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Fig. 3 | Epigenetics & Chromatin

Fig. 3

From: Tat inhibition by didehydro-Cortistatin A promotes heterochromatin formation at the HIV-1 long terminal repeat

Fig. 3

dCA partially inhibits HIV expression in U1 cells. a Schematic diagram of the Tat-TAR feedback loop present in provirus in U1 cells. Tat’s H13L mutation weakens interaction with P-TEFb. b The viral production in U1 cell treated with dCA. Cells were split and treated on average every 3 days in the presence of ART with or without dCA (10 nM). Capsid production was quantified by p24 ELISA. Data are representative of three independent experiments. c The viral production in U1 cell stimulated by SAHA. After 48 days of treatment, cells (highlighted with “⦿/” in panel A) were stimulated with 2.5 µM SAHA for 24 h. Capsid production was quantified by p24 ELISA. Data are average of 3 independent experiments and the error bars represent the SD of 3 independent experiments. d The viral mRNA levels in U1 cell stimulated by SAHA. Samples from panel B were used for RNA extraction, and cDNAs from extracted total RNA were quantified by RT-PCR using primers to the Vpr region. Results were normalized as the number of viral mRNA copies per GAPDH mRNA. Viral mRNA generated in the ART control was set to 100%, and the error bars represent the SD of 3 independent experiments. e Distribution of RNAP II on the HIV genome in U1 cells. ChIP assay to RNAPII was performed using cell samples from panels C-D. Results are represented as the percentage of input and subtracted the background of the isotype IgG control. Data are average of 3 independent experiments, and error bars represent the SD of 3 experiments for each primer set. f MNase protective assays using sample in panels B-C. Error bars represent the SD of 3 independent experiments. g The recruitment of PBAF complex on HIV promoter DNA in as determined by ChIP to BAF180. Results are presented as percent immunoprecipitated DNA over input after isotype IgG control background subtraction. Data are average of 3 independent experiments, and error bars represent the SD of 3 experiments for each primer set. The promoter of GAPDH was used as the control. Statistical significance was determined using unpaired t-test (ns, no significance, *P < 0.05)

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