Figure 5
Effect of hyperglycemia and HIV-1 Nef on caspases and GFAP protein. For in vivo studies, 10 day old and STZ induced diabetic mouse pups were injected with 1 × 107 HIV-Nef infectious particles generated from HIV-1 or SNV based vectors systems. The pups were sacrificed 8 week after the injections. The brain tissue sections from cortex were removed and cellular protein lysates were prepared and loaded (25 μg/lane) onto a sodium dodecyl sulfate-(SDS) gel and electrophoresed, followed by a transfer onto a nitrocellulose membrane. The blots were then probed with antibody specific for whole and activated caspase-3. Panel A. Lane 1, Normal mice brain tissue protein serving as control, lane 2. Non-diabetic mice brain injected with HIV-1 Nef particles. Lane 3, diabetic mice brain tissues, lane 4, diabetic mice brain with SNV based Nef expressing virus, lane 5, diabetic mice brain with HIV-1 Nef expressing virus. Panel B. Astrocytes (U87-MG) were cultured under various glycemic conditions and transduced with HIV-1 Nef expressing viral particles. Forty-eight hours later, cells were lysed and the lysates (25 μg/lane) were loaded onto a SDS gel and electrophoresed, followed by a transfer onto a nitrocellulose membrane. The blots were then probed with antibody specific for whole and activated caspase-3. Lanes-1, expression of procaspase-3 in normal astrocytes transduced with HIV-1 Nef particles, 2-astrocytes treated with 10 mM glucose and HIV-1 Nef virus, 3-astrocytes treated with 15 mM glucose and HIV-1 Nef virus, 4-astrocytes treated with 18 mM glucose, 5-non treated normal astrocytes, 6-astroctes treated with 18 mM glucose and HIV-1 Nef virus. Panel C - Primary human astrocytes exposed to various hyperglycemic conditions and transduced with HIV-1 Nef expressing virus. The cells lysates are probe with GFAP antibody. Lanes: 1-non-treated normal astrocytes, 2-astrocytes treated with 18 mM glucose, 3- normal astrocytes transduced with HIV-1 Nef virus, 4- 10 mM glucose treated astrocytes, 5- astrocytes treated with 15 mM glucose and transduced with HIV-1 Nef virus, 6-astrocytes treated with 18 mM glucose and HIV-1 Nef. Panel D - Brain tissue lysates of diabetic or non-diabetic mice with HIV-1 Nef virus delivered into various regions of the brain. The tissues lysates were probed with antibody against GFAP. Lanes: 1-normal mice brain tissue, 2- diabetic mice brain tissue, 3-non-diabetic mice brain exposed to HIV-1 Nef virus, 4-diabetic mice with HIV-1 Nef virus, 5-diabetic mice with HIV-1Nef virus generated from SNV vectors. Panel E: Hyperglycemic treated and HIV-1 Nef-transduced astrocytic cell lysate probed with antibody specific against HIV-1 Nef protein. Lanes: 1-normal astrocytes, 2- astrocytes transduced with HIV-1 Nef virus, 3- astrocytes treated with 10 mM glucose and transduced with HIV-1 Nef virus, 4- astrocytes treated with 15 mM glucose and transduced with HIV-1 Nef virus, 5- astrocytes treated with 18 mM glucose and transduced with HIV-1 Nef virus, 6- astrocytes treated with 18 mM glucose and transduced with SNV Nef virus. Panel F: Brain tissue lysates of diabetic and non-diabetic mice injected with HIV-1 Nef virus and probed with Nef specific antibody. Lanes: 1- normal mice brain tissues, 2- mice injected with HIV-1 Nef virus, 3- diabetic mice injected with HIV-1 Nef virus, 4- diabetic mice injected with SNV based Nef virus, 5-normal mice injected with SNV-based HIV-1 Nef virus.