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Fig. 6
(A–E) Lateral view schematics of zebrafish embryos at 14 hpi that were injected with membrane-localized mCherry-expressing SK-N-AS NB cells that either differentiated into neurons (red) or not (blue) during time-lapse imaging. (A) Empty vector (EV) or scrambled vector (n = 30 embryos), duplicated from Figure 2B for clarity; (B) ITSN1 short hairpin 1 (sh1) or short hairpin 2 (sh2) (n = 29 embryos combined) (C) EV + 100 μM LY294002, a reversible PI3K inhibitor, on cultured cells for 2 h pre-injection (n = 20 embryos). (D) EV + 100 nM wortmannin, an irreversible PI3K inhibitor, on cultured cells for 24 h pre-injection (n = 19 embryos). (E) EV + 1 μM wortmannin on cultured cells for 24 h pre-injection and 1 μM RA on host embryos post-injection through time-lapse (n = 23 embryos).
(F–I) Cell numbers at 48 and 72 hpi normalized to 15 hpi and their segregation into pROI or aROI groups in tested conditions compared with control SK-N-AS cells: ITSN1-silenced (sh2) SK-N-AS cells (F and G), PI3K inhibitor (either LY294002 or wortmannin)-treated SK-N-AS cells (H and I). Control pROI and aROI values are duplicated from Figure 2J for clarity.
(J) Scatter plot of maximal projection lengths and their segregation into aROI and pROI groups stratified by tested conditions. Control aROI and pROI columns are duplicated from Figure 2I for clarity and are composed of scrambled and empty vector data points.
(K–L′) 9 μm thick optical slices of representative SK-N-AS cells expressing the GFP-AktPH construct in the aROI (K and K′) or pROI (L and L′). Dashed lines denote membrane projections and arrows indicate the base of projections.
(M) Scatter plot of GFP intensity in a 2 μm diameter sphere adjacent to the cell membrane when the maximal projection first forms.
(N) GFP intensity (2 μm diameter sphere) of single representative cells over time in the aROI (red; K and K′), pROI (blue; L and L’), aROI with ITSN1 sh2 (black), or aROI post-treatment with 1 μM wortmannin and 1 μM RA (gray). Horizontal bars denote mean values, with 95% confidence intervals indicated. n.s. p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. ivt, in vitro; ivv, in vivo. Scale bar (A–E), 100 μm. Scale bar (K and L′), 5 μm.
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Reprinted from Developmental Cell, 56(19), Treffy, R.W., Rajan, S.G., Jiang, X., Nacke, L.M., Malkana, U.A., Naiche, L.A., Bergey, D.E., Santana, D., Rajagopalan, V., Kitajewski, J.K., O'Bryan, J.P., Saxena, A., Neuroblastoma differentiation in vivo excludes cranial tumors, 2752-2764.e6, Copyright (2021) with permission from Elsevier. Full text @ Dev. Cell