Fig 2: EBF expression allows pericytes to be distinguished from MSCs. a MSCs isolated from human adipose tissue (MSC-AT) showed, as expected, significantly lower levels of PDGFRß as compared to HBVPs (p < 0.01), with high levels of CD90 that conversely was barely detected in HBVPs (p < 0.01). Of note, CD146 was expressed, albeit at low levels, only in HBVPs (p < 0.01). b The expression levels of the EBF family members in MSC-AT and HBVPs showed that EBF1 was expressed also in MSC-AT, albeit at a significant lower level as compared to HBVPs (p < 0.05). While EBF2 was expressed at a comparable level in both MSC-AT and HBVPs, EBF3 was detected only in MSC-AT, albeit at a very low level (p < 0.01). Of note, EBF4 was detected only in HBVPs, although at a very low level (p < 0.05). These data suggest that the EBF expression profile allows MSC-AT to be distinguished from pericytes. *p < 0.05, **p < 0.01

Fig 3: Intratumor heterogeneity at the single-cell level of a subtype 2 retinoblastoma (RBSC11).a 2D t-SNE plot of 1198 single retinoblastoma cells from one patient. Each dot represents one cell. b Heatmap of top cluster markers (top 20 most upregulated genes per cluster according to fold-change). Representative cluster markers and enriched gene sets are shown. Cluster marker p-values were calculated by hypergeometric tests with BH correction. c Expression of selected genes shown in 2D t-SNE plot (early photoreceptor markers: CRX, OTX2; late cone markers: ARR3, GUCA1C; neuronal/ganglion markers: EBF3, GAP43, DCX; proliferation marker: MKI67; pro-apoptotic marker: BNIP3; macrophage marker: CD14; T-cell marker: CD3D). d CNV profiles inferred from single-cell gene expression. Each row represents the profile of one individual cell. The genes on chromosome 6p overexpressed in the non-malignant cells monocyte/microglia correspond to HLA complex genes and should not be interpreted as CNV in cluster 5. e Upper panel: Diagram summarizing the interpretation of the different clusters of the 2D t-SNE plot. Lower panel: A progression model for this retinoblastoma case based on genomic alterations.

Fig 4: The diversity of Pitx3-expressing midbrain neurons in human embryos. a Immunostaining analysis of PITX3 and TH in human ventral midbrain, on frozen sections. Boxed areas indicate the localization of close-up images. b Network plots of the Ebf3 and Aldh1a1, with green indicating low and red high expression levels. c Close-up views of medial and lateral regions from ventral midbrain of human embryos which correspond to the boxed regions shown in (a). Left sidebar indicates the age of the embryo in weeks. Immunostainings for PITX3, EBF3, and ALDH1A1. Scale bars are 100 μm

Fig 5: EBF1 plays a key role in the pericyte phenotype cell commitment. a Significant downregulation of EBF1 mRNA levels (about 75% decrease; p < 0.01) was obtained in HBVPs by means of siRNA technology, measured by RT-qPCR 24 h after transfection (upper histogram). Immunoblotting performed with protein extracts confirmed the reduction in the synthesis of EBF1 protein (about 50% decrease; p < 0.05). The representative image on the right shows the corresponding EBF1 band at 66 kDa. b Histogram shows that after EBF1 silencing, the expression of EBF3, a marker linked to the MSC phenotype, is significantly increased (p < 0.05). c Silencing of EBF1 does not affect cell proliferation as assessed by the evaluation of the nuclear antigen Ki-67 expression by RT-qPCR, 24 h after seeding cells with or without the addition of different proliferative stimuli, such as the medium from U87 glioblastoma cells cultured under hypoxic conditions or the complete culture medium containing PGS and 10% FBS. d We then investigated whether EBF1 silencing affects the phenotype of the pericytes. Indeed, the histograms show a significant reduction in the main pericyte markers PDGFRβ (p < 0.05) at both the transcriptional (p < 0.05) and protein (p < 0.01) levels. e Additionally, we found a significant reduction in the pericyte marker CD146 (p < 0.01). The expression levels of CD90, a marker linked to a mesenchymal phenotype, did not show a significant reduction. f Of note, the expression levels of VEGF, Ang-1, NG2 and TGF-β, cytokines produced by pericytes during different phases of angiogenesis are significantly reduced in EBF1-silenced HBVPs (p < 0.05). Overall, data confirmed a functional role of EBF1 in pericyte cell fate commitment and functionality. *p < 0.05, **p < 0.01. CTRL (control); siRNA (EBF1-silenced); SCR (scramble, control siRNA); Basal (basal conditions); Hypoxic (hypoxic conditions)