Effect of Hyperglycemia and HIV-1 Nef on Cytoskeleton and Mitochondria of astrocytes. Primary human astrocytes were cultured and exposed to various hyperglycemic conditions for 12 hours as mentioned before followed by washing with 1× PBS. The cells were then fixed with 4% paraformaldehyde for 10 minutes, and washed again with 1× PBS to remove the fixative. The effect of hyperglycemia on the cytoskeleton network (F-actin protein) was observed by staining the cells with phallacidin using protocol provided by the manufacturer, and examined under the fluorescent microscope. Panel A1-A3: A1. Astrocytes grown in normal medium, which served as control was stained with BODIPY phallacidin illustrate the normal cytoskeleton network. A2: Astrocytes treated with 15 mM glucose illustrates loose F-actin network and increased intracellular space indicating the loss of astrocytes. A3. Astrocytes treated with 25 mM glucose indicate significant changes in the cytoskeleton. The F-actin network was expanded and the intracellular space in between the astrocytes was further increased indicating cell death under higher glycemic conditions. B1. Normal astrocytes stained with MitoTracker Red to observe the effect of extracellular HIV-1 Nef recombinant protein on mitochondria. A2. 3 nM Nef protein solution was added into the medium with astrocytes and stained with MitoTracker. A3. Highly polarized mitochondria of primary astrocytes upon exposure to 25 nM of recombinant Nef protein, suggesting that free Nef protein could cause mitochondrial depolarization and ultimately cell death.