Fig 1.
Conessine regulates autophagy.
(A) Chemical structure of conessine. (B) Conessine treatment increased the level of LC3-II and p62. MCF-7 cells were incubated with the indicated concentrations of conessine (0, 2.5, 5, 10, and 20 μM) for 24 h, and the cell lysates were subjected to Western blotting with the indicated antibody. (C) Conessine treatment induced autophagosome formation in HEK293 cells. HEK293 cells stably expressing GFP-LC3 were incubated with the indicated concentration of conessine for 24 h, and the cells were analyzed with confocal microscopy.
Fig 2.
Autolysosomes were not formed by conessine treatment.
(A) Autophagosome formation by conessine. HEK293 cells were transfected with a plasmid encoding mRFP-GFP-LC3. After 24h, the transfected cells were incubated with the indicated concentrations of conessine (0, 2.5, 5, 10, and 20 μM) for 24 h. Cells were analyzed with confocal microscopy. (B) p62 accumulated in autophagosomes after conessine treatment. HEK293 cells were transfected with a plasmid encoding GFP-LC3, and the cells were treated with indicated concentrations of conessine, followed by immunostaining with anti-p62 antibody. Cells were analyzed with confocal microscopy.
Fig 3.
Conessine interferes with autophagic flux.
(A) Conessine treatment suppressed with autophagic flux. MCF-7 cells were treated with either mock or conessine (10 μM) in the presence or absence of Bafilomycin A1 (10 nM). (B) The levels of p62 were analyzed. Autophagic flux experiments were performed in triplicate, and the mean and standard deviations are shown in the graph.
Fig 4.
Conessine treatment induces autophagosome formation in C2C12 cells.
(A) C2C12 cells were transfected with a plasmid encoding GFP-LC3 and cells were treated with either mock or conessine (10 μM). (B) Conessine treatment increased the level of LC3-II and p62. C2C12 cells were incubated with the indicated concentration of conessine for 24 h, and the cell lysates were subjected to Western blotting with the indicated antibody. (C) The levels of LC3-II/LC3-I and p62 were analyzed. The experiments were performed in triplicate, and the means and standard deviations are shown in the graph. (D) Conessine less than 10 μM did not affect proliferation of C2C12 cells. C2C12 cells were treated with the indicated concentration of conessine, and cell viability were measured by MTT assay (E) Conessine treated cells were analyzed with flow cytometry. The percentage of cells at G1, S and G2/M was measured, and is shown in the graph.
Fig 5.
Conessine treatment protects C2C12 myoblast cells from H2O2-induced cell death.
(A) Conessine treatment interferes with H2O2-induced C2C12 cell death. C2C12 cells were treated with the indicated concentration of conessine, followed by H2O2 treatment (4 mM) for 24 h. Relative cell viability was measured by MTT assay. Control versus conessine treatment, * P < 0.01; ** P <0.05 (B) Cellular morphological changes were observed under a phase contrast microscope. (C, D) Apoptotic cells were quantified by counting DAPI-stained nuclei, and apoptotic cells showed nuclei and chromatin condensation. The number of normal cells were shown in graph. At least 160 cells were counted in each samples. Control versus conessine treatment, * P < 0.00005; ** P <0.0005; *** P<0.05. (E) Conessine decreased the sub-G1 cell population, which was induced by H2O2 treatment. C2C12 cells were treated with conessine and H2O2, and the cell cycle was analyzed with flow cytometry. (F) Conessine attenuates H2O2 induced autophagy. C2C12 cells were sequentially treated with conessine and H2O2, and the cell lysates were subject to Western blot with the indicated antibodies. (G) Conessine alleviates H2O2-induced excessive autophagy. C2C12 cells were transfected with GFP-LC3 and sequentially treated with conessine and H2O2. The cells were analyzed by confocal microscopy.