Figure 5: Dynasore-mediated inhibition of VEGF-to-ERK1/2 signalling is independent of DDE (“uncoupling experiment”).

In step 1, HUVECs were treated with vehicle (left side) or dynasore (right side) and stimulated with VEGF (in the presence of vehicle or drug), for 10 min. The scheme (step 1, right side) illustrates that dynasore blocks both dynamin-dependent endocytosis (indicated in the scheme as “DDE blocked”) and the off-targets (indicated in the scheme as “off-targets are inhibited by dynasore”), while dynamin-independent endocytosis (DIE) remains unaffected. Then, the cells were either lysed and subjected to immunoblotting analysis, to test the effect of dynasore on ERK1/2 phosphorylation when the drug is continuously present (see lanes depicted by the long vertical arrow, lanes 2 and 4), or were acid washed (step 2), to remove membrane bound VEGF and dynasore (the effectiveness of acid wash treatment to remove VEGF from plasma membrane is shown in Fig. S4a), and processed to the next step. In step 3, cells were incubated in serum-free medium, for 10 min, to allow full recovery from dynasore, thereby releasing possible off-target effects of the drug (indicated in the scheme as “off-targets: released”, see step 3, right). As there is no ligand in this phase, DDE cannot resume despite removal of the drug (indicated in the scheme as “DDE blocked”). Thus, in lane 5, DDE does not take place throughout the whole experiment, even after removal of the drug (note that this is the main difference with the reversibility experiment shown in Fig. S2a, where VEGF is added after the removal of the drug, which rescues both DDE and the off-targets). Then, the cells were lysed and subjected to immunoblotting analysis using anti-phospho-ERK1/2 antibodies. Since DDE never took place in sample 5 (see above), recovery of the signal in this sample (compare lane 5 with lane 4), upon release of the off-targets, suggests that DDE is not essential for VEGF signalling; thus, the inhibitory effect of dynasore in lane 4 is due to the off-targets. At the bottom of the figure there is a schematic summary of the treatment steps of sample 5. Full-length blots are shown in Fig. S4b.