Figure 7
Activation of the MEK1/2-ERK1/2 signaling pathway suppresses HIV-1 gene expression. HeLa rtTA-HIV(Gag-GFP) cells were pre-treated overnight (~15 h) with/without an inhibitor of MEK1/2 (12 μM U0126, MEKi, white), p38α/β/β2 (15 μM SB203580, p38i, light gray), JNK1/2/3 (1.25 μM SP600125, JNKi, hatched), or NCX (5 μM KB-R7943, NCXi, gray) and treated with a MEK1/2-ERK1/2 activator, anisomycin, to isolate the pathway signal as described (and run in parallel for verification of inhibitor activity) in Figures 6 and 5d–f. Cells were monitored for HIV-1 gene expression by detecting Gag-GFP fluorescence in cell lysates (35 μg) resolved on reducing SDS-PAGE, levels of ERK activation by immunoblotting of phospho- and total-ERK from cell lysates, and extent of viral RNA expression by qRT-PCR of mRNAs extracted. Results were displayed relative to DMSO (+) that were pre-treated with no pathway inhibitor. Statistical comparisons were performed as illustrated (black/gray dashed lines) and described in Methods. (a) Graph of HIV-1 Gag-GFP expression in treated cells (n ≥ 5, mean, s.e.m.) and (b,c and Supplementary Fig. S12c) representative gels of these results. (d) Graph of ERK activation levels in the presence/absence of MEKi and anisomycin (n ≥ 5, mean, s.e.m.). Representative immunoblot of (d) is shown in Supplementary Figure S12a. Gels in (b,c) were run simultaneously and assembled from the same experiment. Stain-free™ total protein staining serves as internal loading control and for normalization of data in (a–d). (e) Graph of the accumulation of US, SS, and MS HIV-1 RNAs in anisomycin treated cells (n ≥ 3, mean, s.e.m.). RNAs were quantified by qRT-PCR as described in Figures 4a,b. Concentrations of MEKi and anisomycin applied in these experiments were predetermined to have little/no impact on total cell density (Supplementary Fig. S12b).
