Fig 2: DVL-enhanced proliferation does not operate through ß-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 ß-CATENIN and non-phosphorylated active ß-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 ß-CATENIN and active ß-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 ß-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.

Fig 3: MCCP cell lines with WT p53 are sensitive to milademetan treatment.(A) MKL-1, WaGa, PeTa, and MS-1 cell lines and (B) MCC PDCLs MCC-301 and MCC 336 were treated with indicated doses of milademetan, and the Cell Titer Glo assay was performed to assess the effect on viability after 3 days of treatment. Each assay was performed in triplicate, and 3 biological replicates were performed. Data are shown as the mean ± SD. (C) MCC MKL-1, WaGa, and PeTa cell lines were treated with 100 nM milademetan, and p53 response was analyzed using WB analysis for the indicated proteins. TBP was used as a loading control. Representative WB of n = 2. PARP (cl.) indicates cleaved PARP.

Fig 4: HXP prevents excessive inflammation after myocardial infarction. (A) Echocardiography parameters: ejection fraction (EF%), and fractional shortening (FS%), measured at 1 week post-MI in PBS-treated and HXP-treated (3 and 9 mg/mL) mice. *p < 0.05. **p < 0.01. n = 10 for each. (B) Representative M-mode echocardiography at 1 week post-MI showing LV end-systolic and end-diastolic dimensions in PBS-treated and HXP-treated (3 and 9 mg/mL) mice. (C) Real-time PCR results showing the relative expressions of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-a in the infarct regions of PBS-treated and HXP-treated (3 and 9 mg/mL) mice at 1 week following MI. Error bars represent SEM of the fold changes in upper ?CT values after normalisation with GAPDH. *p < 0.05. **p < 0.01. (D) Western blots showing the nuclear expression of NF-?B following TNF-a treatment at varying times in adult H9c2 cardiomyocytes. TBP, loading control. (E) Western blots showing the expressions of I?b-a, p-I?b-a and nuclear NF-?B following pre-treatment with HXP for 12 h and subsequent TNF-a treatment for 4 h in adult H9c2 cardiomyocytes. GAPDH: total loading control; TBP: nuclear loading control.

Fig 5: Effects of hoveniae semen seu fructus ethanol (HSFE) extract on (a, b) nuclear translocation of NF-?B p65 and (c) degradation of I?Ba. Cells were seeded with 1.5 × 106 cells/well on a 6-well culture plate and preincubated for 18 h. Then, cells were pretreated with HSFE for 1 h and stimulated with LPS for another (a, b) 1 h or (c) 30 min. At least three independent tests were repeated to ensure reproducibility. ß-Actin and TBP served as the controls for cytosolic and nuclear proteins, respectively. Data in the histograms show protein expression levels relative to those of ß-actin, TBP, or I?Ba. The results shown are representative of three separate experiments. Con: control.