Interrelationship between autophagy and apoptosis in AF cells. Autophagy incidence was obtained by monodansylcadaverine (MDC) staining and apoptosis incidence was assessed by double staining with Annexin V-FITC and propidium iodide. (a) The autophagy incidence and the apoptosis incidence were observed after the cells were treated with the autophagy inhibitor, 3-methyladenine (3-MA). The data are presented as the mean ± standard deviation (SD). *P < 0.01, ** P < 0.001. (b) Representative graphs obtained by flow cytometry analysis after double staining with Annexin V-FITC and propidium iodide. Undamaged cells were stained with negative Annexin V-FITC/PI (bottom left quadrant). After application of 3-MA, the incidence of apoptotic cells increased. (c) Morphologic changes in apoptotic rat annulus fibrosus (AF) cells by Hochest staining. Phase-contrast photomicrograph of rat annular cells cultured in serum-free medium stimulated with IL-1β and 3-MA for 24 hours. Apoptotic cells were characterized by brightly stained with Hoechst 33258 (arrowheads). The results indicate the interrelationship between autophagy and apoptosis. When autophagy is inhibited, the apoptosis incidence is significantly increased.