Fig. 3
Inhibitor of differentiation 4 (ID4) expression in breast cancer cells leads to the activation of an angiogenic programme in macrophages. a Expression matrix representing a panel of angiogenic factors evaluated using TaqMan Low-Density Arrays (TLDA) in macrophages obtained by 1,25-dihydroxyvitamin D3 (VitD3)-mediated differentiation of HL60 cells and subsequently cultured in RPMI medium or in conditioned media (CM) from control (EV) or ID4-overexpressing (ID4) MDA-MB-468 breast cancer cells. b Western blot showing ID4-HA overexpression in MDA-MB-468 cells. c Selected genes modulated in the arrays were evaluated by RT-qPCR in macrophages obtained from VitD3-mediated differentiation of U937 cells and subsequently cultivated in RPMI medium (CTR) or in CM from control (CM si-SCR) or ID4-depleted (CM si-ID4) MDA-MB-468 cells. d Western blot analysis showing the level of ID4 protein after transfection of the indicated small interfering RNAs (siRNAs) in MDA-MB-468 cells. e–g Western blot analysis of ephrin B2 (EphB2), granulin (GRN) and hypoxia-inducible factor (HIF)-1A proteins in differentiated U937 cells cultured in CM si-SCR or CM si-ID4 from MDA-MB-468 cells. h Immunofluorescence analysis of HIF-1A protein performed in differentiated U937 cells cultured in the presence of CM si-SCR or CM si-ID4 from MDA-MB-468 cells. i Western blotting showing the efficiency of vascular endothelial growth factor A (VEGFA) depletion by siRNA transfection in MDA-MB-468 cells used to prepare CM used in experiments shown in (j). j RT-qPCR analysis of the indicated messenger RNAs in U937 macrophages cultivated in the presence of CM from control (si-SCR) or VEGFA-depleted (si-VEGFA) MDA-MB-468 cells. k RT-qPCR analysis of the indicated genes in differentiated U937 cells cultivated in RPMI medium or in CM from MDA-MB-468 cells in the presence of VEGFA blocking antibody (Ab) or a control Ab. Specifically, VEGFA blocking Ab or control Ab were incubated with CM for 30 minutes at room temperature and CM plus Ab was subsequently used to culture U937 cells for 48 hours. Results from at least three biological replicates are shown. Data are presented as mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005 calculated by two-tailed t test