Figure 4: MS-275 enhances the nuclear translocation of NF-κB p65 and enrichment of NF-κB p65 at the transcription start site of IL10 but reduces total NF-κB p65 protein content in LPS/IFNγ-stimulated RAW264.7 macrophages. | Scientific Reports

Figure 4: MS-275 enhances the nuclear translocation of NF-κB p65 and enrichment of NF-κB p65 at the transcription start site of IL10 but reduces total NF-κB p65 protein content in LPS/IFNγ-stimulated RAW264.7 macrophages.

From: HDAC1-3 inhibitor MS-275 enhances IL10 expression in RAW264.7 macrophages and reduces cigarette smoke-induced airway inflammation in mice

Figure 4

After 20 h incubation with MS-275 (1 μM) followed by 1 h LPS/IFNγ stimulation, RAW264.7 macrophages were prepared for immunofluorescence microscopy (A). Green signal represents NF-κB p65 protein, while blue signal visualizes the Hoechst-stained nuclei. MS-275 enhanced the translocation of NF-κB p65 compared to vehicle (LPS/IFNγ and inhibitor solvent-treated) cells, while SAHA (0.41 μM) did not affect the nuclear translocation of NF-κB p65. The presented data are representative images of 3 independent experiments, original magnification 400x. All images were taken under identical instrumental conditions. The effect of MS-275 on NF-κB p65 translocation in LPS/IFNγ-stimulated RAW264.7 macrophages was also analyzed by immunoblotting cell fractions (the blots were cropped; full length blots are shown in Figure S6) (B) and quantified by densitometry (C). As controls for the fractionation PARP-1 (nucleus) and β-actin (cytosol) were used. Data are presented as mean ± SD expressed as fold change compared to control (LPS/IFNγ-treated) cells of 4–5 independent experiments. *p < 0.05 compared to vehicle (LPS/IFNγ and inhibitor solvent-treated) cells. Effects of MS-275 on total NF-κB p65 protein expression in LPS/IFNγ-stimulated RAW264.7 macrophages were analyzed by Western blot (dotted line indicates crop within the same blot; full length blots are shown in Figure S7) (D) and quantified by densitometry (E). Protein levels were normalized against β-actin and control (LPS/IFNγ-treated) cells were set at 100%. Data are presented as mean ± SD of 4 independent experiments and a representative blot is shown in (D). **p < 0.01 compared to vehicle (LPS/IFNγ and inhibitor solvent-treated) cells. qChIP was performed on LPS/IFNγ-stimulated RAW264.7 macrophages treated with MS-275 or vehicle (inhibitor solvent) for 20 h with antibodies against NF-κB p65 (F). Enrichment for IL10 regions −48 bp and −220 bp relative to the transcription start site was analyzed by qPCR. Data are presented as mean ± SEM of 2–3 independent experiments and represent fold enrichment compared to normal rabbit IgG (rIgG). *p < 0.05; **p < 0.01 compared to rIgG.

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