The Journal of Neuroscience, July 6, 2005, ():
Progressive Degeneration of Human Mesencephalic Neuron-Derived Cells Triggered by Dopamine-Dependent Oxidative Stress Is Dependent on the Mixed-Lineage Kinase Pathway
J. Neurosci. Lotharius et al.
25: 6329
Supplemental data
Files in this Data Supplement:
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Supplemental Web Figure 1: Fe2+/METH induces changes in nuclear morphology. LUHMES cells were treated with Fe2+/METH for 6-72 h and stained with Hoechst 33342 to assess nuclear characteristics of apoptosis and with SYTOX green to evaluate plasma membrane integrity. Cells were treated with Fe2+/METH for 72 h in the presence of 250 nM CEP1347. White arrowheads indicate cells with apoptotic nuclei but intact plasma membrane integrity (SYTOX-negative); white arrows indicate apoptotic cells with broken plasma membranes.
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Supplemental Web Figure 2: Fe2+/METH induces caspase 3 processing. A, LUHMES cells treated with Fe2+/METH for 0, 6, 12, 24, 48, and 60 h were stained with an antibody recognizing cleaved caspase 3 (green). Cells were co-stained with Hoechst 33258 (blue) to visualize their nuclear morphology.
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Supplemental Web Figure 3: METH induces a collapse in pH gradients. A, LUHMES cells were stained with the pH sensitive dye acridine orange (25 nM) for 40 min and then treated with 10-1000 μM METH for 40 min. Fluorescence was visualized using a FITC filter. Micrograph on the right is of cells treated with 1 mM METH. B, The average intensity of fluorescent puncta in the cytoplasm was quantified using a Cellomics ArrayScan® instrument. Data are expressed as mean ± SEM of quadruplicate determinations and shown as % of control.
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Supplemental Web Figure 4: Timeline of pathogenic events following exposure of LUHMES cells to Fe2+/METH. Pathogenic events such as oxidative stress, JNK pathway activation, and neurite degeneration precede the actual loss of neurons following Fe2+/METH exposure, which only becomes evident after 72 h. Somewhere between 12-24 h neurites and cell bodies can no longer be rescued from the toxic effects of Fe2+/METH, suggesting a commitment to cell destruction. Note that CEP1347 significantly attenuated, and in most cases completely blocked, all of the deleterious cellular events following exposure to Fe2+/METH.
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Supplemental Web Figure 5: Schematic illustrating the effect of Fe2+/METH on dopaminergic terminals and sites where CEP1347 might exert its neuroprotective action.
