Fig. 4.

SPR protein expression regulates ODC enzymatic activity and cellular proliferation in NB cells. (a) MYCN2 cells were treated with siRNA against SPR using two different transfection protocols, referred to as protocols P-I and P-II, which differ in their voltage settings. Control cells were either transfected with non-targeting scrambled siRNA or mock transfected in the absence of siRNA. P-II was more efficient in SPR knock-down than P-I. Cell lysates were prepared and subjected to SDS-PAGE and Western blot analysis using antibodies specific for ODC, SPR, and GAPDH (loading control). Knock-down of SPR using P-II showed greater reduction of SPR levels than with P-I. (b, c) Equal amounts of cells were seeded and total amount of protein compared 72 hours later using the P-I (b) and P-II (c) transfection protocols (n=4 each). The cellular proliferation was significantly reduced by SPR siRNA in both experimental settings, and a greater anti-proliferative effect was achieved by the stronger knockdown of SPR using the P-II protocol (d, e). ODC enzymatic activity was determined in NB cells in which SPR was down-regulated using transfection protocols P-I and P-II and compared to cells treated with non-targeting scrambled siRNA or mock transfected cells in the absence of siRNA. Specific enzymatic activities are expressed as pmol CO₂/30 min/mg protein. Each sample was measured in triplicates and represents the mean of four independent experiments (n=4). The ODC enzymatic activity was significantly reduced in both sets where SPR was down regulated using siRNA (protocol P-I; P = 0.043 and protocol P-II; P = 0.016).