Figure 6
CSs control HIV-1 gene expression through intracellular signaling. HeLa rtTA-HIV(Gag-GFP) cells were pre-treated with/without pathway inhibitor overnight (~15 h) and treated with ~IC80s of CSs. All results were displayed relative to DMSO (+) with pre-treatment with no pathway inhibitor. (a–c) ERK1/2, MK-2, and JNK1/2/3 are activated upon treatment of cells with CSs (n ≥ 4–6, 3–4, and 3–6, resp., mean, s.e.m.). Graphs quantifying the activation level (phospho/total protein) of each MAP/MAPK by western blot. In (a), the results of pre-treating cells with a MEK1/2 inhibitor (12 μM U0126, MEKi) on ERK1/2 activation is also shown and a representative immunoblot is provided in Supplementary Figure S9c. (d) Representative immunoblots of MAP/MAPK activation levels from (a–c). (e–g) MEK1/2 activation may be involved in CS inhibition of HIV-1 gene expression. The signaling pathway(s) used by a CS to inhibit HIV-1 expression was determined by detecting Gag-GFP fluorescence in cell lysates (~35 μg) by reducing SDS-PAGE after pre-treatment of cells with a MEK1/2 (12 μM U0126, white), p38α/β/β2 (15 μM SB203580/p38i, gray), or JNK1/2/3 (1.25 μM SP600125/JNKi, hatched) inhibitor (n ≥ 5–7, 4–8, and 3–7, resp., mean, s.e.m.). Results were shown relative to DMSO (+). Tubulin immunoblots serve as internal loading control and for normalization of these data. (e) Graph and (f–g) representative gels of these results. Continuous lanes were cropped and assembled from 2 experiments for (f). Statistical comparisons were performed as illustrated (black/gray dashed lines) and described in Methods. Activity of each pathway inhibitor in cells was verified in (a) and Supplementary Figures S9c–g. Concentration of pathway inhibitor and CSs applied were predetermined to have little/no impact on total cell density (Supplementary Fig. S9h).
