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. Author manuscript; available in PMC: 2009 Nov 25.
Published in final edited form as: J Hum Genet. 2009 Jul 24;54(9):516–524. doi: 10.1038/jhg.2009.71

Figure 1. POLG D1135A mutant depletes mtDNA and promotes tumorigenicity in breast cancer cells.

Figure 1

POLG D1135A cDNA was cloned in the tetracycline inducible plasmid pTRE-Tight-BI-AcGFP1. A bicistronic promoter provided the expression of both GFP and POLG simultaneously. Transfected MCF7 Tet-on Advanced cells were treated with 1000 ng/ml doxycycline for up to 5 day and were sorted by FACS. A) GFP fluorescence was used as a guide to sort cells expressing the mutant POLG gene. Mean fluorescent intensity was determined on the FL1 channel of a FACSCalibur flow cytometer. Data represent geometric mean fluorescence intensity. B). MtDNA index in MCF7 Tet-on Advanced cells expressing POLG D1135A. The ratio of mtDNA to nuclear DNA was used as an index for measuring the mtDNA content C). DHE oxidation of MCF7 Tet-on Advanced cells containing POLG D1135A was measured. Mean fluorescence intensity of each treatment group was normalized to day 0 and expressed as fold DHE oxidation + 1 SD. D). Mitochondrial membrane potential was measured by TMRE fluorescence. Data represents mitochondrial membrane potential as a percent of control (day 0) + 1 SD. E). Mitochondrial respiratory activity was measured by the rate of resazurin reduction. F).Tumorigenicity was measured by Matrigel invasion assay.