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. 2013 Oct 30;8(10):e77749. doi: 10.1371/journal.pone.0077749

Figure 5. PKC/IKK inhibition strongly reduces prostratin/ionomycin-induced latent HIV transcription in primary CD4+ T cells.

Figure 5

A and B, evaluating inhibitors of relevant pathway in suppressing NL4-3 luciferase reporter activities in latently infected primary CD4+ T cells. A, latently infected cells pretreated with DMSO (white and black bars), calcineurin inhibitors CsA (dark gray bar) or FK-506 (light gray bar) were incubated in medium that contained no or suboptimal dosages of prostratin (0 to 100 nM, white bars), or contained both prostratin and 1.5 µM ionomycin (black, dark and light gray bars) for 48 h. Either calcineurin inhibitors inhibited the dose-dependent synergistic effect between suboptimal dosages of prostratin and ionomycin on luciferase reporter activities. B, latently infected cells pretreated with DMSO (white and black bars) or various kinase/phosphatase inhibitors (dark, medium and light gray bars) were incubated in media alone (white bar), or media containing anti-CD3/anti-CD28 Dynabeads (1∶1 ratio), or prostratin, in the presence or absence of 1.5 µM ionomycin as indicated for 30 h. All inhibitors suppressed reporter activities induced by TCR crosslinking or prostratin/ionomycin, which involved induction of calcium/calcineurin signaling, but CsA was ineffective against reporter activities induced by prostratin alone. In both A and B, the RLU were normalized based on total protein present in the various cell lysates. All stimulations were performed in triplicate with error bars representing ± standard deviation. Results are representative of experiments performed with cells from three independent donors. C, analysis of prostratin/ionomycin-induced IκBα degradation in primary CD4 cells. PBMC-purified CD4 cells were treated with DMSO or 500 nM CsA for 2 h, stimulated with a suboptimal dose of prostratin at 100 nM in the presence or absence of 1.5 µM ionomycin for 10 min and whole-cell lysate was prepared. Immunoblotting analyses revealed that CsA effectively reduced the effect of stimulus-coupled degradation of cytoplasmic IκBα.