Journal: Scientific Reports
Article Title: HDAC1-3 inhibitor MS-275 enhances IL10 expression in RAW264.7 macrophages and reduces cigarette smoke-induced airway inflammation in mice
doi: 10.1038/srep45047
Figure Lengend Snippet: 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 ) ( 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 ) ( 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.
Article Snippet: Next, sections were incubated overnight at 4 °C with 10 μg/mL anti-human IL-10 rabbit IgG (#HP9016, Hycult Biotech, Uden, The Netherlands).
Techniques: Incubation, Immunofluorescence, Microscopy, Staining, Translocation Assay, Western Blot, Fractionation, Expressing