Extended Data Fig. 9: Loss of mtDNA segregation causes the formation of enlarged nucleoids at mitochondria/ER contact sites and elongates mitochondria.
a) Western blotting of TOP3A and β actin (loading control) in U2OS cells transfected with control or TOP3A siRNAs. Quantification of western blot data is shown in the graph (right), dots represent replicates of independent experiments (N = 3). b) The number of nucleoids larger than 300 nm2 was scored (using confocal images shown in Fig. 8a). siCTRL was compared to siTOP3A siRNAs by unpaired, two-tailed t test (p = 0.0037 for siRNA #1, p < 0.0001 for siRNA #2). N = 11 cells per condition, and data were quantified from one representative experiment of three. c) Airyscan imaging of Ii33-mCherry (ER), Pico Green (DNA), and BFP-Fis1 (mitochondria) of live cells depleted of TOP3A by siRNA (#2). Time-lapse imaging demonstrates mitochondria/ER contacts around enlarged nucleoids. Scale bars = 10 µm and inset scale bars = 1 µm. This experiment was performed three times with similar results. d) Overlap between the ER and mitochondria-localized nucleoids was scored (see Methods). Enlarged nucleoids (>300 nm2) were compared to normal-sized nucleoids (<300 nm2) within the same cells using two-tailed, paired t tests (p < 0.0001 for siCTRL, p < 0.001 for siTOP3A #1, p = 0.0033 for siTOP3A #2). N = 15 cells for siCTRL and siTOP3A #1, N = 14 cells for siTOP3A #2, and data were pooled from three experiments. e) Spinning disk imaging of U2OS cells transfected with the indicated siRNAs, followed by immunofluorescence against DNA and HSP60. Scale bars = 20 µm and inset scale bars = 2 µm. This experiment was performed three times with similar results. f) Quantification of F (see Methods). siCTRL was compared to siRNAs against TOP3A and DRP1 using unpaired, two-tailed t test (p < 0.0001 for siTOP3A #1, p = 0.0003 for siTOP3A #2, p < 0.0001 for siDRP1). Number of cells: N = 20 for siCTRL, N = 18 for siTOP3A #1, N = 15 for siTOP3A #2, N = 14 for siDRP1. Data were quantified from one representative experiment of three. For all graphs, data are reported as mean ± SEM. Source numerical data and unprocessed blots are available in source data.
