Fig. 3

AR deficiency enhances autophagosome formation and maturation in LPS-stimulated macrophages. a BMMs from WT or AR KO mice were treated with or without 500 ng/ml LPS for 16 h and then immunostained for beclin1. Right graph represents quantification analysis of the number of beclin1 + foci per cell. Scale bars = 50 µm. b Immunoblot analysis of beclin1 in BMMs from WT or AR KO mice treated with or without 500 ng/ml LPS for 16 h. c Induction of LC3 + vacuoles in BMMs from WT or AR KO mice treated with or without 500 ng/ml LPS for 16 h and then immunostained for LC3. Right graph represents the quantification analysis of the number of LC3 + puncta per cell. Scale bars = 20 µm. d Immunoblot analysis of LC3 in BMMs from WT or AR KO mice treated with or without 500 ng/ml LPS for 16 h. Densitometric LC3II/LC3I/β-actin ratios are shown below the blot. e Time-course analysis of autophagy in macrophages during the 32 h of LPS stimulation. BMMs from WT or AR KO mice were treated with 500 ng/ml LPS for the indicated durations. Densitometric LC3II/LC3I/β-actin ratios are shown below the blot. f Immunoblot analysis of LAMP1 in BMMs from WT or AR KO mice treated with or without LPS for 16 h. g Confocal microscopy of BMMs from WT or AR KO mice treated with or without 500 ng/ml LPS for 16 h and immunostained for LC3 and LAMP1. Scale bars = 20 µm. Right graphs represent co-localization of LAMP1 and LC3 signals evaluated using Manders’ overlap coefficient. h Ultrastructural analysis of autophagy via transmission electron microscopy in BMMs. WT or AR KO BMMs were treated with or without 500 ng/ml LPS for 16 h. The panel on the right is the enlargement of the outlined area on the left. Scale bars = 1 μm. Right graphs represent the quantification of the number of autophagosomes and autolysosomes per cross-sectioned cell. Data are expressed as the mean ± SD from three independent experiments, *P < 0.05, **P < 0.01