Autophagy incidence under different IL-1β concentrations and serum supplement. (a) Representative graphs obtained by flow cytometry analysis after monodansylcadaverine (MDC) staining. The data showed that the levels of autophagy were relative low when the cells were cultured with 10% fetal bovine serum (FBS). However, with serum withdrawal media, the incidence of autophagy obviously increased. (b) The autophagy incidences of rat annulus fibrosus (AF) cells cultured with or without serum supplement and stimulated with different concentrations of IL-1β. All data are presented as the mean ± standard deviation (SD). *P < 0.01. (c) Activity of cytoplasm lysosome, assessed using a Lyso-Tracker kit. The results indicated that IL-1β did not change the activity of lysosome when rat AF cells were incubated with 10% FBS. Conversely, serum deprivation obviously increased the activity of lysosome. (d) mRNA expression of autophagy-related genes in rat AF cells by real-time PCR analysis when treated with or without serum supplement and IL-1β (20 ng/ml). The mRNA levels of AF cells which were cultured in 10% FBS without IL-1β were used as controls. All data are presented as the mean ± SD. *P < 0.01. Serum deprivation obviously increased the mRNA expression levels of Beclin-1, Bcl-2 and microtubule associated protein 1 light chain 3 (LC3) after 12 hours.