Mitochondrial Ca2+analysis, ceramide sensitivity assay and subcloning test. (A, B) Mitochondrial calcium level was followed in real time by measuring Rhod-5N dye. LPC and HPC were treated with ATP 1 mM and traces trend monitored over time. The diagram explains the maximum uptake of calcium in both clones (*p < 0.05). (C) Mitochondrial mass analysis showed statistical difference between HPC and LPC in term of the size of mitochondrial network (**p < 0.01). (D, E) C2-Ceramide (N-Acetylsphingosine) treatment after 8 and 10 hours respectively: the charts highlighted the number of cells before and after incubation with C2-Ceramide 20 μM in the clones. In both frames of time clones from LPC presented increased sensitivity to apoptosis than HPC (***p< 0.001). (F) Pictures of C2-Ceramide treated (8h) and untreated (control) cells (scale bar: 50 μm). (G) Sub-cloning of HPC and LPC demonstrating that only HPC could regenerate both clone types again.
Repele et al. BMC Cell Biology 2013 14:24 doi:10.1186/1471-2121-14-24