Fig. 1mRNA expression of heme oxygenase-1 (HO-1) in INS-1 cells after 24 hours treatment with different concentration of cobalt protoporphyrin (CoPP) (0 nM, 500 pM, 5 nM, 50 nM, 500 nM, 5 µM, 50 µM). Data are mean±standard error of the 3 separate experiments. aP<0.01 vs. control (0 nM).
Fig. 2Heme oxygenase-1 (HO-1) mRNA expression of INS-1 cells in the presence of 500 nM of cobalt protoporphyrin (CoPP) with different time course (1, 2, 4, 6, and 24 hours). Data are mean±standard error of the 3 separate experiments. aP<0.01 vs. control (0 nM).
Fig. 3Cytotoxicity assay of qualifying cell death in INS-1 cells. 5×105 cells/well were seeded into a 96-well plate, and each well was treated with 10 µL of CCk-8 solution (Cell counting kit-8) for 3 hours at 37℃ containing 5% CO2. Data are expressed as mean±standard error of the 3 separate experiments. aP<0.05 vs. control (0 nM).
Fig. 4Cell death rate in cultured INS-1 cells according to acridine orange (AO)/propidium iodide (PI) staining. (A) AO/PI staining in the presence of mannitol, low glucose (LG; 5.5 mM/L), and high glucose (HG; 27.7 mM/L) concentrations in INS-1 cells. The red-stained cells are dead. (B) Time-dependent course of cell death rate. Image was obtained using a microscope (×200). Data are expressed as mean±standard error of the 3 separate experiments. aP<0.05 vs. LG (72 hours).
Fig. 5Effect of cobalt protoporphyrin (CoPP) on cell death rate in cultured INS-1 cells using acridine orange (AO)/propidium iodide (PI) staining. (A) AO/PI staining in the presence or absence of CoPP (500 nM) in INS-1 cells at different glucose concentrations. The red-stained cells represent dead cells. (B) Effect of CoPP on cell death rate in cultured INS-1 cells using AO/PI staining. Imaging performed with a microscope (×200). Data are expressed as mean±standard error of the three separate experiments. aP<0.05 vs. low glucose (LG), bP<0.01 vs. high glucose (HG).
Fig. 6Intracellular peroxide level in INS-1 cells using flow cytometry. (A) The amount of reactive oxygen species (ROS) was measured to observe the effect of cobalt protoporphyrin (CoPP). (B) Fluorescence microscopy of ROS staining in INS-1 cells. The green staining is DCFDA and the red staining is propidium iodide. Image obtained using a microscope (×200). Data are expressed as mean±standard error of the three separate experiments. aP<0.01 vs. low glucose (LG), bP<0.05 vs. high glucose (HG).
Fig. 7Effect of cobalt protoporphyrin (CoPP) on protein level was determined using a Western blot analysis. (A) The level of heme oxygenase-1 (HO-1) protein. (B) The level of caspase-3 protein. (C) Confocal microscopy for TUNEL staining in INS-1 cells at 72 hours. The green staining is TUNEL and the blue staining is DAPI. Imaging performed with a confocal microscope (×400). Data are expressed as mean±standard error for the 3 separate experiments. aP<0.05 vs. low glucose (LG), bP<0.01 vs. high glucose (HG).
Fig. 8The effect of cobalt protoporphyrin (CoPP) on glucose-stimulated insulin secretion at 24 hours and 72 hours in low glucose (LG; 5.5 mM/L) and high glucose (HG; 25 mM/L) concentrations. Data are expressed as mean±standard error of the 6 separate experiments. aP<0.05 vs. LG.