Fig. 3

CC-99677 inhibited the cytokine production in monocytes and macrophages and reduced the TTP expression through inhibition of MK2. Human monocytes (n = 3) were isolated from the whole blood and were differentiated into macrophages using M-CSF followed by IFNγ. Both monocytes and macrophages were treated with the depicted micromolar concentrations of CC-99677 for 1 h, and the cytokines shown were analyzed after stimulation with 100 ng/mL LPS for 4 or 24 h (a). Lysates from macrophages pretreated with CC-99677 followed by LPS stimulation for 4 h or 24 h were analyzed by capillary electrophoresis and probed with total TTP antibody and GAPDH antibody (b). TNF, IL-6, and IL-1β transcripts were measured at depicted times in macrophages stimulated with LPS and then treated with actinomycin D in the presence of CC-99677 or DMSO (c). Act D, actinomycin D; AUC, area under the curve; DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL, interleukin; IFNγ, interferon gamma; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; MK2, mitogen-activated protein kinase-activated protein kinase-2; TNF, tumor necrosis factor; TTP, tristetraprolin