Figure 4

The decreased extracellular S1009A in response to Aβ1-42 monomers is dependent on increase of intracellular Ca ²⁺ level in human THP-1 monocytes. THP-1 cells were incubated with Aβ1-42 (10 μM), actinomycin (ActD, 100 nM) or cycloheximide (CHX, 1 μM) for 24 hours. Western blot analyses were conducted to determine the effects of actinomycin or cycloheximide on S100A9 release in the conditioned media, as described in Figure 1. Ionomycin and thapsigargin mimicked the Aβ1-42-evoked response. THP-1 cells were incubated with Aβ1-42 (10 μM), ionomycin (2 μM) or thapsigargin (2 μM) for 24 hours. (A) The conditioned media and (B) total cell lysate were examined for S100A9 by western blot analyses. (C) THP-1 cells were pretreated with EGTA (0.5 mM) or BAPTA (10 μM) for 1 hour followed by incubation with either the vehicle only (−) or Aβ1-42 (10 μM) for 24 hours. Western blot analyses were conducted to determine the role of intracellular Ca²⁺ levels in Aβ1-42-mediated reduction of S100A9 release. Ionomycin and thapsigargin, which elevate intracellular Ca²⁺ level, mimicked the Aβ1-42-evoked response, decreasing S100A9 secretion. In contrast, the Aβ1-42-mediated decrease of S100A9 secretion was attenuated by either depletion of extracellular Ca²⁺ with EGTA or chelation of intracellular Ca²⁺ by BAPTA. Representative gels from three experiments are shown. (D, E, F) Quantitative analysis of (A, B, C) showing the levels of S100A9 release. All data are presented as the means ± SEM (n = 3). **P <0.01, versus vehicle treated samples; ^## P<0.01, versus Aβ1-42 treated samples. ActD, actinomycin; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CHX, cycloheximide; EGTA, ethylene glycol tetraacetic acid.