Figure 4: Cholesterol accumulation and its impact on mitochondrial function and architecture.

(A) Measurement of mitochondrial ROS in control and cholesterol treated pancreatic beta cells with or without the pre-treatment of small molecule M1. The cells were stained with Mito SOX red for 30 minutes and analyzed by flow cytometry. (A(i)) Representative histogram showing mean fluorescence intensity of MitoSOX stained cells; Black: Untreated, Unstained, Blue: Untreated stained, Red: Cholesterol treated, Green: M1pre-treated cells treated with cholesterol. (A(ii)) Bar diagram representing MitoSOX staining in untreated, cholesterol treated cells with or without the pre-treatment of small molecule M1.Data are expressed as mean ± SEM of 3 independent experiments. Chol: cholesterol, M1: small molecule M1. (B) Measurement of mitochondrial membrane potential in control and cholesterol treated pancreatic beta cells with or without the pre-treatment of small molecule M1. The cells were stained with JC-1 dye and analyzed by flow cytometry. JC-1 monomers were detected in the FL1 channel (Green). JC-1 aggregates were detected in FL2 channel (Red) and the ratio of FL2/FL1 was calculated for the determination of mitochondrial membrane potential. The data is expressed as fold decrease of FL2/FL1 with respect to cholesterol untreated cells. (C) Untreated and cholesterol treated cells with or without M1 pre-treatment was stained with Mito-Tracker Red and imaged in Leica confocal laser scanning microscope (Germany) using Leica advanced fluorescence suite 2.6.3. Images are representative of three independent experiments (scale: 7.24 μm).