Figure 7
WNT/β-catenin signaling regulates mitochondrial dynamics machinery in PTENWT melanoma cells. (a) Western blot for mitochondrial dynamic genes, MFN1, MFN2, OPA1 and DRP1 in total protein extracts and mitochondrial sub-fractions of A375 control and WNT3A overexpression cells. E-cadherin and α-tubulin were used to judge the purity of the mitochondrial extraction, whereas Ponceau staining was used to judge equal loading across all sample sets. (b) Western blot analysis of MFN1 expression in PTENWT melanoma cells stimulated with carrier control or rWNT3A (50 ng/ml) for 48 h. Densitometry data for this blot are shown in Supplementary Figure 7a. (c) Western blot analysis of MFN1 in A375 WNT3A overexpression cells treated with scrambled and β-catenin siRNA for 72 h. (d) Western blot analysis of MFN1 expression in A375 cells treated with scrambled and PTEN siRNA for 72 h followed by stimulation with carrier control or rWNT3A (50 ng/ml) for 48 h. (e) Quantified Imaris data of mitochondrial size in A375 cells transfected with PTEN siRNA or a scrambled control siRNA for 72 h before stimulation with carrier control or rWNT3A (50 ng/ml) for 48 h. Numbers in each chart represents the percentage of mitochondria that range in size from 0.1 to 10 μM2 for each treatment. *P<0.05 and ***P<0.001 (as compared with control). (f) Reverse transcription (RT)–quantitative PCR (qPCR) for MFN1, MFN2, OPA1 and DNM1L (encoding for DRP1) transcripts in A375 control and WNT3A overexpression cells. mRNA expression levels were normalized based on the expression of three housekeepers, YWHAZ, UBC and ACTB. (g) A375 control and WNT3A overexpression cells were analyzed by western blot for Parkin and PINK1 expression. FL-PINK1 indicted by the 60 kDa band and the cleaved isoform by the 50 kDa band. (h) A375 control and WNT3A overexpression cells were analyzed by western blot for autophagy markers LC3 and p62 (left panel), and following treatment with β-catenin or scrambled siRNA (right panel). For all western blot panels, α-tubulin served as the loading control. Graph shows the ratio of LC3I (18 kDa) to LC3II (16 kDa) in total protein and following treatment with β-catenin or scrambled siRNA in A375 control and WNT3A overexpression cells (calculated from data shown). (i) Representative images of A375 cells following stimulation with carrier control or rWnt3a (50 ng/ml) and Rapamycin (as a positive control103; 200 nM) or control for 48 h. Cells stained with; MitoTracker Deep Red (red), LC3 (green) and Hoechst (blue). Scale bar, 50 μm. Graph shows the number of LC3 puncta, mean shown±s.d. **P<0.01 and ****P<0.0001. (j) Reverse transcription (RT)–quantitative PCR (qPCR) for SQSTM1 (encoding p62) and AXIN2 transcripts in A375 cells following stimulation with carrier control or rWnt3a (50 ng/ml) for 48 h. mRNA expression levels were normalized based on the expression of the housekeeper ACTB. ***P<0.001 and ****P<0.0001.
