Figure 6
ER hyper-oxidation, mitochondrial calcium uptake and mitochondrial ROS generation contributes to cell death by PAC-1. (a) HeLa and MEF DKO cells were treated with 50 or 100 μM of PAC-1 for 24 h. The cells were stained with mitochondrial calcium indicator Rhod-2 as described. Rhod-2 fluorescence analyzed by FACS is shown as histograms. (b) HeLa and MEF DKO cells were either pretreated with 0.05% DMSO or mitochondrial calcium uptake inhibitor, Ru360 (25 μM) or cell-permeant calcium chelator, BAPTA-AM (50 μM) followed by PAC-1 treatment for 24 h. Cell death was quantified by chromatin condensation assay as described (n=3, mean±S.D.). (c) HeLa cells stably expressing mitochondria-targeted roGFP were developed as described. Microscopic image of mitochondrial localization of the probe is shown at × 100 magnification. The emission at 535/30 nm was collected at dual excitation 405/20X and 490/20X in sequential mode using × 60 objective for both untreated and treated (PAC-1 for 12 h) stable cells. The ratio images were generated by dividing 405 nm channel by 490 nm channel on a pixel by pixel basis. The ratio scale is also shown. (d) HeLa cells stably expressing ER-targeted roGFP, roGFP2-iL-KDEL was developed as described. Microscopic image of ER-targeted probe is shown at × 100 magnification. Ratio imaging was carried out as earlier using × 100 objectives. The ratio images were generated by dividing 405 nm channel by 490 nm channel on a pixel by pixel basis. The ratio scale is also shown
