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. 2017 Oct 20;292(50):20720–20731. doi: 10.1074/jbc.M117.797845

Figure 4.

Figure 4.

JNK plays a crucial role in determining H3S10p levels, but this modification barely affects basal Cga gene expression and does not affect H3K9ac. A, αT3-1 cells were serum-starved overnight prior to treatment with 1–10 μm of the JNK inhibitor SP600125 (JNKi) for 6 h before isolation of the histone fraction and Western analysis using antibody to H3S10p, with H3 as loading control. Normalized values relative to the untreated control are shown below the blot, and presented graphically on the right. B, cells were prepared and treated similarly with GnRH and/or SP600125 added 30 min before GnRH, and H3S10p and H3 examined by Western blot analysis as above. NS, p > 0.05 for average H3S10p/H3 levels. C, following treatment with SP600125 (10 μm for 6 h) ChIP analysis was carried out, as described in the legend to Fig. 1, to determine the levels of H3S10p at the Cga promoter/5′ end. H3S10p levels are expressed as fold of the mean level in untreated cells; ***, p < 0.001. D, Cga mRNA levels were measured as described in the legend to Fig. 3B, in cells treated with GnRH for 7 h with or without SP600125 added 30 min beforehand. The mRNA levels are expressed as fold of the mean levels in untreated control cells. Statistical analysis was as described in the legend to Fig. 1. E, similarly qPCR was carried out on cells treated with SP600125 for 12–24 h, and Cga mRNA levels are expressed as fold of the mean levels in untreated cells. Statistical analysis was as described before. F, αT3–1 cells were transfected with control GFP plasmid or dominant-negative CDC42. Cells were lysed 48 h after transfection, and Cga mRNA levels measured and data presented as before. *, p < 0.05. G, ChIP analysis was also carried out for H3K9ac in cells treated with SP600125 for 6 h, and levels associated with the Cga gene are shown after normalization to H3, relative to the mean levels in untreated cells. NS, p > 0.05. H, Western blot analysis was also carried out on SP600125-treated cells, and the effects on global levels of both H3S10p and H3K9ac were evaluated using specific antisera with H3 as control. Average H3S10p/H3 or H3K9ac/H3 levels in JNKi-treated versus untreated cells are indicated: *, p < 0.05; NS, p > 0.05.