Microscopy illustrates a hypoxia-dependent mitochondrial degradation.
Transmission electron microscopy: PANC-1 cells were treated with active and inactive APD-CLD-derivatives under differential oxygen concentrations for 4 hours after which the cells were fixated in glutaraldehyde and analyzed by transmission electron microscopy. White arrows: collapsed mitochondria. Black arrows: regular mitochondria. N = 1. Fluorescence microscopy: MCF7 cells were treated with an APD-CLD-alkyne probe under hypoxia for 6 hours. The probe was conjugated to FAM-N3 via CuAAC. Subsequently, cells were stained with either rabbit anti-TOMM20 Ab (left panel) or rabbit anti-calnexin Ab (right panel) overnight followed by a goat anti-rabbit Ab conjugated with AlexaFluor647. Randomly selected pictures illustrate that the probe has strong co-localization with TOMM20. N = 1. MCF7 cells were treated with 2b, CCCP or DMSO under hypoxia or normoxia for 6 hours. Cells were stained with JC-1 (Cayman Chemical). Randomly selected pictures illustrate a hypoxia-selective loss of membrane potential after treatment with 2b. Viability assay of PANC-1 cells treated with CCCP: PANC-1 cells were treated with CCCP for 48 hours under hypoxia or normoxia and the viability was assessed with CellTiter Blue. The uncoupler did not display hypoxia-selective toxicity by itself. N = 3. Seahorse experiment: One-hour normoxic pretreatment of BxPC3 cells with BE-43547A2 does not alter the mitochondrial respiratory chain activity as measured by oxygen consumption. N = 7.