Figure 7.

Identification of autophagy regulators. (a) Analysis of the P2 factor. P2 factor scores of all compounds are arranged in descending order and plotted on the “Score Distribution” graph. Green in the graph indicates the compounds with a high P2 score, but without reports about autophagy. The rank, name, dose, and score are shown. (b) Structures of the compounds tested in this study. Structures were obtained from MolView (http://molview.org/). (c) Polar charts of response scores of the compounds tested. Response scores are plotted as a bar chart in polar co-ordinates with heatmap. (d) Western blotting analysis of HeLa cells treated with the compounds tested. HeLa cells were treated with the compounds tested at the indicated concentration for 24 h. The whole-cell lysate was analysed by western blotting using anti-LC3 antibody. *LC3-I, **LC3-II. Full-length blots are presented in Supplementary Fig. S7d. (e) Autophagy flux evaluation of GFP-LC3-RFP-LC3ΔG-HeLa cells treated with the compounds tested. HeLa cells expressing GFP-LC3-RFP-LC3ΔG were treated with the compounds tested using the method described in D. GFP and RFP signals were quantified with a Tecan Infinite M200 plate reader and the GFP/RFP ratio was calculated. Each bar represents the mean ± SE, n = 6. Significance test was conducted with the Turkey–Kramer method and only significant differences between DMSO and the tested compounds are shown: ***P < 0.001. (f) Imaging analysis of GFP-LC3-RFP-LC3ΔG-HeLa cells treated with the compounds tested. HeLa cells expressing GFP-LC3-RFP-LC3ΔG were treated with the compounds tested using the method described in D, fixed with 4% paraformaldehyde, stained with TO-Pro-3 iodide, and the fluorescence signals were detected with a TCS SP5 confocal microscope. Green signals indicate GFP (LC3) and blue signals the TO-Pro-3 iodide (nucleus). Scale bars correspond to 50 μm. In (d–f), a representative result of at least two independent experiments is shown.