Mitoblock 6

Chemicals used for the treatment of cells were prepared as followed, with final dilutions made with the respective cell culture medium. FCCP (Abcam, Cambridge, UK), 1 mm in DMSO, MPP⁺(Sigma-Aldrich), 10 mm in water, ionomycin (ab120370, Abcam) 10 mm, and 1 mm in DMSO, 2-deoxyglucose (Sigma-Aldrich) 0.5 m in water, menadione (Sigma-Aldrich), 1.5 mm in DMSO, BAPTA-AM (ab120503, Abcam), 2.5 mm in DMSO, BAPTA (ab144924, Abcam), 1 mm in water, CDDO-Me (Sigma-Aldrich), 1 mm in DMSO, UCF-101 (Sigma-Aldrich), 10 mm in DMSO, MitobloCK-6 (Focus Biomolecules), 5 mm in DMSO, bafilomycin A1 (Calbiochem, San Diego, CA, USA), 100 and 10 μm in DMSO, 17-AAG (ab141433, Abcam), 5 mm and 50 μm in DMSO and MG132 (Sigma-Aldrich), and 10 and 1 mm in DMSO.

HeLa cells were treated with 15 µM APE1 inhibitor III (APE1inhIII) (Merck Millipore, Burlingtion, MA, USA) or the solvent DMSO (Merck Millipore, Burlingtion, MA, USA) for 5 h at 37 °C [28 (link)] before mitochondrial replication imaging protocol (mREP) or total DNA isolation was performed. For scavenging of ROS, HeLa cells were treated for 24 h with either 100 µM mitoTempo (Sigma-Aldrich/Merck, St. Louis, MO, USA) [37 (link)] or N-acetyl-L-cysteine (NAC) (Sigma-Aldrich/Merck, St. Louis, MO, USA) [29 (link),38 (link),39 (link),40 (link)] or water as solvent. To block mitochondrial import of proteins, HeLa cells were treated for 48 h with 10 µM mitoBlock-6 (Focus Biomolecules, Plymouth Meeting, PA, USA). To slow down mitochondrial DNA replication, the HeLa cells were treated for 24 h with 10 µM 2′,3′-Dideoxycytidine (ddC) (Sigma-Aldrich/Merck, St. Louis, MO, USA) [41 (link)].