Fig 1: Effects of 25OHChol on caspase activity in BE(2)-C cells. BE(2)-C cells were treated with Chol or oxysterols at a concentration of 1 µg/mL for 48 h, after which caspase activity was evaluated using flow cytometry and Western blotting. (A) To measure caspase-9 activity, cells were incubated with FITC-LEHD-FMK at 37 °C with 5% CO2 for 1 h, followed by flow cytometry analysis. (B) For assessing caspase-3/7 activity, cells were incubated with TF2-DEVD-FMK under the same conditions for 3 h, and activity was then measured by flow cytometry. (C) In a separate experiment, BE(2)-C cells were treated with 25OHChol at 1 µg/mL, and cell lysates were harvested at 0, 24, and 48 h after treatment. Total protein was analyzed via Western blot to detect caspase-3. The band intensity ratio of cleaved caspase-3/pro-caspase-3 was calculated and the mean values presented as a graph, with data shown as the mean ± SD from three replicates per condition (n = 3). Statistical significance was determined at *** p < 0.001 compared to the control group. (D) BE(2)-C cells were exposed to 1 μg/mL 25OHChol and Z-VAD-FMK at specified concentrations for 48 h. Cell viability was determined using the CCK-8 assay, with data presented as the mean ± SD from three replicates per condition (n = 3). *** p < 0.001 compared to the control group and # p < 0.5, ## p < 0.1, ### p < 0.01 compared to the 25OHChol group.