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Fig. 2 | Translational Neurodegeneration

Fig. 2

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

Fig. 2

CRYAB inhibits autophagy in a mTOR-independent manner. a-b Knockdown of CRYAB in U251 cells enhances the formation of LC3+ dots. U251 cells were transfected with siCRYAB. Some of these cells were also treated with 1 μM rapamycin for 6 h. The cells were immunostained with CRYAB and LC3 antibodies 48 h after transfection. Scale bars: 5 μm (a) and 10 μm (b). c-d Quantitative data in a and b, respectively. Data are presented as the mean ± SEM, c, n = 50 cells, d, n = 40 cells. Unpaired t-test. *P < 0.05. e Treatment with rapamycin does not alter CRYAB knockdown of CRYAB-induced augmentation in the conversion of LC3-I to LC3-II in U251 cells. U251 were transfected with siCRYAB and stimulated with 1 μM rapamycin for 6 h. At 48 h after transfection, the cellular LC3 levels were assessed by western blot. The graph shows a statistical analysis of the conversion of LC3-I to LC3-II in the cells. Data are presented as mean ± SEM, n = 5. f Immunoblot analysis of the cell lysates from U251 cells transfected with siCRYAB. The graph shows a statistical analysis of the expression levels of phosphorylated mTOR, p70S6K and total mTOR. Data are presented as mean ± SEM, n = 3. *P < 0.05. g Immunoblot analysis of the cell lysates from U251 cells overexpressing CRYAB and treated with rapamycin (1 μM, 6 h). Data are presented as mean ± SEM, n = 3. *P < 0.05. h Knockdown of CRYAB-induced enhancement in autophagy does not affect autophagic degradation and autophagosome-lysosome fusion. Western blotting analysis of the conversion of LC3-I to LC3-II in U251 cells transfected with siCRYAB and stimulated with 10 μM CQ for 6 h. Data are presented as mean ± SEM. n = 3. *P < 0.05

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