Suppression of astrocytic autophagy by αB-crystallin contributes to α-synuclein inclusion formation

CRYAB regulates autophagy process in vitro. a Knockdown of CRYAB enhances the conversion of LC3-I to LC3-II in U251 cells. U251 cells were transfected with CRYAB siRNA (siNC: the siRNA of negative control; siCRYAB: the siRNA of CRYAB) and the cellular LC3 levels were assessed by Western blotting 48？h after transfection. Data are presented as mean？±？SEM, n？=？6. Unpaired t-test. **P？<？0.01. b Overexpression of CRYAB inhibits LC3-II formation in U251 cells. The cellular LC3 levels were assessed by western blotting 48？h after transfection. Data are presented as the mean？±？SEM, n？=？3. Unpaired t-test. **P？<？0.01. c CRYAB overexpression in primary astrocytes inhibits LC3-II formation. Primary cultured astrocytes were transfected with lentivirus vector encoding CRYAB. The cellular LC3 levels were assessed by Western blotting 48？h after transfection. Data are presented as mean？±？SEM, n？=？3. Paired t-test. *P？<？0.05. d U251 cells were transfected with siCRYAB in the presence or absence of H2O2 (1？mM, 6？h). The cellular p62 levels were assessed 48？h after transfection using western blotting. Data are presented as mean？±？SEM, n？=？3. *P？<？0.05. e Knockdown of CRYAB leads to a marked increase in the number of autophagic vacuoles. Autophagic vacuoles in U251 cells transfected with siNC or siCRYAB were examined using electron microscopy. The arrows indicate autophagic vacuoles. Scale bars: 0.5？μm. The graph shows a statistical analysis of cytoplasmic occupancy of autophagic vacuoles in the cells. Data are presented as mean？±？SEM. Twenty cells in each group were examined. Unpaired t-test. ***P？<？0.00