Journal: The Journal of Biological Chemistry

Article Title: Exosomes from uninfected cells activate transcription of latent HIV-1

doi: 10.1074/jbc.M117.793521

Figure Lengend Snippet: Exosomes from uninfected cells on latent HIV-1–infected cells increase short RNA levels. A, exosomes from CEM, Jurkat, and U937 cells were isolated using ultracentrifugation prior to addition to Jurkat E4 cells in 0.24-, 0.78-, and 2.4-unit/ml increments (determined using an AchE assay) once per day over the course of 3 days. The E4s were incubated for an additional 48 h prior to harvest, and total RNA was isolated and subjected to RT with a TAR-specific primer. qPCR was performed to quantify the total amount of TAR RNA copies. B, exosomes from CEM, Jurkat, and U937 cells were isolated using ultracentrifugation prior to addition to Jurkat E4 cells in 0.6-, 1.2-, and 1.8-unit/ml (determined using AchE assay) increments. The cells were then incubated for an additional 48 h prior to a GFP assay. Student's t tests compared untreated cells with cells treated with exosomes. In Figs. 1​1​​​​–7 and ​and10,10, all cells were grown in exosome-free medium. **, p < 0.01; ***, p < 0.001. Error bars, S.D.

Article Snippet: GFP assay Jurkat E4 (10 5 ) cells were plated and treated with varying concentrations (0.24, 0.78, and 2.4 units/ml) of CEM-, Jurkat-, or U937-derived exosomes.

Techniques: Infection, Isolation, AChE Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Exosomes from uninfected cells activate transcription of latent HIV-1

doi: 10.1074/jbc.M117.793521

Figure Lengend Snippet: Presence of short RNA transcripts in wild-type HIV-1–infected T cells and monocytes. Exosomes from Jurkat and U937 cells were isolated using ultracentrifugation prior to being added to ACH2, U1, or OM10.1 cells once per day over the course of 3 days. The cells were then allowed to incubate for an additional 48 h prior to harvest. Total RNA was isolated and then subjected to RT with a primer specific for TAR. qPCR was performed to quantify the total amount of TAR RNA copies. A, U1 cells were treated with 2.79, 13.95, and 41.85 milliunits/ml U937 exosomes. B, U1 cells were treated with 2.90, 14.5, and 43.5 milliunits/ml Jurkat exosomes. C, ACH2 cells were treated with 2.42, 12.10, and 36.30 milliunits/ml U937 exosomes. D, ACH2 cells were treated with 1.75, 8.75, and 26.25 milliunits/ml Jurkat exosomes. E, OM10.1 cells were treated with 2.42, 12.10, and 36.30 milliunits/ml U937 exosomes. F, OM10.1 cells were treated with 1.75, 8.75, and 26.25 milliunits/ml Jurkat exosomes. All exosome concentrations were determined by an AchE assay. Student's t tests compare exosome-treated cells with untreated cells. Error bars, S.D.

Article Snippet: GFP assay Jurkat E4 (10 5 ) cells were plated and treated with varying concentrations (0.24, 0.78, and 2.4 units/ml) of CEM-, Jurkat-, or U937-derived exosomes.

Techniques: Infection, Isolation, AChE Assay

Journal: The Journal of Biological Chemistry

Article Title: Exosomes from uninfected cells activate transcription of latent HIV-1

doi: 10.1074/jbc.M117.793521

Figure Lengend Snippet: Presence of increased RNA pol II and Cdk9 on HIV-1 genome when treated with exosomes from uninfected cells. Exosomes from Jurkat and U937 cells were isolated by ultracentrifugation and added to ACH2 (A) and U1 (B) cells under cART treatment. The exosomes were added at a concentration of 0.403 millunits/ml once per day for 3 days. Cells were allowed to incubate for an additional 48 h. The cells were cross-linked prior to ChIP assay utilizing antibodies for phosphorylated RNA polymerase II (Ser-2/5), Cdk9, and IgG. DNA was then quantified using qPCR. The primers for the PCR were NF-κB1–2F and TAR +59-R (supplemental Fig. 2). Student's t test was used to compare samples with the IgG control. Error bars, S.D.

Article Snippet: GFP assay Jurkat E4 (10 5 ) cells were plated and treated with varying concentrations (0.24, 0.78, and 2.4 units/ml) of CEM-, Jurkat-, or U937-derived exosomes.

Techniques: Isolation, Concentration Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Exosomes from uninfected cells activate transcription of latent HIV-1

doi: 10.1074/jbc.M117.793521

Figure Lengend Snippet: TAR-gag is a long non-coding RNA. ACH2 cells and U1 cells were pretreated for 5 days with a cART mixture. One milliliter of cell supernatant was collected and treated with nanotraps (NT), specifically a 30% NT080/082 slurry, prior to rotating for 16 h at 4 °C to concentrate exosomes. Jurkat and U937 cells (3.5 × 106; 0.75 and 1.0 ml, respectively) were treated with 1 ml of either ACH2 or U1 supernatants or the concentrated exosomes from the infected cells (emphasized by orange boxes). Jurkat (A) and U937 (B) were incubated for 72 h prior to harvest for Western blotting using p24 and pr55 combined antibodies. Whole-cell extract (WCE) was used as a positive control for Western blots.

Article Snippet: GFP assay Jurkat E4 (10 5 ) cells were plated and treated with varying concentrations (0.24, 0.78, and 2.4 units/ml) of CEM-, Jurkat-, or U937-derived exosomes.

