Figure 3

Ca²⁺ influx/PKC/Erk1/2 signaling pathway is involved in the SP6616-mediated β-cell protection. (a) INS-832/13 cells were incubated with SP6616 (10 μM) in the presence or absence of STZ (0.4 mM) for 24 h, and the cell lysate was analyzed by western blot assay using the corresponding antibodies. (b) Relative protein levels of p-Erk1/2/Erk1/2 and p-Akt/Akt in a. (c) INS-832/13 cells were incubated with SP6616 (1, 5, 10 μM) in the presence or absence of STZ (0.4 mM) for 24 h, and the cell lysate was analyzed by western blot assay using p-Erk1/2 and Erk1/2 antibodies. (d) Relative protein levels of p-Erk1/2/Erk1/2 in c. (e) After INS-832/13 cells were incubated with SP6616 (10 μM) and STZ (0.4 mM) in the presence or absence of U0126 (10 μM) for 24 h, the cell lysate was analyzed by western blot assay using p-Erk1/2 and Erk1/2 antibodies. (f) Relative protein levels of p-Erk1/2/Erk1/2 in e. (g) INS-832/13 cells were transfected with Kv2.1 N or EGFP, and incubated with STZ (0.4 mM) and SP6616 (10 μM) or STZ alone, then the cell lysate was analyzed by western blot using p-Erk1/2 and Erk1/2 antibodies. (h) Relative protein levels of p-Erk1/2/Erk1/2 in g. (i) INS-832/13 cells were incubated with SP6616 (1, 5, 10 μM) in the presence or absence of STZ (0.4 mM) for 24 h, then intracellular Ca²⁺ level were monitored by Fluo-8 AM fluorescence dye. Data were shown as the AUC of intracellular Ca²⁺ level. (j) INS-832/13 cells were incubated with SP6616 (1, 5, 10 μM) in the presence or absence of STZ (0.4 mM) for 24 h, and the cell lysate was analyzed by western blot using p-PKC and PKC antibodies. (k) Relative protein levels of p-PKC/PKC in j. (l) INS-832/13 cells were incubated with SP6616 (10 μM) and STZ (0.4 mM) in the presence or absence of GFX (20 μM) for 24 h, and the cell lysate was analyzed by western blot using corresponding antibodies. (m) Relative protein levels of p-PKC/PKC in l. (n) Relative protein levels of p-Erk1/2/Erk1/2 in l. All data were obtained from three independent experiments and presented as means±S.E.M. (*P<0.05, **P<0.01, ***P<0.001; ns, no significance)