Figure 6

Changes of LC3 with cytokine cocktail or IL-1β stress in primary tri-cultures. (A) Representative LC3 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 for 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 LC3 (green), CD68 for microglia (red), GFAP for astrocytes (blue), and DAPI for nuclei (cyan) in tri-cultures. (C) Fluorescence intensity of LC3 staining in neurons, astrocytes, and microglia quantified using ImageJ software in confocal images. (D) Co-labeling of LC3 (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 (Olympus IX-81). A white square represents a magnified ROI. Two-dimensional fluorograms display the intensity and distribution of different colored pixels within the magnified ROI in the merged image. Scale bars, 42 μm.^*** P <0.001 compared to control; ^δδδ P <0.001, compared to IL-1β with Aβ42; ^### P <0.001 compared to Baf; ^&&& 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; ^σσσ P <0.001 compared to Baf with IL-1β; ^γγγ P <0.001 compared to Baf with IL-1β 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.