Fig 2: Role of ROS in MeHg-induced JNK-signaling activation and apoptosis in RIN-m5F cells. Cells were treated with MeHg (2 µM) for different time intervals in the absence or presence of NAC (1 mM) and Trolox (100 µM) for 1 h prior to addition of MeHg. (A) ROS generation was determined by flow cytometry using the fluorescent probes: DCF and DHE. H2O2 (50 µM) was used as a positive control. (B) Protein expression of caspase-3, procaspase-12, CHOP, and XBP-1s and (C) phosphorylation of JNK1/2 protein were examined using Western blot analysis. (D) In addition, RIN-m5F cells were treated with MeHg (2 µM) for 1h in the absence or presence of the specific inhibitor of JNK (SP600125; 10 µM) for 1 h prior to addition of MeHg. ROS generation was detected by flow cytometry using the fluorescent probe: DCF and DHE. Data in A and D are presented as mean ± SD of six independent experiments assayed in triplicate. * p < 0.05 compared to vehicle control; # p < 0.05 compared to MeHg treatment. Results shown in B and C are representative images of at least three independent experiments. ß-actin was used as loading control.

Fig 3: JNK-mediated signaling plays a critical role in MeHg-induced β-cell apoptosis. RIN-m5F cells were treated with MeHg (2 μM) for different time intervals in the absence or presence of specific inhibitors of JNK (SP600125; 10 μM), ERK1/2 (PD98059; 10 μM), and Akt (LY294002; 5 μM) for 1 h prior to the addition of MeHg. (A) Caspase-3 activity was measured using a Caspase-3 Activity Assay Kit. (B) Cell viability was detected using MTT assay. (C) Phosphorylation levels of JNK1/2 protein were examined using Western blot analysis. (D) Mitochondrial membrane potential (MMP) depolarization was detected using flow cytometry. (E) Protein expression of caspase-3 and -7, procaspase-12, CHOP, and XBP-1s was examined using Western blot analysis. (F) RIN-m5F cells were pretreated with JNK inhibitor (SP600125; 10 μM) and antioxidant NAC (1 mM) for 1 h and then exposed to MeHg (2 μM) for 4 h. Insulin secretion stimulated by 20 mM glucose was detected by an insulin immunoassay kit. Data in A, B, D, and F are presented as mean ± SD of six independent experiments assayed in triplicate. * p < 0.05 compared to vehicle control; # p < 0.05 compared to MeHg treatment alone. Results shown in C and E are representative images of at least three independent experiments. β-actin was used as loading control.