Journal: Genes & Development
Article Title: Fatty acid uptake activates an AXL–CAV1–β-catenin axis to drive melanoma progression
doi: 10.1101/gad.351985.124
Figure Lengend Snippet: Oleic acid induces β-catenin–CAV1 interaction and nuclear localization. ( A ) Immunofluorescence of IGR39 cells using antibodies against the indicated proteins in cells exposed to 100 μM oleic (OA). Scale bars: top six panels, 50 µm; bottom three panels, 20 µm . ( B ) Western blots for the indicated proteins from fractionated IGR39 cells treated or not with 100 μM OA over time. GAPDH and TBP were used as markers of the cytoplasmic and nuclear fractions. ( C ) Immunofluorescence of IGR39 cells for the indicated proteins after exposure or not to 100 μM OA or palmitic acid (PA) for 6 h. Scale bars, 25 µm. ( D ) Western blot of fractionated IGR39 cells treated with oleic acid for the indicated times. ( E ) Luciferase reporter assays in the indicated cell lines showing the ratio of luciferase activity from the TOP:FOP-flash β-catenin activity reporters in melanoma cells exposed to 100 μM OA or PA for the indicated times. LiCl (10 mM) was used as a positive control. N = 3. Error bars indicate SEM. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001; one-way ANOVA statistical test. ( F ) Western blot for the indicated proteins immunoprecipitated from IGR39 nuclear extract using anti-β-catenin antibody from cells treated or not with 100 μM OA or PA for the indicated times. The blot shows both immunoprecipitation (IP) and flowthrough (FT). ( G ) Western blots for the indicated proteins from fractionated IGR39 cells treated or not with 100 μM OA for the indicated times and transfected with either control or CAV1-specific siRNA. ( Left ) Nuclear fraction. ( Right ) Cytoplasmic fraction.
Article Snippet: Specific siRNA oligonucleotides for β-catenin and control siRNA were obtained from Qiagen, Caveolin-1 and SRC siRNA were from Santa Cruz Biotechnology, and SRC siRNA were from Dharmacon, Inc.
Techniques: Immunofluorescence, Western Blot, Luciferase, Activity Assay, Positive Control, Immunoprecipitation, Transfection, Control
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