FIGURE 5.

Effect of ER stress on the ΔΨ_m in L. major. L. major cells (10⁷/ml) were incubated with the potential sensitive probe JC-1 (6 μm) for 15 min at 25 °C to assess ΔΨ_m after treatment with 0.2% DMSO or TM (20 μg/ml) and analyzed by flow cytometry with excitation at 488 nm. Emission was detected at 530 nm for monomer and 590 nm for J-aggregate. A drop in ΔΨ_m was identified as a change in JC-1 properties from forming J-aggregates (emission at 590 nm) at high ΔΨ_m to forming J-monomers (emission at 530 nm) at low ΔΨ_m. The nearly complete monomer was induced by treating cells with 50 μm CCCP, an uncoupler of mitochondrial respiration, 15 min prior to addition of JC-1. Blank in dot plots indicates blank cells (cells without JC-1); +JC1 indicates 0.2% DMSO-treated cells stained with JC-1 as control; +TM indicates 8-h TM-treated JC-1-stained cells; +NAC indicates NAC-preincubated and 8-h TM-treated JC-1-stained cells; +Verapamil indicates verapamil-preincubated, 8-h TM-treated JC-1-stained cells, and +CCCP indicates CCCP-treated JC-1-stained cells. Data are representative of at least three independent experiments. B, time-dependent analysis of 590/530 values of DMSO-treated control cells, cells treated with 20 μg/ml TM, cells preincubated with NAC and Ca²⁺ channel inhibitor verapamil, and cells treated with CCCP. Data are representative of means ± S.D. of three independently performed experiments. The asterisks indicate the level of statistical significance (0.05). C, time-dependent measurement of intracellular ATP levels in DMSO-treated control cells and cells treated with TM or TM-treated but preincubated with NAC and verapamil. ATP concentration is expressed as nanomoles of ATP/10⁶ cells. Values were average ± S.D. of three independent experiments.