Journal: Science Advances
Article Title: Histone decrotonylation plays a distinct role in HIV latency
doi: 10.1126/sciadv.aec0149
Figure Lengend Snippet: ( A and B ) Peripheral resting CD4 + T cells were isolated from eight PWH on ART and treated for 24 hours with the following compounds: PMA (200 ng/mL), ionomycin (2 µM), SAHA (350 nM), RGFP966 (2.5 µM), mod-RGFP966 (2.5 µM), and citarinostat (5 µM). (A) The cell-associated HIV gag levels were measured by droplet digital PCR (ddPCR), with results shown individually for each donor, including triplicate ddPCR data. (B) A combined summary of the triplicate ddPCR results from all donors ( n = 8) is presented as averages. PMA 200 ng/mL; ionomycin 2 µM; SAHA 350nM; RGFP966 5µM; mod-RGFP966 5µM; citarinostat 5µM. ( C ) Cell-free HIV gag levels in the culture supernatant from three donors treated with 350 nM SAHA and 5 µM citarinostat were measured. ( D ) Brain tissue from NHP or participants with HIV was mechanically dissociated to generate a single-cell suspension, followed by Percoll gradient separation. CD3 + T cells were depleted using a CD3 selection kit. Brain MG were isolated from the CD3-negative fraction by CD11b + selection. Brain MG were cultured ex vivo for 1 to 2 weeks to allow recovery and attachment before being used in the LRA study. ( E ) Brain MG from participants with HIV were treated with DMSO (control), 40 mM NaCr, 350 nM SAHA, or 10 μM RGFP966 ( n = 4). ( F ) In a separate experiment, cells were treated with DMSO or 5 μM citarinostat ( n = 5). ( G ) Brain MG from NHPs were treated with 5 μM citarinostat or DMSO control ( n = 6). After 24 hours, the drugs were removed, and cells were cultured for another 6 days. Cells and culture supernatants were harvested for HIV LTR RNA [(E) and (F)] or SIV RNA (G) measurement by ddPCR. A combined summary of the triplicate ddPCR results is presented as averages. Error bars represent SD; * P < 0.05; ** P < 0.01; *** P < 0.001.
Article Snippet: For human samples, CD11b MicroBeads, human and mouse (Miltenyi Biotec) were used, while for NHP samples, CD11b MicroBeads, NHP (Miltenyi Biotec) were used.
Techniques: Isolation, Digital PCR, Single Cell, Suspension, Selection, Cell Culture, Ex Vivo, Control
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![( A and B ) Peripheral resting CD4 + T cells were isolated from eight PWH on ART and treated for 24 hours with the following compounds: PMA (200 ng/mL), ionomycin (2 µM), SAHA (350 nM), RGFP966 (2.5 µM), mod-RGFP966 (2.5 µM), and citarinostat (5 µM). (A) The cell-associated HIV gag levels were measured by droplet digital PCR (ddPCR), with results shown individually for each donor, including triplicate ddPCR data. (B) A combined summary of the triplicate ddPCR results from all donors ( n = 8) is presented as averages. PMA 200 ng/mL; ionomycin 2 µM; SAHA 350nM; RGFP966 5µM; mod-RGFP966 5µM; citarinostat 5µM. ( C ) Cell-free HIV gag levels in the culture supernatant from three donors treated with 350 nM SAHA and 5 µM citarinostat were measured. ( D ) Brain tissue from NHP or participants with HIV was mechanically dissociated to generate a single-cell suspension, followed by Percoll gradient separation. CD3 + T cells were depleted using a CD3 selection kit. Brain MG were isolated from the CD3-negative fraction by <t>CD11b</t> + selection. Brain MG were cultured ex vivo for 1 to 2 weeks to allow recovery and attachment before being used in the LRA study. ( E ) Brain MG from participants with HIV were treated with DMSO (control), 40 mM NaCr, 350 nM SAHA, or 10 μM RGFP966 ( n = 4). ( F ) In a separate experiment, cells were treated with DMSO or 5 μM citarinostat ( n = 5). ( G ) Brain MG from NHPs were treated with 5 μM citarinostat or DMSO control ( n = 6). After 24 hours, the drugs were removed, and cells were cultured for another 6 days. Cells and culture supernatants were harvested for HIV LTR RNA [(E) and (F)] or SIV RNA (G) measurement by ddPCR. A combined summary of the triplicate ddPCR results is presented as averages. Error bars represent SD; * P < 0.05; ** P < 0.01; *** P < 0.001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8052/pmc13068052/pmc13068052__sciadv.aec0149-f8.jpg)