Figure 7. Rotenone-enhanced mammosphere formation is ablated by treatment with Doxycycline or Mito-tempo. (A) MCF7 cells were pre-treated for 48 hours with Rotenone (from 1 to 4 nM) as a monolayer. Then the cells were harvested by trypsinization and re-plated under low-attachment conditions for the mammosphere assay. Note that pre-treatment of MCF7 monolayers with rotenone at either 1 or 2 nM stimulated mammosphere formation. (B) As in panel (A), except the MCF7 monolayers were pre-treated for 48 hours with Rotenone (1 nM). Then, the cells were re-plated under low-attachment conditions. Note that further treatment with i) an inhibitor of mitochondrial biogenesis (Doxycycline; 50 μM) or ii) a mitochondrial-based antioxidant (Mito-tempo; 100 μM), is sufficient to block Rotenone-enhanced mammosphere formation.