Dox provokes morphological and functional defects to mitochondria. Mitochondrial morphology in mice treated with saline (CTRL) or Dox. (A) Representative electron micrograph (5800× magnification) of heart muscle derived from saline (0.9%) and Dox (20 mg/kg) treated mice, with magnified insets below, scale bar 2 µm. (Left panel) Saline-treated mice exhibit normal mitochondrial morphology. (Right panel) Dox-treated mice displaying abnormal mitochondria with disrupted cristae and electron dense granules consistent with calcium deposits (red arrows). (B) Mitochondrial morphology assessed by epifluorescence microscopy using mitochondrial outer membrane protein Tom 20 (Red) and nuclear DNA (blue, Hoechst 33258), scale bar 10 µm. (C) Representative images of mitochondrial calcium uptake by Rhod-2AM (red fluorescence, see Section 2 for details), scale bar 10 µm. (D) Mitochondrial permeability transition pore (mPTP) opening assessed by using Calcein-AM in the presence of cobalt chloride, loss of green fluorescence indicates mPTP opening, scale bar 10 µm.