BioMed Research International / 2016 / Article / Fig 2

Research Article

HMBA Enhances Prostratin-Induced Activation of Latent HIV-1 via Suppressing the Expression of Negative Feedback Regulator A20/TNFAIP3 in NF-κB Signaling

Figure 2

Prostratin enhances HMBA-stimulated HIV-1 transcriptional elongation by augmenting HMBA-induced P-TEFb activation. (a) Prostratin augments HMBA-induced P-TEFb activation. F1C2 (Cdk9-f) cells, a HeLa-based cell line stably expressing Cdk9-Flag, were treated as indicated, followed by low-salt extraction to yield low-salt faction (LSF). The anti-Flag immunoprecipitates of LSF were analyzed by Western Blot (WB) for the P-TEFb-bound HEXIM1 in LSF, which represents the level of inactive 7SK snRNP complex. (b) Prostratin enhances HMBA-induced P-TEFb recruitment on HIV-1 promoter. HIV-LTR-Luc cells with indicated treatment were assayed by anti-Cdk9 chromatin immunoprecipitation (ChIP) for the enrichment of P-TEFb on the promoter of HIV-LTR-Luc. Data from three independent experiments were averaged and presented as fold enhancement compared to untreated cells. (c and d) Inhibiting P-TEFb activation or recruitment only blocks H+P-induced transcriptional elongation of HIV-1. HIV-LTR-Luc cells were pretreated with flavopiridol (FVP) or histone deacetylase inhibitor TSA, followed by H+P treatment as indicated. The isolated RNA were analyzed by qRT-PCR for the initiation and elongation transcripts of HIV-LTR-Luc as in Figure 1(e).

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