OBJECTIVE To investigate the nuclear proteomes in mitochondrial DNA (mtDNA)-depleted A549 cells (Rho0 cells) and their parental cells (Rho+ cells),and to learn more about the nuclear responses to mitochondrial dysfunction.METHODS The nuclear proteomes of Rho and Rho + cells were characterized by two dimensional electrophoresis (2-DE) and SELDI-TOF ProteinChip technologies,the differentially expressed protein- spots were identified by MALDI-TOF mass spectrum (MS),the nucleophosmin and P53 expression were detected by Western blotting assay,and the mitochondrial memhrane potential (MMP) was measured by the laser scanning confocal microscope.RESULTS 2-DE results showed 11 protein-spots were down-regulated and 21 protein-spots were up-regulated in Rho0 cell nuclei.SELDI-TOF MS analysis with NP20 ProteinChips revealed 4 protein-peaks decreased in Rho0 cell nuclei.One down-regulated protein-spot was identified as elF-6,and 4 up-regulated proteinspots were identified as nucleophosmin,SFRS1,SFRS3 and hnRNP G,respectively.The increased expression of nucleophosmin in Rho0 cells was verified by Western blotting.Based on the clues from proteomic analysis,P53 expression in Rho0 cells was higher than in Rho + cells,and MMP was consistent in Rho + and Rho0 cells.CONCLUSION mtDNA-depletion induces nuclear proteome alteration.Rho0 cells can be used as a model to study the crosstalk between mitochondrion and nucleus.

Objective: To investigate the effect of manganese chloride (MnCl₂) or 1-methyl-4-phenylpyridinium (MPP ⁺) on oxidative stress and autophagy in human neuroblastomaSK-N-SH cells and the mechanism of the neurotoxicity of manganese. Methods: SK-N-SH cells were treated with MnCl₂ or MPP⁺ at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, and 2.0 mmol/L for 24 hours, and MTT assay was used to measure cell viability. The cells weretreated with MnCl₂ or MPP⁺ at doses of 0.125, 0.25, and 0.5 mmol/L for 24 hours, and flow cytometry was used to measure the content of reactive oxygen species (ROS) in cells, a laser scanning confocal microscope was used to observe autophagosome in cells, and Western blot was used to measure the expression of autophagy-related proteins P62 and LC3-II/LC3-I. Results: Compared with the control group, the 0.0625-2.0 mmol/L MnCl₂ and 0.125-2.0 mmol/L MPP ⁺ treatment groups had significant reductions in the viability of SK-N-SH cells, and the 0.25-2.0 mmol/L MnCl₂ treatment groups had significantly lower viability than the groups treated with the same doses of MPP⁺ (all P<0.05) . Compared with the control group, the 0.125-0.25 mmol/L MnCl₂ and 0.125-0.5 mmol/L MPP⁺ treatment groups had significant increases in the content of ROS, and the 0.25-0.5 mmol/L MPP⁺ treatment groups had significantly higher content of ROS than the groups treated with the same doses of MnCl₂ (all P<0.05) . Compared with the control group, the 0.25-0.5 mmol/L MnCl₂ andMPP⁺ treatment groups had significant increases in autophagy-related proteins LC3-II/LC3-I and significant reductions in P62 expression; the 0.125-0.5 mmol/L MPP⁺ treatment groups had significantly higher LC3-II/LC3-I than the groups treated with the same doses of MnCl₂, and the 0.125 and 0.25 mmol/L MPP ⁺ treatment groups had significantly lower P62 expression than the groups treated with the same doses of MnCl₂ (all P<0.05) . Conclusion Both MnCl₂ and MPP⁺ can induce oxidative stress and autophagy in SK-N-SH cells, and MPP⁺ has a significantly greater inductive effect on autophagy of SK-N-SH cells than MnCl₂.