Fig. 4

PKC412 reactivation is not associated with chromatin modulation. a Western blotting detection of acetylated histone H3 levels in latently infected cells. ACH2 cells were mock treated or treated with PKC412 (0.5 μM), butyrate (5 mM) or VOR (330 nM) and the cells were collected at different time points. Western blotting analysis was performed with anti-acetyl-histone H3 and anti-histone H3 antibodies. b The diagram shows the positions of the nucleosomes bound to the HIV-1 LTR and the location of the primers used for the real-time PCR in the ChIP assay (upper panel). Cells treated with or without PKC412 were assayed by ChIP with an anti-acetyl-histone H3 antibody. Immunoprecipitation with IgG served as the negative control. Cells treated with butyrate or VOR were used as the positive control. Real-time quantitation of the fold change relative to the negative control is shown (lower panel). c, d ACH2 cells were treated with PKC412 (0.5 μM) or the methyltransferase inhibitor 5-Azac (5 μM) for 24 h and the methylation levels of the H19 imprinted control region (ICR) and the HIV-1 LTR U3 region were detected by real-time PCR using specific primers following MeDIP ChIP with anti-5-methlcytosine (c). HIV-1 productions were measured with an HIV-1 p24 ELISA kit (d). Error bars represent variations between duplicate samples and the data are representative of results obtained in two independent experiments. The results were significant (* p < 0.05 and ** p < 0.01) compared to the mock treated cells