Fig 2: Mutation of the NLS sequence in DVL1 or DVL3 leads to reduced proliferation. (A) Schematic of the three conserved domains (DIX, PDZ, DEP) in DVL proteins, together with the positions of the nuclear localisation sequence (NLS) and the nuclear export sequence (NES), together with the changes to key amino acid residues to destroy the NLS (red text). (B) Representative images of proliferating C25 DVL1+, DVL1-mNLS+, DVL3+, DVL3-mNLS+ and control (Ctrl) myoblasts, with incorporated EdU (red), immunolabelled for GFP (green) from the lentivirus (correlated with DVL expression) and DAPI nuclei counterstain (blue). EdU pulse was performed 24 h after seeding stably overexpressing C25 myoblast lines. Scale bar represents 100 µm. Quantification was performed for 4 biological replicates, and significant difference was calculated using a One-way ANOVA with Dunnett’s post hoc test, comparing each group with the control, where an asterisk denotes p < 0.05 and 3 asterisks denotes p < 0.001. (C) Representative images of DVL3+ (left) or DVL3-mNLS+ (right) C25 myoblasts. Red arrows point to an unusually round, and an uncharacteristically elongated, DVL3-mNLS+ myoblast.

Fig 3: (a) Cytoplasmic localization of DVL1 in intracranial meningioma, with moderate to high expression. The specimen was taken from a 70-year-old female patient who was diagnosed with atypical (grade II) meningioma. (b) Nuclear localization of DVL1 in intracranial meningioma, with high expression. The specimen was taken from a 58-year-old female patient who was diagnosed with atypical (grade II) meningioma. Magnification: 200×, scale bar: 200 μm.

Fig 4: 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.

Fig 5: DVL1 and DVL3 require nuclear localisation to enhance proliferation in ARMS cells. (A) Representative images of stable proliferating DVL1+, DVL1-mNLS+, DVL3+, DVL3-mNLS+ and control (pUltra) RH30 ARMS cells with incorporated EdU (red) and a DAPI nuclear counterstained (blue). Scale bar represents 100 µm. Quantification was performed for 3 biological replicates, and a significant difference was calculated using a student’s T-test, where an asterisk denotes p < 0.05 comparing the two groups connected with a line and # denotes p < 0.05 compared to control. (B) Representative images of proliferating DVL3+, DVL3-ΔDIX+, DVL3-ΔPDZ+, DVL3-ΔDEP+ or control (pUltra) RH30 cells with EdU incorporation visualised (red) and a DAPI nuclear counterstained (blue). Scale bar represents 100 µm. Quantification was performed for 4 biological replicates, and significant difference calculated using a One-Way ANOVA, comparing each group against the control, where an asterisk denotes p < 0.05, and three asterisks denotes p < 0.001.