685642.fig.004a
(a)
685642.fig.004b
(b)
685642.fig.004c
(c)
685642.fig.004d
(d)
Figure 4: Effects of cytopiloyne on calcium mobilization, DAG generation, and PKCα activation. (a) After Fura 2-AM loading, RIN-m5F cells were stimulated with 16.7 mM glucose (HG) and cytopiloyne (CP) at 7, 14, and 28 μM. The level of intracellular calcium, as shown by the 340/380 nm ratio, was detected using a fluorescence spectrophotometer. (b) RIN-m5F cells were stimulated with glucose, cytopiloyne (CP), PMA, and glimepiride (GLM). Total cell lipids and their commercial standards, DAG and cholesterol (CHL), were resolved on a silica thin layer plate. The quantity of DAG and cholesterol in each sample is replotted into histograms. (c) RIN-m5F cells were incubated with vehicle (NS), cytopiloyne (CP, 7, 14, and 28 μM), PMA (1 μM), and 16.7 mM glucose (HG). Membrane proteins of each sample were subjected to Western blot with anti-PKCα and anti-actin antibodies. (d) RIN-m5F cells were incubated with vehicle, cytopiloyne (28 μM), and PMA (1 μM) in the absence or presence of EGTA (10 μM) and nimodipine (Nimo, 1 μM). Total proteins were subjected to Western blot with anti-PKCα (t-PKCα) and anti-phospho-PKCα (p-PKCα) antibodies.