Figure 3
CSC-induced concentration-dependent signalling: translocation of AIF into the nucleus and permeabilisation of lysosomes. (a) Shows the result of annexin V/propidium iodide staining and FACS analysis of CSC-treated P53 knock down and BCL-XL-overexpressing cells. The cells were incubated with 50 and 100 μg/ml CSC for 48 h. (b) Shows immunofluorescence pictures of cellular AIF distribution in CSC-treated endothelial cells for 24, 48 and 72 h. Green fluorescence is specific for AIF and the nucleus is stained with TOPRO-3 (shown in blue). (b) Shows also immunofluorescence pictures of endothelial cells (controls and CSC incubated cells) incubated with an isotype control (negative control; cells stained with FITC rabbit anti-human IgG isotype). (c) FACS-based analysis of lysosomal integrity. Endothelial cells were incubated with 50 or 100 μg/ml CSC for 24, 48 and 72 h and lysosomal integrity was analysed using the LysoTracker dye. (d) Shows western blot analysis of LC 3 (autophagy marker). HUVECs were incubated with 50 or 100 μg/ml CSC for 24, 48 and 72 h. All experiments were performed in triplicates and were repeated at least three times. Mean values±S.D. are shown. Asterisks indicate significant differences (*P<0.05; **P<0.01) compared with the controls. MFI=mean fluorescence intensity
