685642.fig.005a
(a)
685642.fig.005b
(b)
685642.fig.005c
(c)
685642.fig.005d
(d)
685642.fig.005e
(e)
Figure 5: Cytopiloyne-mediated insulin secretion and expression are abolished by a dominant-negative mutant and a PKCα inhibitor. (a) RIN-m5F cells were grown in medium with 16.7 mM glucose (HG) or 3.3 mM glucose (LG) in the presence of PMA (1 μM), GF109203X (GF, 3 μM), and cytopiloyne (CP, 28 μM). The insulin level in the supernatants was determined using an ELISA kit. (b) RIN-m5F cells were transfected with 5 μg of pHACE-PKCα DN (+) or pcDNA3 (−) plasmid and grown in medium supplemented with 16.7 mM (HG) or 3.3 mM glucose (LG) in the presence of PMA and cytopiloyne. The insulin level was determined as described in (a). The expression level of dominant-negative HA-tagged PKCα (DN) and an internal control, actin, in the transfected cells was determined by Western blot using anti-HA and anti-actin antibodies. (c) RIN-m5F cells were transfected with phINS-Luc and pRL-TK plasmids. The cells were grown in medium with 16.7 mM (HG) or 3.3 mM glucose (LG) in the absence and presence of PMA, GF109203X, and cytopiloyne. The activity of the insulin promoter (pINS) in fold was measured using dual luciferase assays. (d) RIN-m5F cells were transfected with phINS-Luc and pRL-TK plus 5 μg of pHACE-PKCα DN (+) or pcDNA3 (−) plasmids. The cells were grown in medium with 16.7 mM (HG) or 3.3 mM glucose (LG) in the absence or presence of PMA and cytopiloyne. Insulin promoter activity expressed as fold change relative to vehicle-treated control was measured using dual luciferase assays. The expression level of dominant-negative HA-tagged PKCα (DN) and actin in the transfected cells was determined using Western blot and anti-HA and anti-actin antibodies. (e) RIN-m5F cells were grown in medium with 3.3 mM glucose (LG) and/or cytopiloyne (CP, 28 μM) in the presence of EGTA (10 μM) or nimodipine (Nimo, 1 μM). The insulin level in the supernatants was determined.