Figure 5

MSU induces endogenous LC3 conversion. (A) Expression levels of LC3-I and LC3-II after MSU stimulation. Confluent OBs were cultured in α-MEM with 10% FBS and were stimulated with 0.1 to 1 mg MSU for 24 hours. Reactions were stopped, and cells were prepared to perform immunoblot analysis with anti-LC3-I/LC3-II and anti-β-actin antibodies. (B) Kinetic effects of MSU. OBs were cultured as in (A) and stimulated by 0.5 mg MSU for the indicated times. (C) Effects of 3-methyladenine (3-MA), wortmannin (W), and spautin-1 on MSU-induced conversion of LC3. Cells were pretreated with vehicle (CONT), 3-MA 6 μM, W 50 nM, or spautin-1 (10 μM) for 30 minutes. The cells were then stimulated with 0.5 mg MSU for 24 hours in the absence or in the continuous presence of 3-MA, W, or spautin-1. Immunoblots are representative of four different donors (3-MA, W), and of three different donors (spautin-1). (D) Autophagosome localization in GFP-LC3-transfected OBs. OBs were transfected with GFP-LC3 plasmid and then incubated with vehicle (Control) or 0.5 mg MSU for 4 hours before analysis with confocal microscopy (×200 magnification). (E) Effects of Dynasore on MSU-induced conversion of LC3. Confluent OBs were pretreated with vehicle (CONT) or 80 μM Dynasore for 10 minutes, and then incubated with vehicle or 0.5 mg MSU for 24 hours. Data are expressed as arbitrary densitometric units normalized by β-actin levels; LC3-II conversion induced by MSU was the ratio of LC3-II levels of MSU-stimulated cells over LC3-II levels of vehicle-stimulated cells. The effect of Dynasore is presented as mean value ± SEM of percentage inhibition of LC3-II conversion. Statistical analysis was performed by using the paired two-tailed t test (n = 3 different donors); *P < 0.05.