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Fig. 4 | Epigenetics & Chromatin

Fig. 4

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

Fig. 4

dCA does not inhibit HIV expression in ACH-2 cells. a Schematic representation of the Tat-TAR feedback loop present in provirus in ACH-2 cells. b The viral production in ACH-2 cell treated with dCA. Cells were split and treated on average every 3 days in the presence of ART with or without 10 nM dCA. Data are representative of three independent experiments. c The viral production in ACH-2 cell treated stimulated by SAHA. After 60 days, 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 level in ACH-2 cell treated with 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 Nef 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 ACH-2 cell. ChIP assay was performed using cells samples from panels B-C. 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. f The chromatin structure of the HIV LTR in U1 cells (samples from panel B-C) investigated by MNase protective assays. 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. h H3 occupancy on HIV promoter DNA determined by ChIP to H3. Results are presented as percent immunoprecipitated DNA over input, after isotype IgG control background subtraction. The ORF of GAPDH was used as the control. Data are average of 3 independent experiments and error bars represent the SD of 3 experiments for each primer set. Statistical significance was determined using unpaired t-test (ns, no significance, *P < 0.05)

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