Figure 3. Role of GSK3β in HIV⁺_sup ± morphine-mediated cell death.

(A) Cells were repeatedly imaged for 72 h after treatments. Digital images show the same cells/fields at 0, 24 and 72 h (white arrowheads indicate dead cells that were alive in previous image). (B) Cells were assessed for viability at 6 h intervals in digital images. Findings were reported as the average percentage of neuronal survival as a proportion of pre-treatment neuron count ± SEM. Significance was analyzed by repeated measures ANOVA and Duncan’s post hoc test; n = 3 separate experiments (at least 150 neurons per treatment group). HIV⁺_sup significantly reduced neuronal survival (*p < 0.05 vs. Control), which was significantly augmented by morphine co-treatment (^$p < 0.05). HIV⁺_sup ± morphine-mediated effects were partially, but significantly, reversed by VPA (^#p < 0.05). VPA also effectively negated HIV⁺_sup-morphine interactions. (C) At 72 h after treatments, cells were fixed, permeabilized, and labeled for TUNEL and Hoechst 33342. The rate of neuronal apoptosis was reported as the average percentage of TUNEL(+) cells ± SEM. Significance was analyzed by one-way ANOVA and Duncan’s post hoc test; n = 3 separate experiments. HIV⁺_sup significantly increased the percentage of TUNEL(+) cells (*p < 0.05 vs. Control), and morphine augmented the effect (^$p < 0.05). HIV⁺_sup ± morphine-mediated effects were partially, but significantly, reversed by VPA co-treatment (^#p < 0.05), and VPA negated interactions between HIV⁺_sup and morphine. Control = control_sup; HIV = HIV⁺_sup; Mor = morphine; VPA = valproate.