Fig. 5

Aplidin and analogs do not induce canonical unfolded protein response but downregulate vascular maturation factors VASH1 and DKK3. Three different branches of UPR, the master regulator GRP78/HSP5A, the IRE1a/XPB-1 s and PERK/CHOP/DDIT3 pathway were analyzed. In contrast to thapsigargin (10 nM), bortezomib (5 nM), Aplidin (5 nM) and analogs (10 nM) did not induce HSP5A gene expression (a) and splicing XPB1 within 24 h (b). c Only thapsigargin was able to increase GRP78 and XPB1 s protein as analyzed after 72 h by Western Blot. d DDIT3/CHOP was significant elevated on gene expression after 24 h in bortezomib, Aplidin and analog-treated cells. Aplidin and derivates did not display such an inductive response as observed for thapsigargin. e For analysis of vascular maturation factors HUVECs were incubated with bortezomib (5nM), Aplidin™ (5nM) or Aplidin analogs (each 10 nM) Western Blot analysis of VASH1 protein in cytosolic extracts of bortezomib, Aplidin™ and Aplidin analog-treated endothelial cells. Aplidin™ and analogs downregulated VASH1 and KDR protein levels after 24 h. f DKK-3 release from endothelial cells was reduced upon treatment with bortezomib, Aplidin™ or PM01215 and PM02781 after 72 h, as determined by a sandwich ELISA specific for human DKK-3. The means of one treatment group were compared to untreated control. The level of significance for the analysis was set at p < 0.05