Combinations of pro-inflammatory cytokines elevate endogenous APP levels in mouse primary astrocyte cultures. (A-C) Cultured wild-type C57BL/6J mouse primary astrocytes were stimulated with the indicated pro-inflammatory agents (alone and combinations) for 24 (A), 48 (B), or 96 h (C). Cell lysates were then prepared and analyzed for APP, GFAP, and β-actin by immunoblot. Upper panels show APP immunoblot images and lower histograms represent quantifications of APP immunoblot signals expressed as percent of vehicle control. The mature APP band at 130 kDa and the immature APP band at 110 kDa are indicated by the arrowheads. GFAP and β-actin immunosignals served as loading controls. Note that cytokine combinations including TNF-α and IFN-γ were generally more potent at increasing endogenous APP levels in astrocytes over time in culture, raising APP levels to ~300% of control. (D) C57BL/6J mouse primary astrocyte cultures were stimulated with TNF-α, IFN-γ, TNF-α+IFN-γ, or vehicle control for the indicated times and analyzed for endogenous APP mRNA levels by TaqMan quantitative RT-PCR. Histograms represent quantifications of APP mRNA levels expressed as percent of vehicle control. Note that only TNF-α+IFN-γ-stimulated astrocytes at 96 h exhibited a statistically significant increase in APP mRNA. Statistical analysis for A-D was performed by two-tailed t-test based on a normal distribution of the data. Significance indicates comparison to individual vehicle control within each measurement and each time point (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). Error bars, standard error of the mean (SEM).