Figure 5. Identification and quantification of autophagosomes in the joints of GlcN treated and food restricted mice.

GFP-LC3 transgenic mice were treated as described in Figure 4. Mice were perfused with PBS, and the knee joints were harvested and sagittal vibratome-cut sections (70 μm) were stained with Rhodamine Phalloidin, anti-LC3 far red and Hoechst 33342 to label F-actin, LC3 and nuclei, respectively, and then analyzed by confocal microscopy. A, Representative images of GFP-LC3 signal in response to GlcN treatment are shown. A merged fluorescence image is shown in the bottom panel; GFP-LC3 (green), LC3 far red (red), F-actin (red), nuclei (blue). Two sets of control mice were included as described in Figure 4. B, Quantitative analysis of vesicles (autophagosomes) in chondrocytes, including the total number of vesicles per cell and the total area of vesicles per cell (μm²). GlcN treatment: * = P < 0.01 vs. vehicle; Starvation: ** = P < 0.05 vs. non starvation. Vehicle indicates standard diet without GlcN. Values represent average ± SEM of four animals per group. Magnification: 63x.