Fig. 5. The silencing of CPX I reduces the stimulus-induced secretion. The effect of siRNA CPX I on the secretory function in response to glucose and leucine was evaluated in (a) INS1 and (b) ßTC3 cells. Cells (5x10⁵) were seeded in 24-well plates 1 day before transfection. Cells were transiently co-transfected with the plasmid encoding hGH as a reporter gene for secretion and with siRNA CPX I or pSUPER. Two days after transfection, the cells were cultured in 2 mM of glucose for 24 hours. Cells were then preincubated in KRBH for 60 minutes in 2 mM glucose (basal condition) and were subsequently incubated for 45 minutes in KRBH either under basal condition or with 20 mM glucose (plus 10 µM foskolin and 100 µM IBMX) or 20 mM leucine (plus 2 mM of glutamine). The amount of hGH released into the buffer and remaining inside the cells under basal and stimulatory conditions was determined by ELISA. Results were expressed as a percentage of hGH content. In the absence of siRNA CPX I, INS1 and ßTC3 cells were able to secrete hGH in response to glucose and leucine by 5-fold and 3-fold, respectively. By adding siRNA CPX I, cells were still able to secrete hGH in response to glucose and leucine compared to basal condition but the secretory response was significantly decreased by about 50% compared to cells transfected with pSUPER. (c) The 50% decrease of hGH secretion was also observed in response to 24 mM KCl in INS1 cells. (d) The decrease of stimulus-induced secretion by the silencing of CPX I occurs in a dose-dependent manner. The figure shows the mean ± s.e.m. of at least three independent experiments measured in triplicate (* P<0.01, ***P<0.001).