Subsequently, PCLS were stimulated with LPS and IFN in continued presence of the inhibitors, with 10 ng/mL LPS ((Mm00443258_m1), (Mm00434228_m1), (Mm00446190_m1), (Mm04208136_m1), (Mm00440502_m1) (Mm00434174_m1) and (Mm99999915_g1) were purchased as Assay-on-Demand (Applied Biosystems). 2.4. acetyltransferases by MG149 correlates with inhibition of histone acetylation in murine precision-cut lung slices, inhibition of acetylation was observed using an LC-MS/MS based assay on histone H4 res 4-17, which contains the target lysine Busulfan (Myleran, Busulfex) of KAT8. Following up on this, upon treatment with MG149, reduced pro-inflammatory gene expression was observed in lipopolysaccharide and interferon gamma stimulated murine precision-cut lung slices. Based Busulfan (Myleran, Busulfex) on this, we propose that 6-alkylsalicylates such as MG149 have potential for development towards applications in the treatment of inflammatory lung diseases. model for lung inflammation. An advantage associated with the use of these type of organ slices is that the amount of required experimental animals can be reduced (18). Since promoting roles for lipopolysaccharide (LPS) and interferon gamma (IFN) have been described in asthma and COPD, as reviewed by Boorsma et al. (19), a combined stimulus of LPS and IFN was selected as an inflammatory stimulus in PCLS. Open in a separate window Fig. 1 Chemical structure of MG149 Here, we report the kinetics of inhibition of the MYST HAT family member KAT8 by MG149, and a calculation of the inhibitory constant Ki of MG149 for KAT8. The inhibition of HATs by MG149 could be correlated to inhibition of histone acetylation in murine PCLS upon MG149 treatment, as determined by a mass spectrometry based analysis. This inhibition was observed on histone H4 res 4-17, made up of H4 K16 which is the target of KAT8. Finally, we report reduced pro-inflammatory gene expression upon treatment with MG149 in murine PCLS. Taken together, this indicates that 6-alkylsalicylates such as MG149 have potential for development towards applications in the treatment of inflammatory lung diseases. 2.?Materials and methods 2.1. General Reagents and Materials All chemicals and reagents were purchased from Sigma Aldrich (St. Louis, Missouri, USA) unless otherwise stated. MG149 was purchased from Axon Medchem (Groningen, The Netherlands). The purity of Busulfan (Myleran, Busulfex) MG149 was assessed by HPLC, MS, and NMR by Axon Medchem and was 99%. Suberoylanilide hydroxamic acid (SAHA) was purchased from Selleckchem (Huissen, The Netherlands). The purity of SAHA was assessed by HPLC, MS, and NMR by Selleckchem and was 99%. 2.2. Precision-cut lung slices Precision-cut lung slices (PCLS) were prepared and cultured as described previously (20). All experiments were performed according to national guidelines and Busulfan (Myleran, Busulfex) upon approval of the experimental procedures by the local Animal Care and Use committee of Groningen University, DEC number 6962A. Viability of MG149 treated PCLS was assessed by the amount of lactate dehydrogenase (LDH) released by the tissue slices into the culture medium. The measurements were performed as described previously (20). LDH release from the PCLS into the incubation medium was plotted relative to maximal LDH release, as determined by lysing 3 slices with 1% Triton X-100 for 30 min at 37C at the start of the experiments. 2.3. Gene expression analysis in PCLS by RT-q-PCR For gene expression analysis, PCLS were pre-treated with MG149 at 5 or 10 M for 16 hrs. Inhibitor stocks were prepared in DMF and were further diluted in culture medium. Vehicle treatment constituted of pre-treatment with 0.2% DMF for PCLS, for 16 hrs. Subsequently, PCLS were stimulated with LPS and IFN in continued presence of the inhibitors, with 10 ng/mL LPS ((Mm00443258_m1), (Mm00434228_m1), (Mm00446190_m1), (Mm04208136_m1), (Mm00440502_m1) (Mm00434174_m1) and (Mm99999915_g1) were purchased as Assay-on-Demand (Applied Biosystems). 2.4. Lysine acetyltransferase 8 (KAT8) inhibition assays Activity of the HAT lysine acetyltransferase 8 (KAT8) was measured using chemical detection of coenzyme A (CoASH) after fluorescent labelling, as described previously (21). 2.5. Histone extraction and Micro BCA? Protein Assay For the histone extractions, PCLS were treated with the HAT inhibitor MG149 at 5 or 10 M, in single treatments or in combination with 2 M SAHA. PCLS were incubated with the inhibitors for 16 hrs. Subsequently, PCLS were stimulated with LPS and IFN in continued presence of the inhibitors as described above for the Rabbit Polyclonal to ABCF1 gene expression analysis. Inhibitor stocks were prepared in DMF and were further diluted in DMEM culture medium. Vehicle treatment constituted 0.2% DMF. PCLS were lysed in ice-cold Dulbeccos Phosphate-buffered Saline (DPBS; Gibco by Life Technologies) supplemented with protease inhibitors (#88266, Thermo Scientific, Rockford, IL, USA) and sodium butyrate (1.
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