Fig. 2.

Mitochondria self-generate hypoxia but are nonessential for directed migration towards oxygen. a PCR amplification of mtDNA D-loop region and hnRNPA1 nuclear DNA in wt MCF10A and rho0 cells demonstrating the effective depletion of mtDNA from rho0 cells. b Oxygen consumption rate (OCR) of wt and rho0 cells before and following addition of (a) oligomycin D (0.5 µM), (b) FCCP (1 µM), (c) antimycin A 0.5 µM + rotenone (0.5 µM) (mean ± SD). This experiment demonstrates that rho0 cells are indeed unable to respire. c Tracking and redistribution of rho0 (red) and wt (green) cells set under confinement and treated or not with antimycin A (AA) or oligomycin D (OD) demonstrate that hypoxia generation is required for directed migration under confinement. Top panels: cell trajectories. White dotted-lines indicate the border of the cell cluster at 0 h. Bottom panels: relative distribution of MCF10A cells at the edge of the cluster at 0 h and 48 h post-confinement. Scale bar, 500 µm. d Tracking of confined rho0 (red) and wt cells (green) either alone or combined (1:1 ratio) for 24 h. Upper right: magnification of the framed regions. This experiment demonstrates that rho0 cells that do not respire are still capable of aerotactic migration provided that they are located within the oxygen gradient generated by wt cells. Scale bars; 1 mm (500 µm for magnification panels). e Mean values of directionality and speed from (d) (mean ± SD; n = 3 independent experiments). ***P < 0.001 by two-tailed Student’s t-test. +C confined