Figure 2

SP6616 improves pancreatic β-cell dysfunction by inhibiting Kv2.1 channel. (a) After 2-h incubation with glucose-free KRB buffer, INS-832/13 cells were incubated with SP6616 (1, 5, 10 μM), ScTx-1 (100 nM) or glibenclamide (0.5 μM) in the presence of 16.8 mM glucose in KRB buffer, and insulin secretion was then detected by AlphaLISA insulin kit. (b) INS-832/13 cells were transfected with Kv2.1N or EGFP (control), and incubated with glucose-free KRB buffer for 2 h. The cells were stimulated with SP6616 (10 μM) or ScTx-1 (100 nM) in KRB buffer with 16.8 mM glucose, and insulin secretion was detected. (c) INS-832/13 cells were incubated with different concentrations of SP6616 (1, 5, 10 μM) in the absence or presence of STZ (0.4 mM) for 24 h, and then MTT assay was conducted. (d) INS-832/13 cells were treated with SP6616 (1, 5, 10 μM) and STZ (0.4 mM) for 8 h, and the cell lysate was then analyzed by western blot assay using caspase 3 antibody. (e) Relative protein levels of cleaved caspase 3/caspase 3 in d. (f) INS-832/13 cells were treated with SP6616 (1, 5, 10 μM) and STZ (0.4 mM) for 8 h, and then caspase 3/7 activity was detected. (g) INS-832/13 cells were transfected with Kv2.1N or EGFP, and incubated with SP6616 (10 μM) and STZ (0.4 mM) for 24 h, followed by MTT assay. (h) Intracellular Ca²⁺ level in INS-832/13 cells was monitored by Fluo-8 AM fluorescence dye. The cells were pre-incubated in KRB buffer for 2 h and then the plate was loaded on FlexStationII384. The baseline fluorescence signal was measured for the first 20 s, and then stimulated with 16.8 mM glucose in the presence of SP6616 (10 μM) or ScTx-1(100 nM). These data were shown as AUC of intracellular Ca²⁺ change. (i) The intracellular Ca²⁺ assay was conducted as (h) in calcium-free HBSS buffer. (j) The intracellular Ca²⁺ assay involving nifedipine was conducted as h. 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)