Figure 3.

Analysis of effects of nemorosone on mitochondrial membrane potential ΔΨ_m and cytosolic calcium concentration [Ca²⁺]_c in pancreatic cancer cells and fibroblasts. (A) Cells were treated with the indicated concentrations of nemorosone or vehicle for 4 h and stained with the voltage-dependent dye JC-1 prior to fluorescence microscopy. Intact mitochondria fluoresce red while cells with atteunuated mitochondrial membrane potential ΔΨ_m exhibit cytosolic green fluorescence. Images are representative of three independent experiments with similar results. (B) Kinetic analysis of ΔΨ_m upon nemorosone treatment. Cells were prestained with JC-1 and subsequently treated with 20 µM nemorosone. Fluorescent images were recorded every 10 s after onset of treatment. Development of relative red and green fluorescence as well as red/green fluorescence ratio is shown as mean ± SD of three independent experiments. (C) Analysis of nemorosone-induced changes in cytosolic calcium concentration [Ca²⁺]_c in MIA-PaCa-2 cells and human dermal fibroblasts (HDF). Cells were loaded with the calcium-sensitive Fluo-4 dye and subsequently treated with the indicated concentrations of nemorosone or vehicle after recording a baseline for 30 s (black arrows). Development of Fluo-4 fluorescence relative to baseline fluorescence (F/F₀) is shown as mean of three independent experiments. DMSO, dimethylsulphoxide.