Determining drug mechanism of action in Calu6 cells in vitro. A, Increased retention of the fluorescent cytotoxic metabolite of banoxantrone, AQ4, as oxygen concentration decreased, with (B) example images shown for cells exposed to 10 µmol/L of banoxantrone differing oxygenation conditions. C and D, Clonogenic survival assays showed that banoxantrone became increasingly cytotoxic with reduced oxygenation whereas the impact of atovaquone on survival was not affected by oxygen level. E, A total of 10 µmol/L atovaquone completely ablated OCR under normoxia while banoxantrone had no effect. F, Under hypoxia (3% O2), OCR ablation was observed at 1 µmol/L atovaquone and while a trend toward reduced OCR was observed for banoxantrone this was not significant.