Fig. 5.

Overexpression of APP and accumulation of β-amyloid in human primary skeletal myotube cultures exposed to IL-1β (10 ng/ml) and/or IFN-γ (300 U/ml) for 24–72 h. (A) A 24 h exposure to IL-1β, alone or in combination with IFN-γ, induced enhanced staining for APP (Alexa-594, red). Grey-scale analysis of the same experiment demonstrates a significant increase of the staining intensity upon IL-1β, and even more in combination with IFN-γ. (B) Immunoblot analysis of APP demonstrates an increased level of protein expression upon 24–72 h of exposure to IL-1β in combination with IFN-γ. Protein loading in each lane is confirmed by detection of β-actin. (C) Staining for β-amyloid reveals an enhanced intracellular accumulation upon 48 h of exposure to IL-1β, particularly in combination with IFN-γ (Alexa-488, green). Grey-scale analysis of the same experiment demonstrates a significant increase of the staining intensity upon IL-1β, and even more in combination with IFN-γ. (D) After 48 h of exposure to IL-1β and IFN-γ, intracellular aggregation of β-amyloid is evidenced by thioflavin-S, which is statistically significant as revealed by grey-scale analysis of the same experiment. Photos taken by a CCD-camera using a conventional fluorescent microscope with a 10× (A and C) or 20× (D) objective. All photomicrographs for the analyses in this Figure have been acquired with the same settings of camera and microscope; all data are representative of at least three experiments with similar results. Scale bars represent 100 µm in A, C and 60 µm in D.