Figure 5
WNT3A-mediated effects in PTENWT melanoma cells are β-catenin dependent. (a) Western blot analysis of β-catenin expression levels in A375 WNT3A overexpression cells following transfection with β-catenin siRNA or scrambled control siRNA for 72 h. α-tubulin served as the loading control. (b) Fold-change in cell migration in A375 WNT3A overexpression cells treated with scrambled or β-catenin siRNA. (c) Fold-change in CS activity in A375 WNT3A overexpression cells treated with scrambled or β-catenin siRNA. (d) Fold-change in lactate secretion from A375 WNT3A overexpression cells treated with scrambled or β-catenin siRNA, normalized to total protein concentration. (e) Fold-change in lactate secretion from PTENWT cells treated with rWNT3A (50 ng/ml)±rDKK1 (50 ng/ml) or carrier treatment, normalized to total protein concentration. (f) Quantified Imaris data for A375 overexpression cells treated with rWNT3A (50 ng/ml)±rDKK1 (50 ng/ml) or carrier control. Numbers in each chart represents the percentage of mitochondria that range in size from 0.1 to 10 μM2 for each treatment. For panels (b–f), mean shown±s.d. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 (as compared with control). (g) A375 overexpression cells stained with MitoTracker Green (10 nM; green) and counterstained with Hoechst (blue). siRNA treatment as described above and in main text. Example images shown. Scale bar, 10 μm. (h) Quantified Imaris data for A375 overexpression cells transfected with β-catenin siRNA or a scrambled control siRNA. Numbers in each chart represent the percentage of mitochondria that range in size from 0.1 to 10 μM2 for each treatment. ****P<0.0001. (i) PANTHER analysis of proteins identified by MS-IP with increased (upper panel) and decreased (lower panel) binding to β-catenin. See main text for experimental detail of the MS-IP approach used. (j) Western Blot for enzymes identified by MS-IP analysis; LDHA, LDHB, PFKB. MFN1 was used as a positive control (see later text and Figure 7). Parental lines were stimulated with carrier or 50 ng/ml rWnt3a, 48 h before analysis.
