Cell culture
Reagents
RNA purification and quantitative reverse transcription PCR
Oligonucleotide transfection
Melanoma coculture with adipocytes
Invasion assays
Quantification of IL-6
Immunofluorescence
Plasmids and site-directed mutagenesis
Transfection and luciferase reporter assay
Western blot analyses
Wound scratch assay/migration assay
Oil red O staining
XTT cell proliferation assay
Mice and histopathology analysis
Human samples and histopathology analysis
mRNA profiling analysis
Proliferation and invasion gene signature analysis
Signaling pathway analysis for WM1716 P value calculations
Gene expression analysis
Gene set enrichment analysis
Kaplan-Meier analysis
Cells were synchronized by serum starvation for 18 hours and then released for 10 hours. Cells (1 × 105 cells per sample) were fixed in cold 70% ethanol at 4°C overnight and permeabilized with 0.1% Triton X-100. To analyze DNA content, samples were treated with propidium iodide (0.05 mg/ml) (P4170; Sigma-Aldrich) and RNase A (ribonuclease A) solution (0.1 mg/ml) (R4642; Sigma-Aldrich) at room temperature for 30 min and were analyzed immediately by flow cytometry using a BD FACSCalibur flow cytometer. Data were gated by using Kaluza analysis software.