Extended Data Fig. 7: Characterization of primary mouse cerebral cortex ECs (cECs) from male WT and 16p11.2^df/+ mice.

a, Representative images and quantifications of immunocytochemical staining for cEC-specific markers GLUT-1, eNOS and VE-Cadherin, showing no difference between WT and mutant cECs isolated at P14 (blue: DAPI). b, Assessment of apoptosis in P14 cEC cultures. The Caspase-3/7 green assay revealed normal apoptotic rates in 16p11.2^df/+ cECs. c, qPCR validation on cEC RNA using mouse VEGFR-2, CD31 and eNOS as markers (a no reverse transcriptase control was used as negative control). d, Assessment of endothelial gene enrichment using RNAseq data normalized to a publicly-available database from Dr. Ben A. Barres lab, Stanford University, USA (Zhang et al. 2014, PMID 25186741; http://www.brainrnaseq.org/). e, Assessment of neuronal contamination using RNAseq data (as in d). A very low level of contamination was achieved. Examples given are from cortical endothelial cells (cECs) isolated from male mice at P14. f, Confirmation of cEC 16p11.2 haploinsufficiency by RNAseq. Mapping of fold change (FC) to 7qF3 locus genes confirms a ~50% decrease in gene expression levels at both P14 and P50. Data are mean ± s.e.m in a (VE-Cadh.) and c,d,e, whisker boxes (min to max, center line indicating median) in a (eNOS, GLUT1), or mean with individual values in b. CTL, control; WT, Wild-Type. For RNAseq, n = 3-4 biological replicates per group (2 mice per replicate).