LPS stimulation induced autophagy in HMrSV5 cells. (A) Western blot analysis of Beclin-1 and LC3-II in HMrSV5 cells treated with LPS at various concentrations for 12 hours or 1 μg/ml LPS for the indicated time periods. β-actin was used as a loading control. (B) Densitometric anaysis of the blots showing the ratios of Beclin-1 and LC3-II to β-actin. (C) Transmission electron microscopy (TEM) of LPS-induced autophagy. Single-membrane phagosomes were seen in image 1. Image 2 shows typical double-membrane autophagosomes. Image 3 and 4 show multilayer structures. n, nucleus; av, autophagic vacuole; white arrows, single-membrane compartments; black arrows, double-membrane or multilayer structures. Scale bars: image1: 0.5 μm; image 2, 3 and 4: 200 nm. (D) Autophagic vacuoles were labeled with monodansylcadaverine (MDC, blue). Scale bars: 20 μm. (E) Graphs display quantitation of the number of autophagosomes per cross-sectioned cell (left panel) and the number of MDC-labeled autophagosomes per cell (right panel). Data are mean values ± SD (n ≥3). *p < 0.05 (vs. control); **p < 0.01 (vs. control).