Techniques: Infection, Incubation, Western Blot, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Exosomes from uninfected cells activate transcription of latent HIV-1

doi: 10.1074/jbc.M117.793521

Figure Lengend Snippet: The addition of uninfected exosomes to wild-type HIV-1–infected cells increases long transcript levels. A, exosomes from Jurkat and U937 cells were isolated using ultracentrifugation prior to addition to ACH2 and U1 cells at concentrations of 26.25 milliunits/ml (lane 2), 43.50 milliunits/ml (lane 4), and 41.85 milliunits/ml (lane 5) (all were determined by an AchE assay). The exosomes were added once per day for 3 days prior to incubation for another 48 h. The cells were then harvested; total RNA was isolated and subjected to RT with a primer specific to the 3′-end of the HIV-1 genome. qPCR was performed with gag-specific primers to quantify the levels of genomic mRNA. B, exosomes from Jurkat and U937 cells were isolated using ultracentrifugation before addition to ACH2 and U1 cells at concentrations of 26.25 milliunits/ml (lanes 4 and 6) and 41.85 milliunits/ml (lanes 5 and 7), respectively. The exosomes were added once per day for 3 days prior to incubation for another 48 h. The cells were then harvested and lysed, and the resulting lysates were run on a gel, transferred, and subjected to Western blotting for the presence of pr55 and p24. β-Actin was used as a control. Student's t tests compare exosome-treated ACH2 cells with untreated ACH2 cells, whereas a second t test compares exosome-treated U1 cells with untreated U1 cells. Whole-cell extract (WCE) was used as positive control for Western blots. Molecular weight (MW) is shown in lane 3. Error bars, S.D.

Article Snippet: GFP assay Jurkat E4 (10 5 ) cells were plated and treated with varying concentrations (0.24, 0.78, and 2.4 units/ml) of CEM-, Jurkat-, or U937-derived exosomes.

Techniques: Infection, Isolation, AChE Assay, Incubation, Western Blot, Control, Positive Control, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Exosomes from uninfected cells activate transcription of latent HIV-1

doi: 10.1074/jbc.M117.793521

Figure Lengend Snippet: Presence of short RNA transcripts in wild-type HIV-1–infected T cells and monocytes treated with cART. Exosomes from Jurkat and U937 cells were isolated using ultracentrifugation. The aforementioned exosomes were added to either ACH2 cells or U1 cells under cART treatment. cART consisted of an equal-parts (10 μm) mixture of indinavir (IDV), lamivudine (3TC), tenofovir disoproxil fumarate (TDF), and emtricitabine (FTC). A, the exosomes were added at concentrations of 1.60 milliunits/ml (lanes 2 and 4) and 1.88 milliunits/ml (lanes 3 and 5) once per day over the course of 3 days. The cells were allowed to incubate an additional 48 h prior to harvest; their total RNA was isolated and subjected to RT with a TAR-specific primer. qPCR was performed to quantify the total amount of TAR RNA copies. B, OM10.1 cells were treated with exosomes derived from Jurkat cells. The concentration of exosomes added to each sample was 0.107 milliunits/ml (lane 2), 0.535 milliunits/ml (lane 3), or 1.60 milliunits/ml (lane 4). C, OM10.1 cells were treated with exosomes derived from U937 cells. The concentration of exosomes added to each sample was as follows: 0.125 milliunits/ml (lane 2), 0.626 milliunits/ml (lane 3), or 1.88 milliunits/ml (lane 4). All exosome concentrations were determined by an AchE assay. Student's t test compares exosome-treated ACH2 cells with untreated ACH2 cells, whereas a second t test compares exosome-treated U1 cells with untreated U1 cells. A third t test compares exosome-treated OM10.1 cells with untreated OM10.1 cells. Error bars, S.D.

Article Snippet: GFP assay Jurkat E4 (10 5 ) cells were plated and treated with varying concentrations (0.24, 0.78, and 2.4 units/ml) of CEM-, Jurkat-, or U937-derived exosomes.

Techniques: Infection, Isolation, Derivative Assay, Concentration Assay, AChE Assay

Journal: The Journal of Biological Chemistry

Article Title: Exosomes from uninfected cells activate transcription of latent HIV-1

doi: 10.1074/jbc.M117.793521

Figure Lengend Snippet: Effect of uninfected exosomes on long RNA transcripts in wild-type HIV-1–infected cells under cART conditions. Exosomes from Jurkat and U937 cells were isolated using ultracentrifugation prior to addition to ACH2 (A) and U1 cells (B); cells were under cART treatment, which consisted of an equal-parts (10 μm) mixture of IDV, 3TC, TDF, and FTC. Concentrations of exosomes were 1.60 milliunits/ml (lane 2) and 1.88 milliunits/ml (lane 3). Exosomes were added once per day for 3 days. Cells were allowed to incubate for an additional 48 h. The cells were then harvested; total RNA was isolated and subjected to RT with a primer specific to the 3′-end of the HIV-1 genome. RT-qPCR was performed to quantify the levels of genomic RNA. Student's t test was performed to compare exosome-treated lanes with untreated lanes. Experimental results in B (lane 3) were run on a plate different from that used for lanes 1 and 2 and thus cannot be statistically compared with lane 1. Error bars, S.D.

Article Snippet: GFP assay Jurkat E4 (10 5 ) cells were plated and treated with varying concentrations (0.24, 0.78, and 2.4 units/ml) of CEM-, Jurkat-, or U937-derived exosomes.

Techniques: Infection, Isolation, Quantitative RT-PCR