asterisk denotes p < 0.05, two asterisks p < 0.01 and 3 asterisks p < 0.001. (B) Representative images of proliferating C25 myoblasts after knockdown of each DVL isoform for 48 h, immunolabelled for β-TUBULIN (green) to show cell morphology, with nuclei counterstained with DAPI (blue). Incorporated EdU is also visualised (red). Scale bar represents 100 µm. (C) Quantification cell circularity (0 equates to a straight line, 1 to a perfect circle) and size in proliferating C25 myoblasts after knockdown of each DVL isoform for 48 h, displayed as violin plots. Red line demarcates mean. Statistically significant differences were assessed using a One Way ANOVA with a Dunett’s post hoc test, comparing each knockdown individually to the control (SiCtrl). N = 135 cells from 3 independent experiments. Three asterisks denote p < 0.001. (D) Quantification of the percentage of human C25, 16U and 54–6 myoblasts that had incorporated EdU after knockdown of each DVL isoform. More than 200 nuclei were analysed for each of N = 3–6 wells. (E) Crystal violet staining of C25 myoblasts after DVL knockdown after 24 or 96 h of proliferation. (F) Quantification of crystal violet incorporation in C25 myoblasts after DVL knockdown after 24, 72 and 96 h of proliferation. N = 4. Data is represented as mean ± SD. Statistical differences were assessed using a One-Way ANOVA with Dunett’s post hoc test, comparing each knockdown group with the control, for each time-point, where two asterisks denote p < 0.01 and three asterisks p < 0.001.