Fig. 2.

Treatment with BAFi's specifically modulates BAF target genes, and displaces the BAF complex from the latent HIV-1 LTR.

(Panel a) Knockdown of BAF complex was obtained by nucleofecting J-Lat 11.1 cells with siRNA targeting the BAF complex specific subunit BAF250a. RNA levels of BAF250a were quantitated by RT PCR analysis, whereas protein levels of BAF250a as well as of the other subunits of the complex were determined by Western blot. RT-PCR analysis of J-Lat 11.1 cells after BAF250a knockdown (Panel b) or treatment with BAFi's A01 (1 μM), A11 (1 μM) and C09 (1 μM) for 18 h (Panel c). The effect of BAFi's treatment was monitored analyzing the changes in gene expression of HIV-1 LTR driven genes (GFP, GAG) and BAF target genes (TLR2, MMP9, IFNß, IL6, IL10, SOCS3). A set of BAF independent genes (CYCA2, MIP26, ß-ACTIN, ß-2-MICROGLOBULIN) was used as control. RT-qPCR data are presented as mean ± SD. (Panel d) BAFi treatment results in displacement of the BAF complex-specific subunit BAF250a from DHS1 and nuc-1 regions of the HIV-1 LTR. J-Lat 11.1 cells untreated or treated with BAFi's at indicated concentrations were subjected to ChIP using antibodies specific for the BAF250a subunit. Input and immunoprecipitated DNA were analyzed by PCR using primer pairs specific for the HIV-1 LTR nuc-0, DHS1, and nuc-1 regions and for a control region located upstream of the Axin gene promoter. ChIP results are presented as percent of immunoprecipitated material over input, error bars represent the standard deviation of three independent experiments. * indicates the level of significance at p < 0.05.