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Fig. 3 | BMC Cancer

Fig. 3

From: The Aplidin analogs PM01215 and PM02781 inhibit angiogenesis in vitro and in vivo

Fig. 3

Aplidin analogs induce oxidative stress and activate JNK in human endothelial cells. a Generation of reactive oxygen species (ROS) was analyzed by CellRox®Green, a novel fluorogenic probe for measuring oxidative stress in living cells. The cell-permeant dye is weakly fluorescent while in a reduced state and exhibits bright green photostable fluorescence upon oxidation by ROS. Aplidin™ (5nM) and Aplidin derivatives (10 nM) were added to endothelial cells with or without the antioxidant N-acetyl-cysteine (NAC), and ROS in mitochondria monitored (green signals) after 6 h. H202 served as positive control (30 μM, 2 h of incubation). Nuclei were stained with NucBlue. Bars indicate 10 μm. b In vitro proliferation in presence of antioxidative N-acetyl-cysteine (NAC) was analyzed by real-time proliferation in xCELLigence system. Endothelial cells were pre-incubated for 1 h with antioxidative NAC (25 μM) or medium alone and then stimulated with H202, Aplidin™ (5nM) or Aplidin derivatives (PM01215, PM02781; 10 nM each). In comparison to H202-treated cells, Aplidin analog-treated cells could not be rescued by NAC, indicating a terminal growth arrest. c HUVECs were starved overnight and preincubated with drugs for 60 min, then stimulated with standard culture medium supplemented with VEGF, bFGF (100 ng/mL each) and 10 nM of each drug or DMSO (0.1 %) as control. Cytosolic extracts were analyzed by Western Blot for phosphorylation of ERK and AKT after 20 min. Phosphorylation of c-Jun N-terminal kinases (JNK) was analyzed in starved endothelial cells treated with PM01215 and PM02781 for 5 min. In comparison to DMSO-treated cells, Aplidin analogs increased JNK phosphorylation. GAPDH and tubulin served as internal controls for loading and equal protein transfer

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