Suppression of HIF-α subunits in RCC10 and RCC4 CCRCC cell lines decreases NMU expression. A) VHL Type 2C mutants suppress HIF and NMU: Retroviral RCC10 and RCC4 cell pools expressing either wild-type VHL (VHL 30), a type 2C mutant VHL (either VHL L188V or VHL V84L) or an empty vector were prepared as described previously . Expression of VHL was confirmed by Western blotting. Analysis of HIF-α subunit expression showed that levels of both HIF-1α and HIF-2α were suppressed in cell pools expressing either wild-type VHL or type 2C mutant VHL. NMU expression was significantly decreased in RCC10 and RCC4 cell lines expressing either wild-type or type 2C mutant VHLs, but not an empty vector control. B) Suppression of HIF via siRNA of HIF-α subunits results in down-regulation of NMU: RCC10 and RCC4 cells were transfected with a control siRNA (firefly luciferase) or siRNA oligos targeting HIF-1α or HIF-2α using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA). (Oligos: Firelfly Luciferase 5'-CGUACGCGGAAUACUUCGAdTdT-3' (sense), 5'-AAGCUAAAGGUACACAAUUdTdT-3' (antisense); HIF-1α 5'-CUGAUGACCAGCAACUUGAdTdT-3' (sense), 5'-UCAAGUUGCUGGUCAUCAGdTdT-3' (antisense); HIF-2α 5'-CAGCAUCUUUGAUAGCAGUdTdT-3' (sense), 5'-ACUGCUAUCAAAGAUGCUGdTdT-3' (antisense). Cells were plated in duplicate and harvested 48 hours post transfection for either RNA or protein. Western blotting for HIF-1α and HIF-2α confirmed selective and efficient knock-down of the targeted HIF-α subunit. Real-time RT PCR showed that HIF-α knock-down significantly suppressed NMU expression in both RCC4 and RCC10 cells. *p < 0.01, using Student's t test.