Endothelial cells produce the vasoactive peptide apelin. (A) Mass spectrometry analysis of the brain microvascular endothelial cell (hEC) secretome identified 22 peptides and proteins specific to endothelial cells. (B) Apelin peptide coverage (37%) is indicated in red on the full-length sequence. (C) RT-PCR for APLN and GAPDH is shown for hEC and glioblastoma patient-derived cells with stem properties (GSCs) #1, #4, #9 and #12 RNA total cell lysates. (D) Apelin secretion in mitogen-free control media (MF), and in conditioned media (CM) prepared from GSC#1, human brain microvascular EC (hEC), mouse macrovascular EC (mEC) and orthotopic mouse brain tumour-isolated EC (tEC). Apelin secretion was measured in CM from hEC cultured in acidified medium (pH 6.8) or control conditions (pH 8.2). Data are representative of n ≥ 2 with mean ± SEM. Red dashed lines indicate the minimum sensitivity range of APLN detection. (E) Confocal analysis of SOX2 (green) + PECAM (red), APLN (green) + PECAM (red), APLN (green) + NESTIN (red), NESTIN (green) + APLNR (red) in glioblastoma clinical samples. Nuclei are shown in blue (DAPI). Arrowheads and arrows indicate APLNR/NESTIN and APLN/PECAM-double positive cells respectively. Scale bars = 25 µm. Data are representative of n = 4 newly diagnosed patient samples. All panels are representative of n = 3, unless specified.