DIDS induces Annexin V staining in a dose- and time-dependant manner.

<p>(<b>A</b>) Summary of Annexin V fluorescence from neurons treated for 2 hrs in 96-well microplates as indicated. (<b>B</b>) Sample confocal microscopy images of Annexin V (green) and DAPI (blue) fluorescence from neurons treated as indicated for 2 hrs. (<b>C</b>) Summary of Annexin V fluorescence from neurons treated for 24 hrs in 96-well microplates as indicated. (<b>D</b>) Sample confocal microscopy images of Annexin V (green, and arrows) and DAPI (blue) fluorescence from neurons treated as indicated for 24 hrs. Images are representative of 4 separate experiments. Data are mean ± SEM. Asterisks (*) indicated significant difference from normoxic controls; bars indicate significance between treatments (<i>p</i><0.05). AFU = artificial fluorescence units.</p

DIDS treated cells retain plasma membrane integrity despite ATP depletion and an apoptotic phenotype.

<p>DIDS treatment induced an apoptotic phenotype and also dose-dependant [ATP] depletion from neurons, but membrane integrity was preserved. (<b>A</b>) Sample TEM images of neurons treated as indicated for 24 hrs. TEM experiments were repeated 2 times and 10–20 cells were examined from each treatment group. (<b>B</b>) Summary of neuronal [ATP] versus [DIDS] (40 or 400 µM) following 24 hrs treatment, normalized to untreated controls at <i>t</i> = 0. ATP luciferase experiments were repeated 5 times. (<b>C</b>) Summary of neuronal propidium iodide (PI) uptake vs. [DIDS] following 24 hrs of treatment, normalized to untreated controls. PI exclusion experiments were repeated 4 times. [DMSO] = 0.4%. Data are mean ± SEM. Asterisks (*) indicate significant difference from normoxic controls; bars indicate significance between treatments (<i>p</i><0.05).</p

DIDS upregulates apoptotic pathways in neurons.

<p>(<b>A</b>) Sample Western blots of apoptosis-related protein expression from neurons treated as indicated for 6 hrs. (<b>B–D</b>) Summaries of fold-change in cytochrome C (<b>B</b>), caspase 3 (<b>C</b>), and JNK3 (<b>D</b>) protein expressions from (A) normalized to α–actin expression in the same sample. Data are mean ± SEM from 3 separate experiments for each protein and treatment. Asterisks (*) indicate significant difference from untreated controls (<i>p</i><0.05).</p

DIDS (400 µM) causes mitochondrial swelling and cristae reduction.

<p>DIDS treated mitochondria were longer, fewer in number, and had markedly reduced cristae density. (<b>A</b>) Sample TEM images of mitochondria from neurons treated as indicated for 24 hrs. Arrow indicates “ballooning” of outer mitochondrial membrane (OMM). (<b>B</b>) Summary of mitochondrial morphology-related parameters from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060804#pone-0060804-g004" target="_blank">Fig. 4A</a>. (<b>C</b>) Summary of cristae morphology-related parameters from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060804#pone-0060804-g004" target="_blank">Fig. 4A</a>. Data are mean ± SEM. Asterisks (*) indicate significant difference from normoxic controls (<i>p</i><0.05).</p

DIDS (400 µM) treated nuclei exhibit apoptotic chromatin condensation, nuclear envelope shrinkage and fragmentation of the nuclear membrane into apoptotic bodies.

<p>(<b>A</b>) Sample TEM images of nuclei from neurons treated as indicated for 24 hrs. Arrows indicate condensed chromatin beads. Inset: DAPI-stained chromatin distribution in healthy (ACSF) and apoptotic (DIDS-treated) 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). (<b>D</b>) Summary of cleaved lamin A expression normalized to DAPI-stained nuclei following 24 hrs treatment. (<b>E</b>) Sample immunohistochemical (IHC) fluorescent confocal microscopy images of cleaved lamin A (red) and DAPI (blue) staining in neurons from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060804#pone-0060804-g005" target="_blank">Fig. 5D</a>. Lamin A IHC experiments were repeated 4 times. Data are mean ± SEM. Asterisks (*) indicate significant difference from normoxic controls (<i>p</i><0.05).</p

DIDS causes RNA and DNA degradation in a dose-dependant manner.

<p>(<b>A&B</b>) Summaries of the effect of ACSF±DMSO±DIDS (40 or 400 µm) on total RNA content (RNA_tot) (A) or RNA integrity (RIN) (B) from neurons treated for 1, 2, 4, 8 or 24 hrs as indicated. RNA_tot was normalized to untreated samples. RNA experiments were repeated 4 times at each time point. (<b>C</b>) Summary of the ratio of terminal transferase dUTP nick end-labeling (TUNEL)-positive neurons relative to DAPI-stained nuclei from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060804#pone-0060804-g006" target="_blank">Fig. 6D</a>. (<b>D</b>) Sample confocal microscopy images of TUNEL (green, arrows) and DAPI (blue) fluorescence from neurons treated as indicated for 24 hrs. TUNEL experiments were repeated 4 times. Data are mean ± SEM. Asterisks (*) indicate significant difference from normoxic controls; bars indicate significance between treatments (<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

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