Figure 4

The NMU pathway is functionally active in renal cancer cells and contributes to a migratory and invasive phenotype. A) Western blotting showed that NMUR1 is expressed in CCRCC cells. B) NMU peptide induces a calcium flux in renal cancer cells: RCC cell cultures were loaded with a calcium sensitive dye (fluo-4 AM; Invitrogen, Carlsbad, CA) and washed with HBSS. A fluorometric imaging plate reader (FLIPR; Molecular Devices, Sunnyvale, CA) recorded fluorescence (λEX = 488 nm, λEM = 540 nm) before and after robotic addition of 60 μL of NMU peptide (final concentration = 300 μM) or a vehicle control. A representative trace from RCC10/VHL cells is shown. RCC10, RCC4 and RCC4/VHL cells all showed similar results. Significance was determined by analysing peak fluorescence induction in NMU treated and untreated cells from five independent experiments. C & D) Constitutive expression of NMU promotes cell migration and invasion: Retroviral cell pools expressing either NMU or an empty vector were prepared in RCC10/VHL cells. Cells were serum-starved, fluorescently labelled (migration assay only) and plated onto the upper compartment of FluoroBlok transwell migration chambers (C) or matrigel BioCoat transwell invasion chambers (D). Media with 5% FCS was added to the lower compartment. After 9 hours (migration assays) or 24 hours (invasion assays) cells were fixed. Invasion membranes were stained with Giemsa. Cells were counted in five fields. RCC10/VHL cells expressing NMU showed significantly more migration and invasion than empty vector control cells (*p < 0.05, Student's t test, three independent experiments).