Figure 3: MS-275 enhances LPS/IFNγ-induced NF-κB p65 activity and increases NF-κB p65 acetylation and histone acetylation in RAW264.7 macrophages.

RAW-Blue macrophages were subjected to 1 μM MS-275 or 0.41 μM SAHA for 20 h and stimulated with LPS/IFNγ the last 4 h of the experiment. Treatment of RAW-Blue cells with HDACi followed by LPS/IFNγ significantly increased NF-κB activity (A). Data are presented as mean ± SD of 3–4 independent experiments and control (LPS/IFNγ-treated) cells were set at 100%. **p < 0.01, compared to vehicle (LPS/IFNγ and inhibitor solvent-treated) cells. For detection of NF-κB p65 K310 acetylation, RAW264.7 macrophages were incubated with HDACi for 20 h, and lysed. Effects on NF-κB p65 K310 acetylation were studied by Western blot (the blots were cropped; full length blots are presented in Figure S3) (B) and quantified by densitometry compared to p65 as loading control (C). Data are presented as mean ± SEM expressed as fold change compared to vehicle (LPS/IFNγ and inhibitor solvent-treated) of 5–6 independent experiments. *p < 0.05, compared to vehicle (LPS/IFNγ and inhibitor solvent-treated). RAW264.7 histones were resolved by SDS-PAGE and blotted for detection of histone acetylation (blot was cropped; full length blot is shown in Figure S4) (D). Histones H3 and H4 were excised from the gel and subjected to LC-MS/MS analysis (E). MS-275 or SAHA increased acetylation on one peptide of histone H3 (res. 18–26: KQLATKAAR) and one of histone H4 (res. 4–17: GKGGKGLGKGGAKR). Other peptides were not affected. Data are presented as mean ± SD of 3–5 independent experiments. ^#p < 0.001 compared to vehicle (inhibitor solvent-treated). No significant differences were observed between untreated and vehicle-treated cells (Figure S5).