0 μg/ml LPS and a time point of 12 hours were chosen for further experiments. Figure 2 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. Vactosertib price 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; PLX-4720 nmr 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). Autophagosome formation could be confirmed further by fluorescence microscopic analysis of GFP-LC3 cells. HMrSV5 cells were transiently transfected with plasmids encoding GFP-LC3 and then incubated

with 1.0 μg/ml LPS for 12 hours. It was observed that the transiently transfected cells exhibited characteristic fluorescent punctate GFP-LC3 (LC3-II) while green fluorescence of control cells remained cytosolic and diffuse (Figure 3). Figure 3 Induction or inhibition of autophagy by pharmacological agents. Cells transiently transfected with the GFP-LC3 plasmid were treated with combination of drugs: control, LPS (1.0 μg/ml), LPS + 3-methyladenine (3-MA, 10 mM), LPS + wortmannin (Wm, 50 nM), or LPS + Polymyxin B (PMB, 100 μg/ml). (A) Autophagosomes were defined as GFP-LC3

puncta. DAPI was used to label nuclei (blue). Scale bars: 20 μm. Arrows indicate punctate Liothyronine Sodium GFP-LC3 (green). (B) Graph displays the percentage of cells with GFP-LC3-positive autophagosomes. **p < 0.01 (vs. control), ##p < 0.01 (vs. LPS). Monodansylcadaverine (MDC), a specific marker for autolysosomes [24], was also applied to confirm the induction of autophagy in treated HMrSV5 cells. As shown in Figure 2D, only basal levels of autophagy were observed in control cells, while increased number of vesicles as well as their size, which was indicated by the characteristic MDC staining, could be seen in the cells treated with LPS (Figure 2D and E, right panel). Transmission electron microscopy (TEM) demonstrated that after exposure of LPS for 12 hours, the number of canonical double-membrane autophagosomes in HMrSV5 cells was significantly higher than that of control cells (Figure 2C and E, left panel).