IS induces extensive autophagic vacuolization of neuronal cytoplasm and depletes [ATP] in neurons and astrocytes.

<p>IS-treated neurons and oligomycin-treated neurons and astrocytes exhibit extensive cytoplasmic vacuolization, organelle digestion, and [ATP] changes characteristic of autophagy. (<b>A</b>) Sample TEM images of neurons (upper panels) and astrocytes (lower panels) treated as indicated for 24 hrs. Arrows indicate vacuoles. (<b>B</b>) Summary of vacuole density in the cytoplasm by volume. TEM experiments were repeated 2–3 times and 10–20 cells were examined from each treatment group. (<b>C</b>) Summary of change in [ATP] with time from neurons (open symbols) and astrocytes (closed symbols) treated as indicated through 48 hrs from >10 different experiments. (<b>D</b>) Summary of fold-change in protein expression from western blot analysis of PARP cleavage in samples treated for 6 hrs. Changes were normalized to α–actin expression in the same sample. (<b>E</b>) Sample western blots from (D). Blots are representative of 3 separate experiments. Data are mean ± SEM. Asterisks (*) indicate significant difference from untreated controls (<i>p</i><0.05). Treatments: control (DMEM/F12), ischemic solution (IS), 2.5 µm staurosporine (STS), and 10 µm oligomycin A (Oligo A).</p

IS upregulates autophagy and apoptotic pathways in neurons and astrocytes.

<p>(<b>A</b>) Sample Western blots of autophagy- and apoptosis related protein expression from neurons (left panels) and astrocytes (right panels) treated as indicated for 6 hrs. (<b>B</b>) Summary of fold-change in protein expressions from (A) normalized to α–actin expression in the same sample. Data are mean ± SEM from 3 separate experiments for each protein, cell type, and treatment. Asterisks (*) indicate significant difference from untreated controls (<i>p</i><0.05).</p

IS-treated nuclei exhibit apoptotic chromatin condensation, nuclear envelope fragmentation into apoptotic bodies, and laddered DNA cleavage.

<p>(<b>A</b>) Sample TEM images of nuclei from neurons (upper panels) and astrocytes (lower panels) treated as indicated for 24 hrs. White arrows indicate condensed chromatin beads and fragmenting nuclei. (<b>B</b>) Summary of nuclear volume density from samples in (A). (<b>C</b>) Summary of chromatin volume density from samples in (A). Note: nuclei were too fragmented in oligomycin A-treated astrocytes for quantification. (<b>D</b>) Sample western blots of lamin A cleavage in samples treated as indicated for 6 hrs. Blots are representative of 3 separate experiments. (<b>E</b>) Summary of fold-change in protein expression from (D) normalized to α–actin expression in the same sample. (<b>F&G</b>) Sample conventional agarose gel electrophoresis gel images of DNA fragmentation from neurons (F) and astrocytes (G) treated as indicated for 0, 12, 24, or 48 hrs. Gels are representative of 4 separate experiments. Data are mean ± SEM. Asterisks (*) indicate significant difference from untreated controls (<i>p</i><0.05).</p

IS induces apoptotic Annexin V translocation in neurons and astrocytes.

<p>(<b>A</b>) Sample paired DIC (left panels) and Annexin V and DAPI (green and blue fluorescence, respectively; right panels) confocal microscopy images of neurons and astrocytes treated as indicated for 24 hrs. Images are representative of 4 separate experiments. (<b>B</b>) Summary of the ratio of Annexin V-positive stained cells to DAPI-stained nuclei. (<b>C</b>) Summary of Annexin V fluorescence from neurons or astrocytes treated in 96-well microplates as indicated for 24 hrs. Data are mean ± SEM. Asterisks (*) indicated significant difference from untreated controls (<i>p</i><0.05).</p

IS causes mitochondrial fission and membrane rupture.

<p>(<b>A</b>) Sample TEM images of mitochondria from neurons (upper) and astrocytes (lower) treated as indicated for 24 hrs. (<b>B</b>) Sample western blots of apoptosis inducing factor (AIF) and cytochrome C (Cyto C) release from samples treated as indicated for 6 hrs. Blots are representative of 3 separate experiments.</p