Figure 1

Melanoma cells exhibit increased apoptosis in response to WNT/β-catenin signaling. (a) Panel of chosen melanoma cells describing their associated genetic backgrounds for BRAF, NRAS and PTEN status. (b) Hetero- and homozygous mutations in the PTEN gene, result in depleted PTEN and phosphorylated-AKT (P-Akt) expression. In all, 10 μg of whole-cell lysate of PTEN^WT and PTEN^Mut lines were blotted for PTEN, AKT and P-AKT (t308) expression. α-Tubulin served as the loading control. (c) Ratio of the protein expression of AKT relative to P-AKT in individual melanoma cells and the average ratio of PTEN^WT and PTEN^Mut cell lines. Calculated from data presented in b. (d) WNT3A induces β-catenin-transcription factor/lymphoid enhancer-binding element reporter activity in melanoma cells. Results were normalized to the inverted promoter reporter plasmid (FOPFlash) after 24 h stimulation with carrier or rWNT3A (50 ng/ml). (e) Western blot analysis of WNT3A expression levels in A375 and A2058 control and WNT3A overexpression cells. Heat shock protein 90 (Hsp90) served as a loading control. (f) Stable overexpression of WNT3A in melanoma cells induces WNT/β-catenin signaling as measured using the TOPFlash β-catenin-transcription factor/lymphoid enhancer-binding element reporter. Results normalized to FOPFlash after 24 h of serum starvation. (g) Stable WNT3A overexpression reduces melanoma cell numbers. Live population cell counts of A375 and A2058 control, and WNT3A overexpression cells were carried out over 36 and 21 days, respectively. A paired Student’s t-test was carried out on the total cell number, as indicated (*). (h) WNT3A increases apoptosis in melanoma cells. Fold-change in Apo-TRACE staining following 48 h serum starvation in A375 and A2058 control, and WNT3A overexpression cells. Fluorescence was normalized to total protein concentration. For all panels, mean±s.d. *P<0.05 or **P<0.01.