Figure 1. Identification of alkaloid compounds with autophagic activities.

a) Detection of GFP-LC3 puncta from compound-mediated autophagy in HeLa cells. Cells were transiently transfected with the EGFP-LC3 plasmid for 24 h and then treated with DMSO (-ve Ctrl), 300 nM rapamycin (+ve Ctrl) or the indicated compounds at their respective IC₅₀ concentrations for an additional 24 h. Bar chart represents the quantitation of autophagic cells. The percentages of autophagic cells were calculated as the number of cells with GFP-LC3 puncta (≥10 puncta/cell) divided by the total number of GFP-positive cells in the same field. b) Chemical structures of the four selected alkaloids, liensinine, isoliensinine, dauricine and cepharanthine. c) Endogenous expression of LC3-II in HeLa cells. Cells treated for 24 h with liensinine (20 μM), isoliensinine (10 μM), dauricine (10 μM) or cepharanthine (10 μM) were visualised by fluorescence microscopy after staining with an LC3-II antibody followed by TRITC-conjugated anti-mouse secondary. d) Autophagic effect of alkaloids in various types of cancer and normal cells. MCF-7, PC3, Hep3B, A549, H1299 and LO2 cells were transiently transfected with the EGFP-LC3 plasmid for 24 h and then treated with DMSO (Ctrl), liensinine (20 μM), isoliensinine (10 μM), dauricine (10 μM) or cepharanthine (10 μM) for 24 h. Fluorescence images were captured at 60× magnification; scale bar, 15 μm.