Fig. 5

Modulation of granulin (GRN) expression affects the angiogenic potential of macrophages. a Western blot analysis of GRN in U937 cells transfected with a GRN expression vector or an empty vector (EV) and cultured in the presence of RPMI medium or conditioned media (CM) from MDA-MB-468 cells. b RT-qPCR analysis of GRN messenger RNA levels in the same experimental conditions described in (a). c CM from the indicated experimental conditions were injected into the perivitelline space of zebrafish embryos, and neovascular response originating from the developing subintestinal plexus was evaluated. Injection of PBS alone or PBS supplemented with recombinant vascular endothelial growth factor A (rhVEGFA) was used, respectively, as negative and positive controls. Spikes sprouting from subintestinal plexus were counted in at least 42 embryos per condition. Graph shows the distribution of the population of embryos evaluated for each condition. Representative images are shown in (d). Significance was evaluated by one-way analysis of variance followed by Sidak’s multiple comparisons test using GraphPad software (GraphPad, La Jolla, CA, USA). ***P < 0.0005. e Western blot analysis of GRN in U937 cells transfected for 8 hours with control small interfering RNA (si-SCR) or three different GRN-targeting siRNAs (si-GRN_#1,_#2,_#3) and subsequently cultured in the presence of CM from MDA-MB-468 cells for 72 hours. f and g Tube-formation assays involving EA.Hy926 endothelial cells performed in the presence of CM from the conditions indicated in (e). RPMI medium, supplemented (rhVEGFA) or not (RPMI) with recombinant VEGFA, was used as a positive or negative control, respectively. Data are presented as mean ± SEM. *P < 0.05, **P < 0.005 calculated by two-tailed t test