Losmapimod

PD0325901 (Chemscence, Monmouth Junction, NJ), a MEK inhibitor; Gö 6983 (Abcam, Cambridge, UK), a protein kinase c (PKC) inhibitor; MK-2206 (Selleck, Houston, TX), a protein kinase b (AKT) inhibitor; Losmapimod (Selleck), a p38α/β mitogen-activated protein kinase (p38 MAPK) inhibitor; SP600125 (Selleck), a c-jun N-terminal kinase (JNK) inhibitor; Saracatinib (Selleck), a proto-oncogene tyrosine-protein kinase Src (SRC) inhibitor and JAK Inhibitor I (Santa Cruz Biotechnology, Dallas, TX), a janus kinase (JAK) inhibitor, were dissolved in dimethyl sulfoxide (DMSO) (Sigma, ST. Louis, MO) and added to the culture medium. Pure DMSO was used as the negative control.

To study the effect of p38 MAPK activation and Adam 17 signaling pathway, BM progenitor cells and splenic DCs were isolated and enriched with the Easy Sep mouse hematopoietic progenitor cell isolation kit and pan DC kit, respectively (Stem Cell Technology). 200,000 cells were cultured in 96-well plates overnight in Iscove's modified Dulbecco's medium supplemented with 10% (vol/vol) FCS, 2- mercaptoethanol (50 μm), sodium pyruvate (1 mM), penicillin (100 U ml⁻¹ l) streptomycin (100 μg ml⁻¹), mouse GM-CSF (20 ng ml⁻¹) and human Flt3L-Ig (100 ng ml⁻¹). Cells were incubated in presence or absence of the selective inhibitor losmapimod (20 μM, GW856553X, Selleckchem) or Adam17 inhibitor (50 μM, Tocris). Cultures were stimulated with 5 μg ml⁻¹ of phorbol 12-myristate 13-acetate and ionomycin (PMA) and 5 μg ml⁻¹ ionomycin (Sigma-Aldrich, St. Louis, MO, USA) for 15 min before fixation and permeabilization with BD Phosflow Perm Buffer III (BD Biosciences) or for EdU staining. Cells were stained with mouse antibodies against CD11c-APC-Cy7 (N418), CD11b-BV650 (M1/70), PDCA1-FITC (1A8), MHCII-PE-Cy7 (M/114) from BioLegends and p38 MAPK-Alexa Fluor-647 (pT180/pY182) (BD Biosciences) and acquired by flow cytometry. p38 MAPK phosphorylation were assessed on gated population of LSK⁺CD115⁺ Flt3⁺ (CDPs) cells or CD11c⁺ CD11b⁺ DCs.