Fig. 6. Effect of up and down regulation of tomosyn-1 expression on exocytosis. (A) INS-1E cells were transiently co-transfected with a plasmid encoding hGH and with the empty pSUPER vector (control), the pEGFP-rat-tomosyn-1, siRNA-i or siRNA-a. After 3 days, the cells were incubated under basal condition or in the presence of stimulatory concentrations of glucose, K⁺, forskolin and IBMX. After 45 minutes, the incubation medium was collected. The fraction of hGH released by the cells under basal and stimulatory conditions were measured by ELISA. None of the constructs used in this study affected basal secretion (data not shown). Stimulated exocytosis was defined as the ratio of basal hGH release to the amount of hGh secreted in the presence of glucose, K⁺, forskolin and IBMX. The results are the mean of at least three independent experiments measured in triplicates. (B) INS-1E cells were transiently co-transfected with the pSUPER-GH plasmid, encoding both hGH and either the empty pSUPER vector (control) or the siRNA-a, and with either pEGFP or pcDNA3-mouse-tomosyn-1. As described above, cells were incubated under basal condition or in the presence of stimulatory concentrations of glucose, K⁺, forskolin and IBMX for 45 minutes. The incubation medium was then collected and the fraction of hGH released by the cells was measured by ELISA. The results are the mean of at least three independent experiments measured in triplicates.