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. 2019 Jun 24;9:9177. doi: 10.1038/s41598-019-45641-x

Figure 1.

Figure 1

ECDD-S27 is a potent autophagic flux inhibitor. (a,b) Screening of natural product-derived compounds for their autophagy modulating activity. Raw264.7 macrophages were transfected with cDNAs encoding RFP-GFP-LC3B. At 48 h post transfection, cells were treated with DMSO (negative control), starvation (positive control), or natural product-derived compounds (50 µM) for 4 h. Cells were then fixed and analyzed by HC image analysis to quantify the number of total LC3B puncta per cell. The dashed line represents 3 S.D. above that of the mean of the DMSO treated control. ECDD-S27 was identified as the most effective compound to increase the number of total LC3B puncta per cell from the screen. Representative images of the HC image analysis with boundary of cells (right panels). Bar 5 µm. (c,d) ECDD-S27 inhibits autophagic flux. Raw264.7 macrophages expressing RFP-GFP-LC3B were treated with DMSO, starvation, or 50 µM of ECDD-S27 for 4 h. Cells were then processed for confocal microscopy analysis and the number of RFP+GFP+-LC3B (autophagosomes) and RFP+GFP−-LC3B (autolysosomes) puncta per cell was quantified. Only puncta ≥0.3 µm in size were counted. Data are the means ± SEM from at least three independent experiments. At least 30 cells per condition per independent experiment were quantified; *p < 0.05 and ***p < 0.001, all relative to the DMSO control, were determined by one-way ANOVA with a Tukey’s multiple comparison test. Bar 5 µm. (e–g) LC3-II and p62 immunoblots confirmed inhibition of autophagic flux by ECDD-S27. Raw264.7 macrophages were treated with DMSO with or without bafilomycin A1 or ECDD-S27 at the indicated concentrations for 4 h. Representative images cropped from the same blot are shown and full images are included in the supplementary information. The intensities of LC3-II, p62, and Actin were quantified using ImageJ. The graphs showed densitometric analysis of p62/Actin and LC3-II/Actin levels. The IC50 value of ECDD-S27 in inhibiting autophagic flux was ≤ 0.016 µM. Data are mean ± SEM; *p < 0.05, all relative to the DMSO control from three independent experiments, was determined by one-way ANOVA with a Tukey’s multiple comparison test.