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.