Figure 1. Loss of MARK4 affects IL-1β production under NLRP3 inflammasome activation.

(a) Mark4 expression under unprimed and LPS primed conditions in mouse bone marrow-derived macrophages (BMDM). Cytoplasm proximity ligation assay (PLA) signal per cell cytoplasm is used for quantification. Mean±s.e.m. for all the cells taken from 5 to 8 different views at × 40 magnification for each group. Experiments have been repeated three times. Scale bar, 10 μm. (b) siRNA of MARK4, NLRP3 or ASC caused reduction of ATP induced IL-1β production in THP-1 cells. (c) shRNA of MARK4 caused reduction of IL-1β induced by NLRP3 stimuli, including monosodium urate (MSU) and cholesterol crystals (CC) in THP-1 cells. Mock represents macrophages without further stimulation. (d) BMDM derived from Mark4 KO mice exhibited selectivity towards NLRP3 activating stimuli. Mean±s.e.m., three to four independent experiments combined (b–d), comparisons of the two different groups were analysed by unpaired t test. NS was considered as not statistically significant. *P<0.05, **P<0.01 and ***P<0.001 were considered as statistically significant (a–d). (e) Representative western blots of caspase-1 (Casp1) and IL-1β in the supernatant (Sup.) in Mark4^+/+ and Mark4^−/− macrophages activated with indicated stimuli. Cell lysates were used as controls to indicate equal amount of cells for analysis.