Journal: Aging cell

Article Title: Sod2 overexpression preserves myoblast mitochondrial mass and function, but not muscle mass with aging.

doi: 10.1111/j.1474-9726.2009.00477.x

Figure Lengend Snippet: Fig. 5 Measurement of reactive oxygen species (ROS) in myoblasts. (A) Superoxide in mitochondria measured using MitoSox. Left: for reference, wild-type young myoblasts were treated with the increasing concentrations of rotenone (*P < 0.05 compared to wild-type young; **P < 0.05 compared to aged wild-type). (B) Intracellular ROS including hydrogen peroxide (H2O2) and hydroxyl radical (HO·) measured using CM-H2DCFH fluorescence. Left: for reference, intracellular ROS of wild-type young myoblasts treated with increasing concentrations of BSO (*P < 0.05 compared to wild-type young; **P < 0.05 compared to aged wild-type).

Article Snippet: The mitochondrial fraction was extracted from myoblasts grown to confluency on collagen-coated 150-mm dishes using a commercial mitochondria extraction kit (Imgenex, San Diego, CA, USA).

Techniques:

Journal: Journal of HIV and AIDS

Article Title: HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons

doi: 10.16966/2380-5536.176

Figure Lengend Snippet: HIV-1 and HIV-Tat induced mitochondrial DNA damage in human cortical neurons and in PBMC. A) Human PBMC cells were first infected with HIV-1 or with heat inactivated HIV-1, for 5 days. Conditioned media from PBMC cells were used to treat neuronal cells for 24 hours, and total cellular DNA was isolated. QPCR was performed for both the mitochondrial DNA (complete mtDNA in 16.2 kb, or short mtDNA in 0.2 kb, used for the normalization; top panels) and β-globin nuclear DNA (nDNA) fragments (bottom panels). mtDNA and nuclear DNA damage was calculated and demonstrated as a Relative Amplification and as a Lesions/10 kb (right panels). Control cultures were incubated in media with rotenone, a drug that causes mtDNA damage. Representative images from agarose gel depicting the decrease in amplification of the 17.7-kb β-globin fragment (nuclear DNA or nDNA), (bottom panels) and the 16.2-kb mitochondrial fragment (top images). The decrease in relative amplification is shown by graphs. The data are expressed as the mean ± CD from a minimum of two biological experiments in which PCRs for mitochondrial (mtDNA) or nuclear DNA (nDNA) were performed in triplicates per experiment. Asterisks *indicate that the difference between treated or untreated samples is statistically significant (p<0.05). B) mtDNA damage in human PBMC cells infected with HIV-1 or heat inactivated HIV-1, for 5 days. The data are expressed as the mean ± SD (n=6) from two independent experiments in triplicates. p24 ELISA in PBMC cells infected with HIV-1 JR-FL strain to show efficient infection. ELISA was performed in triplicates and p-value was calculated using Student’s t-test. Asterisks *indicate that the difference between infected or uninfected samples is statistically significant (p<0.05).

Article Snippet: Mitochondrial extracts were prepared from 50 × 10 6 cells using mitochondrial extraction kit (Imgenex).

Techniques: Infection, Isolation, Amplification, Control, Incubation, Agarose Gel Electrophoresis, Enzyme-linked Immunosorbent Assay

Journal: Journal of HIV and AIDS

Article Title: HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons

doi: 10.16966/2380-5536.176

Figure Lengend Snippet: A-C: HIV-1 Tat and VPR-induced mitochondrial DNA damage in human primary neuronal cells. Cortical neurons were transfected with pECFP or pECFP-Tat (A) or VPR (B) for 48 hours. Total cellular DNA was isolated. QPCR was performed for both the mitochondrial (top bars) and nuclear β-globin fragments (bottom bars). The mtDNA and nuclear qPCR results (in fold change) are compared with control. The data represent the average of two independent experiments using triplicated samples). Asterisks *indicate that the difference between infected or uninfected samples is statistically significant (p<0.05). C) Tat deletion mutants were used in transfection of neuronal cells to identify Tat-responsive region in mtDNA damage induction. Graphical representation of Tat domains and deletion mutants (top panel). Cellular localization of each Tat- deletion mutant in transfected cells is shown on the right for each Tat variant. D) Tat deletion variants were studied in the mtDNA damage induction assays, and SOD and ROS activity. Table summarized data shown on the bottom.

Article Snippet: Mitochondrial extracts were prepared from 50 × 10 6 cells using mitochondrial extraction kit (Imgenex).

Techniques: Transfection, Isolation, Control, Infection, Mutagenesis, Variant Assay, Activity Assay

Journal: Journal of HIV and AIDS

Article Title: HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons

doi: 10.16966/2380-5536.176

Figure Lengend Snippet: Mitochondrial DNA damage in human primary neuronal cells, induced by recombinant HIV-1 Tat peptide or adenoviral VPR. Cortical neurons were incubated with recombinant Tat or heat inactivated Tat (A) and adenoviral VPR (B) for 48 hours. Total cellular DNA was isolated, QPCR was performed for both the mitochondrial (top bars), and nuclear β-globin fragments (bottom bars). The mtDNA and nuclear qPCR results (in fold change) are compared with control. The data represent the average of two independent experiments (in triplicates for each sample). The analysis graph showed that compared with control, Tat induced nuclear DNA damage, while mitochondrial DNA damage was significantly lower, but both, mitochondrial and nuclear DNA damage were higher in cells incubated with VPR than controls (adeno-null treated cells).

