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Mitochondria were depleted by culturing MIA PaCa-2 cells in the presence of 100 ng/mL ethidium bromide for 6–8 weeks. The growth culture media were supplemented with 4.5 mg/mL glucose, 50 μg/mL uridine, and 100 μg/mL pyruvate to compensate for respiratory metabolism defects. After establishing Rho0 cells, the media was supplemented with 50 μg/mL uridine for cell growth and expansion. To validate the successful generation of Rho0 cells, OCR and extracellular acidification rate (ECAR) levels were tested using a Seahorse XF96 Analyzer (Agilent).
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