Fig. 2

Characterization of APE1 WT and mitochondrial targeted (MTS-APE1) clones. a Western blot analysis of total (TCE), nuclear (NCE), and mitochondrial (MCE) protein extracts of control (SCR), APE1 shRNA (shRNA), APE1 WT, and MTS-APE1 HeLa clones. Endogenous APE1 is efficiently silenced by a shRNA clone, while ectopic 3xFLAG-tagged APE1 is present only in APE1 WT and MTS-APE1 clones. While in the APE1 WT clone the ectopic protein localizes into both the nuclear and mitochondrial compartments, in MTS-APE1 clones the signal is mostly present in the MCE. Moreover, ectopic MTS-APE1 pre-protein (*) is processed into the mitochondrial matrix where the MTS is removed (**), leaving the active APE1 form. Anti-LSD1 antibodies and anti-ATP5A proteins were used as nuclear and mitochondrial markers, respectively, while anti-Actin as loading control. Full-length blots are presented in Supplementary Figure 2. b mtDNA damage was quantified in each clone. The silencing of APE1 causes a significant increase in the damage detected, while the re-expression of the ectopic APE1 WT or MTS-APE1 restores the basal mtDNA damage. The error bar represents the standard deviation of three independent experiments. (*: p < 0.05). c Representative images of clonogenic assay on APE1 shRNA and stable KI clones. In the zoomed squares it is possible to appreciate the size difference between APE1 WT and MTS-APE1 colonies. d The graphs report the relative number of colonies (left panel) and the colonies’ dimension (right panel) of shRNA, APE1 WT, and MTS-APE1 clones compared to the control clone (SCR). Data reported are the mean of four independent biological replicates. (*: p < 0.05; **: p < 0.01; ***: p < 0.001)