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. 2020 Sep 24;37:101736. doi: 10.1016/j.redox.2020.101736

Fig. 2.

Fig. 2

Analysis of the influence of Endog expression in cell proliferation and ROS production in the Rat-1 cell line, and its dependence on the abundance of mitochondrial DNA. A) Equal number of Rat-1 control cells (NT, not transduced), scrambled-transduced (Scr) cells and cells transduced with either of two independent Endog silencing constructs were seeded in duplicates and the number of cells after 48 h was counted. Data are expressed as the number of cell cycles completed in 48 h in the presence or absence of the ROS scavenger NAC (0.5 mM). All values from 3 independent experiments are plotted plus mean ± SD. B) MitoSOX™ fluorescence was quantified by flow cytometry in preparations of the same experimental groups as in (A). C) Rat-1 cells were pre-treated with EtBr to reduce the mitochondrial DNA (mtDNA) content (see Materials and Methods section for details). Then, normal cells and EtBr-treated cells (EtBr) were transduced with scrambled (Scr) or two independent Endog-specific silencing constructs (Endog shRNA1, 2). Western Blot of EndoG assessing the efficacy of the silencing constructs and COXIV expression assessing the effect of EtBr treatment on mtDNA depletion, performed with samples of control and EtBr-treated cells not transduced (NT) or transduced with the different constructs. GAPDH was used as loading control. D) Equal number of control and EtBr-treated Rat-1 cells were seeded, left not transduced (NT) or transduced with scrambled (Scr) or Endog-directed silencing constructs (Endog shRNA1 and 2) in duplicate plates/treatment and counted after 48 h. Data are expressed as the number of cell cycles completed in 48 h. All values from 3 independent experiments in duplicates are plotted plus mean ± SD. E) MitoSOX™ fluorescence was quantified by flow cytometry in preparations of control not transduced cells (NT) or EtBr-treated cells not transduced (NT) or transduced with scrambled (Scr) or Endog-directed silencing constructs (Endog shRNA1 and 2). Values of four independent experiments are plotted plus mean ± SD. Two-way ANOVA was used to analyze the influence of Endog expression and NAC treatment (A, B) or EtBr treatment (D) and the interaction between them in cell proliferation and ROS production. Ns: not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.