Figure 5

Changes of p62 with cytokine cocktail or IL-1β stress in primary tri-cultures. (A) Representative p62 and β-actin immunoblots from cell lysates of primary tri-cultures exposed or not to 20 μM Aβ42 pretreated or not with 210 nM C16 in serum-free medium and treated with a cytokine cocktail of 200 pg/mL of IL-1β, TNF-α, and IL-6 or with IL-1β alone for 48 hours, treated or not with an autophagic flux inhibitor bafilomycin A1 (Baf) at 50 nM 24 hours before cell lysis. Densities were quantified using GeneTools software. Data of each protein were reported to data of the corresponding β-actin. The results are expressed as arbitrary units (control set at 100%) and are mean ± SEM from six independent experiments in duplicate. (B) Co-labeling of the autophagic receptor p62 (green), CD68 for microglia (red), GFAP for astrocytes (blue), and DAPI for nuclei (cyan) in tri-cultures. (C) Fluorescence intensity of p62 staining in cells quantified using ImageJ software in confocal images. (D) Co-labeling of the autophagic receptor p62 (green), Lyso-ID (red), and DAPI for nuclei (cyan) in tri-cultures. In (B) and (D), all images were from a compilation of the entire z-series sections acquired by confocal microscopy. A white square represents a magnified ROI. Two-dimensional fluorograms display the intensity and distribution of different colored pixels within the magnified ROI. Scale bars, 42 μm. ^*** P <0.001 compared to control; ^§§§ P <0.001 compared to Aβ42; ^†† P <0.01, ^††† P <0.001 compared to IL-1β; ^δ P <0.05, ^δδδ P <0.001 compared to IL-1β with Aβ42; ^## P <0.01, ^### P <0.001 compared to Baf; ^&& P <0.01, ^&&& P <0.001 compared to Baf with Aβ42; ^‡‡‡ P <0.001 compared to Baf with cocktail; ^ϵϵϵ P <0.001 compared to Baf with cocktail and Aβ42; ^£££ P <0.001 compared to IL-1β treatment in microglia by one-way ANOVA with a Newman-Keuls multiple comparison test. Baf, bafilomycin A1; DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; ROI, region of interest.