Figure 3

Effect of EtOH±HIV-1 on astrocyte toxicity, morphology and mitochondrial function. Astrocytes were exposed to EtOH (50 mM) for 5 days and treated with HIV-1 for 24 h. Metabolic activity (a), proliferation (b) and apoptosis (c) were assessed by MTT, BrdU incorporation and DNA fragmentation assays, respectively. All experiments were conducted using three independent astrocyte donors analyzed in multiple replicates and cumulative data normalized to controls are shown as fold changes. Statistical analyses were performed using one-way ANOVA with Bonferroni post hoc test for multiple comparisons (*P<0.05, **P<0.01 and ***P<0.001). In parallel, immunostaining was performed for glial fibrillary acidic protein (GFAP, red) and DAPI (nuclei, blue); (d–g; control (d), HIV-1 (e), EtOH (f) and EtOH and HIV-1 (g)). To monitor the mitochondrial permeability transition pore (mPTP) opening using calcein/CoCl₂ (cobalt chloride) assay, astrocytes (control, h) were incubated with EtOH (50 mM, j) and HIV-1 (i) or in combination (k) for 24 h. Calcein (green) co-localized with Mitotracker (MTR, red) represents closed mPTP (arrowhead, yellow), while loss of green fluorescence represents opening of mPTP (arrow). Original magnification, ×200.