Figure 9

Changes in autophagic factors in microglia. Representative immunoblots show the immunoreactivity of (A) p62, (B) LC3-I, LC3-II, and β-actin from cell lysates of primary microglia pretreated or not with 210 nM C16 and exposed or not to 20 μM Aβ42 in serum-free medium containing or not 200 pg/mL of IL-1β for 48 hours. The autophagic flux inhibitor bafilomycin A1 (Baf) at 50 nM was added or not 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 (percentage of the control set at 100%) and are mean ± SEM from six independent experiments in duplicate. ^*** P <0.001 compared to control; ^§§ P <0.01, ^§§§ P <0.001 compared to Aβ42; ^$ P <0.05, ^$$ P <0.01 compared to Baf; ^†† P <0.01, ^††† P <0.001 compared to Aβ42 with IL-1β and Baf by one-way ANOVA with a Newman-Keuls multiple comparison test. Co-labeling of the autophagic receptor (C) p62 (green) or (D) LC3 (green), Lyso-ID (red), and DAPI for nuclei (cyan) in microglia. All images were from a compilation of the entire z-series sections acquired by confocal microscopy (Olympus IX-81). For (A) and (B), only merged images were selected. 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. Baf, bafilomycin A1; DAPI, 4′,6-diamidino-2-phenylindole; ROI, region of interest.