Figure 2.

Compound E4 promoted autophagy flux and lysosomal biogenesis. (A) E4 dose-dependently increased the level of LC3B-II compared with vehicle control (0.1% DMSO). The expression of LC3-II in N2a cell after being treated with different concentration of E4 for 24 h were detected by Western blotting. Relative intensity of LC3B-II is normalized to that of β-actin/ACTB. Data are presented as mean ± SEM of three replicates in a representative experiment (B). The expression of LC3-II in N2a cell after being treated with indicated concentration of E4 and chloroquine (CQ) for 24 h were detected by Western blotting. Relative intensity of LC3B-II is normalized to that of β-actin/ACTB. Data are presented as mean ± SEM of 3 replicates in a representative experiment. (C) N2a cells were treated with vehicle control (0.1% DMSO), E4 (1 μM), or CQ (20 μM) for 12 h and then stained with LysoTracker Red DND-99 (50 nM) for 30 minutes. Fluorescence intensity of treated cells as measured by fluorescence microscopy. The numeric data are presented as means ± SEM from 3 independent experiments. (D) After the treated with vehicle control (0.1% DMSO), E4 (1 μM) or CQ (20 μM) in N2a cells that transfected with tf-LC3 plasmids for 16 h, the fluorescence signal was captured by fluorescence microscopy and representative images are shown. Scale bar: 15 μm. (E) E4 treatment increase LAMP1, CTSD levels in a dose-dependent manner as compared to vehicle control (0.1% DMSO). After N2a cells treated with E4 and positive control Torin1 (250 nM) at indicated concentration for 24 h. The expressions of LAMP1, pro-CTSD, mature-CTSD were detected by Western blot assay and quantified. (F) CF-7 cells were treated with E4 (1 μM) for 16 h. mRNA transcript abundance was assessed by real-time PCR using specific primers for the indicated genes. Relative quantification is presented as means ± SEM of 3 independent experiments. * p < 0.05, ** p <0.01, *** p < 0.001, ^## p < 0.01.