Figure 4.

Effects of γ-PGA on inflammatory cytokine and NLRP3 expression levels. BMDMs (2 × 10⁶ cells per well for qPCR and 1 × 10⁶ cells per well for immunoblotting) were treated with the indicated γ-PGA concentrations with/without LPS (10 ng/ml). Pro-IL-1β, TNFα, IL-6, IL-1α, NLRP3, IL-10 and IL-1rn transcript levels were analyzed using SYBR green-based quantitative real-time PCR (a) and general PCR analyzed by 2% agarose gel electrophoresis (b). The data represent the mean±s.d. of three independent experiments. (c) Effects of γ-PGA on pro-IL-1β and TNFα translational levels in dose- or time-dependent manners were detected by immunoblotting. The effect of a TLR4 signaling inhibitor (TAK-242) on γ-PGA-mediated cytokine induction was elucidated with BMDMs treated with γ-PGA (0.4 mg/ml) or LPS (10 ng/ml). Pro-IL-1β and TNFα were analyzed using SYBR green-based quantitative real-time PCR (d) and general PCR analyzed by 2% agarose gel electrophoresis (e). All data shown are representative of at least two independent experiments. BMDMs, bone marrow-derived macrophages; IL, interleukin; γ-PGA, poly-gamma-glutamic acid; LPS, lipopolysaccharide; TNFα, tumor necrosis factor-α TLR4, Toll-like receptor 4.