Fig. 4

Aplidin analogs induce premature senescence by induction of the cell cycle inhibitor p16^INK4A. a HUVECs (n = 3) were stimulated for 24 h with the compounds, after which total RNA and protein was extracted. Gene expression was analyzed by real-time PCR and protein expression by SDS-PAGE and Western Blot. Aplidin™ (5 nM) and Aplidin derivatives (10 nM) induced p16^INK4A gene expression. TP53 gene expression was induced neither by bortezomib (5 nM) nor by Aplidin (5 nM) or derivatives (10 nM). P27^KIP1 gene expression was induced only by bortezomib. b Western Blot analysis of p16^INK4A protein after treatment with bortezomib, Aplidin™ and derivatives (72 h). c Upregulation of p53 and cell cycle inhibitors p21 and p27 after treatment with the proteasome inhibitor bortezomib (24 h). Aplidin™ and derivatives did not induce p53, p21 or p27 proteins, even after 72 h of incubation (data not shown). Tubulin alpha served as loading control. d Staining for senescence-associated beta galactosidase in human endothelial cells treated for 72 h with Aplidin™ (5 nM) and analogs (10 nM each). In comparison to control Aplidin analog-treated cells show an increased number of blue (SA-β- gal ^positive) cells, like the positive control H₂O₂. Differences between the means of one treatment group and untreated control were analyzed. The level of significance for the analysis was set at p < 0.05. Bars indicate 20 μm