Article Snippet: Mitochondrial extracts were prepared from 50 × 10 6 cells using mitochondrial extraction kit (Imgenex).

Techniques: Recombinant, Incubation, Isolation, Control

Journal: Journal of HIV and AIDS

Article Title: HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons

doi: 10.16966/2380-5536.176

Figure Lengend Snippet: Titration of doses with Tat peptide or rotenone, and mtDNA damage and SOD activity. Mitochondrial DNA damage in human primary neuronal cells induced by increased concentrations of Tat 101 FL peptide from 0.5 to 50 ng, or 0.036 to 3.6 nM (A) , or by various concentrations of rotenone as specified (20to 500 nM), as a positive control (B) . Cortical neurons were incubated with various concentrations of Tat and rotenone, and a total cellular DNA was isolated and QPCR was performed for the mitochondrial and nuclear fragments. Graphs demonstrate mtDNA damage, SOD activity in panel A, and mtDNA damage and nuclear DNA damage in panel B.

Article Snippet: Mitochondrial extracts were prepared from 50 × 10 6 cells using mitochondrial extraction kit (Imgenex).

Techniques: Titration, Activity Assay, Positive Control, Incubation, Isolation

Journal: Journal of HIV and AIDS

Article Title: HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons

doi: 10.16966/2380-5536.176

Figure Lengend Snippet: Titration of incubation time with Tat peptide or rotenone, and recovery assays. Mitochondrial DNA damage in human primary neuronal cells induced by Tat peptide at different times of incubation (A, B) or rotenone (C). A) Cortical neurons were incubated with Tat at different times of incubation (0 to 48 hours), as specified. Total cellular DNA was isolated and QPCR was performed for the mitochondrial and nuclear genes. Results for mtDNA and nDNA damage are shown in right panels. B) SOD activity in neurons for same time points (0 to 48 hours) of Tat treatment. C) mtDNA damage in neurons caused by Rotenone treatment of cells at different time points from 0 to 48 hours (top panels). Recovery assays from rotenone-caused mtDNA damage tested at 24 to 48 hours of rotenone removal (and replacement with a fresh media 24 h post-treatment) shown in bottom panels.

Article Snippet: Mitochondrial extracts were prepared from 50 × 10 6 cells using mitochondrial extraction kit (Imgenex).

Techniques: Titration, Incubation, Isolation, Activity Assay

Journal: Journal of HIV and AIDS

Article Title: HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons

doi: 10.16966/2380-5536.176

Figure Lengend Snippet: Effect of Tat on mitochondrial energy metabolism in human neurons. A) qRT-PCR assays were performed for genes involved in mitochondrial bioenergetics, using Mitochondria Energy Metabolism Arrays. Table on the bottom shows genes that represent all five Complexes (I to V). B) To confirm changes in ATP gene expression, ATP determination assay was performed in Tat treated neurons. Human primary neurons were treated with Tat, heat inactivated Tat (h.i.) or rotenone for 48 hours. The mitochondrial pellet was lysed in RIPA buffer, then the lysates were analyzed for ATP levels using ATP-luciferase assay. ATP levels were decreased significantly both in Tat or rotenone treated cells. Asterisks indicate that the difference between treated or untreated samples is statistically significant (p<0.05). Data represent the mean ± SD (n=6); *-p=0.05, **-p=0.01, ***-p=0.001. C) Treatment of neuronal cells with Tat (left panels) or transfection of cells with Tat expressing plasmid (right panels) causes inhibition of gene expression of mtDNA damage repair 8-oxoguanine DNA glycosylase-1 (OGG1), an enzyme that can repair 8-oxoG DNA lesion, studied by qRT-PCR. Our data demonstrate that Tat causes inhibition of OGG1 gene expression (70% or 60% a), which will interfere with the mtDNA damage repair mechanisms.

Article Snippet: Mitochondrial extracts were prepared from 50 × 10 6 cells using mitochondrial extraction kit (Imgenex).

Techniques: Quantitative RT-PCR, Gene Expression, Luciferase, Transfection, Expressing, Plasmid Preparation, Inhibition

Journal: Journal of HIV and AIDS

Article Title: HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons

doi: 10.16966/2380-5536.176

Figure Lengend Snippet: Tat increases the levels of apoptotic proteins and dysregulates mitochondrial proteins cellular localization in human neurons. A) Quantitative western blot assay was performed for PINK1 and PKM proteins in lysates from Tat treated neuronal cells. PINK1 and PKM are localizing at higher levels at the membranes of injured/damaged mitochondria. Cell fractionation was performed using mitochondrial isolation kit to separate mitochondrial, cytoplasmic and nuclear fractions. COX IV was used as a mitochondrial fraction control; Grb2 and Tubulin were used as cytoplasmic fraction purity and a loading control, and Lamin A-as a nuclear fraction control. B) Tat expression in transfected neurons activates apoptotic Bax and Bad and inhibits outer membrane VDAC in mitochondrial fraction, and leads to the translocation of apoptotic Cytochrome C from mitochondria to cytoplasm. C) Images of neuronal cells with or without Tat.

Article Snippet: Mitochondrial extracts were prepared from 50 × 10 6 cells using mitochondrial extraction kit (Imgenex).

Techniques: Western Blot, Cell Fractionation, Isolation, Control, Expressing, Transfection, Membrane, Translocation Assay