Figure 4.

SREBF2 overexpression enhances autophagosome accumulation but inhibits autophagy clearance. (A and B) Embryonic cortical and hippocampal neurons isolated from WT and SREBF2 mice were treated with 10 nM rapamycin (RM) or 5 μM Aβ for 24 h. Shown are representative confocal images of neuronal-enriched cultures double immunostained for LC3B (red) and LAMP2 (green) (A) and for SQSTM1 (red) and LAMP2 (green) (B). Insets show a 3-fold magnification of the indicated region. (C) Neurons were pretreated with 5 μM wortmannin or 0.5 mM GSHee for 30 min prior autophagy induction with 5 μM Aβ for 24 h. Shown are representative confocal images of  double immunolabeling with antibodies against to LC3B (red) and LAMP2 (green). Nuclei were stained by DRAQ5 (blue fluorescence). Scale bars: 50 μm. Quantification of the average number of LC3B or SQSTM1 puncta per cell was assessed using ImageJ software and depicted in the corresponding graphs (15 to 20 cells analyzed per genotype and experimental condition from a pool of at least 4 images). The Pearson correlation coefficient was used as a measure of colocalization of Alexa fluor 488 signals (LC3B) with Alexa fluor 555 signals (LAMP2). **P< 0.01.