Fig. 3S.TAP and RGC-like characteristics and bipotentiality of CN-spheroids. (a) Tumor spheroids formed in ITSFn and DMEM-FBS media. (b) Up: Representative images of RT-PCR shows expression of NSC makers: GFAP, Nestin, BLBP, CD133, SOX2, hTERT. In particular, ITSFn-derived CN-spheroids express higher level Nestin, BLBP, CD133, and SOX2. Bottom: Densitometry measurement of each genes relative to GAPDH. (c~h) Immunofluorescence staining of CN-spheroids. Nestin+/BLBP+(c), PSA-NCAM+/BLBP+(d), Nestin+/GFAP-delta+(e). GFAP-delta+/Tuj1+ shows mixed population of RGC and neuroblasts (f). TAP characteristics were confirmed by EGFR+/MASH+ cells (g), SOX2+ cells (h). (i, m) CN-spheroids were attached on the dish and cultured up to 4 weeks. Phase contrast view of ITSFn- (i) and DMEM-FBS-(m) cultured CN tumor sphere shows migration and differentiation of cells at the edge of the sphere. (j~l) CN-spheroids cultured in ITSFn differentiated mostly into neuronal cells (j). (n~p) DMEM-FBS media induced more glia differentiation of the CN-spheroids (n). (q, r) Ca2+ imaging (lower left panels) and immunofluorescence staining (lower right panels) of CN-spheroids cultured in ITSFn (left) and DMEM-FBS (right) media. Cells were pre-loaded with Fura 2. AM, washed and pre-incubated for at least 10 min prior to the addition of KCl (133 mM) for 60 sec. (s) The time of KCl addition is indicated by red. The level of Ca2+ responses to high K+ was almost two folds in CN-spheroids in ITSFn as compared to DMEM-FBS. Error bars indicate SEM. (t) Up: Representative images of RT-PCR shows expression of NSC makers: GFAP, Nestin, BLBP, CD133, SOX2, hTERT. Bottom: Densitometry measurement of each genes relative to GAPDH. Original full-length images of b and t are presented in Supplementary Scale bars, 100 µm (c, g, j). All data presented as mean +/- S.D. Two way ANOVA followed by Bonferroni correction and t-test analysis was done. A p value of <0.05 (*) or 0.01 (**), 0.001(***), and ns: not significant was used to denote statistical significance. Three biological samples (N=3) were used for the experiments.
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