Figure S7.

Interfering with GPC3-Unc5 interaction impacts on neuroblastoma cell migration properties; structural discussion of Unc5 complexes, related to Figure 7

(A) UMAP visualization of single-cell data from neuroblastoma tumors (Dong et al., 2020).

(B) Quantification of Unc5A-D, Unc5D alone, and GPC3 transcripts for each cell type.

(C) Western Blot analysis of GPC3 and Unc5D proteins in SY5Y and C3A cells.

(D) Q-RT-PCR analysis of GPC3 mRNA expression in SY5Y cells, 24 h after transfection, using GPC3 siRNA or scr.siRNA as a control.

(E) Representative images (left) and quantification (right) of transwell assays measuring the migratory properties of SY5Y:GFP cells transfected with either scr or GPC3 siRNA. ^∗∗∗∗:p < 0.0001. Student T test with Welsch correction.

(F) Anti-Myc western blot showing the secretion levels of Myc-tagged nanobody constructs used in Figure 7G. Supernatants of transfected SY5Y cells were analyzed. We find that both constructs are secreted effectively.

(G) Scheme of the in ovo graft experimental paradigm describing the experiments presented in Figures 7C–7H. NB: neuroblastoma.

(H) Illustrations of the phenotypic classification quantified in Figures 7C–7H. Neural crest-derived structures were labeled with an anti-HNK1 antibody. Human NB cells were detected with an anti-mito antibody (in red) and transfected NB cells with GFP (in green). Nuclei were stained with Hoechst. “1” points at isolated cells; “2” points at tumor masses. NT: Neural Tube; Ao: Dorsal aorta; DRG: Dorsal Root Ganglia; No: Notochord. Scale bar: 200 μm.

(I) Two of the four rUnc5DIgIgTSP chains in the complex with hGPC3core are shown as ribbons, colored according to the rainbow (N-terminus = blue, C-terminus = red). The rest of the complex is shown as transparent surface (gray).

(J) rUnc5DIgIgTSP in complex with FLRT2 and Latrophilin3 (Jackson et al., 2016). The two Unc5D chains are highlighted as rainbow ribbons.

(K) Superpositions of Alpha-fold models of the rUnc5D Ig2-TSP1-TSP2 region, after MD simulation, suggests flexibility in the TSP1-TSP2 linker.

(L) The ‘in cis’ model of hGPC3-rUnc5D was created using Alpha-fold, MD simulation and MODELLER. GPC3: shades of blue, Unc5D: shades of green. We have not included intracellular domains.

(M) As panel L, but showing a potential ‘in trans’ configuration where GPC3 and Unc5D are expressed on adjacent cells.

(N) Schematic summarizing the Unc5/GPC3 expression levels and putative interactions in the cortical and neuroblastoma models presented in this manuscript. Expression of Unc5D and GPC3 is color-coded from green (high Unc5D/ low GPC3) to blue (low Unc5D, high GPC3). Cells colored in cyan indicate co-expression of both receptors.

N: neuron, AP: apical progenitor, VZ: ventricular zone, NB: neuroblastoma cell, DNT: dorsal neural tube