Fig 2: Upregulation of the transcription factors POU3F2 and NTF3 during neuronal differentiation of NT2D1. a The protocol for neuronal induction of NT2D1 cells is schematized. b ß3-tubulin staining for neuronal cells in NT2D1 cells untreated (non) and treated with neuronal induction medium at the indicated time points. c Quantification of ß3-tubulin-positive cells. d Immunoblotting analysis for POU3F2, POU3F3, ß3-tubulin, and NTF3 in NT2D1 cells untreated or treated with neuronal induction medium at the indicated time points. The values show the expression relative to that of untreated cells (to which a value of 1 was assigned). e Microarray analysis showed that neuronal induction for 6 h increased the expression of NTF3 and GADD45 in NT2D1 cells. f NTF3 mRNA expression after neuronal induction was analyzed by real-time PCR. The levels of mRNA were calculated as the relative expression compared with that of non-induced NT2D1 cells. GAPDH mRNA was used as a control. *p < 0.05; ***p < 0.001. g Phospho-TrkC (Tyr820) staining in treated and untreated NT2D1 cells. Values are presented as mean ± SEM of three independent experiments for c and f

Fig 3: SLITRK3 overexpression potentiates the effect of NTF3 treatment to increase CSCs and NTRK3 phosphorylation. (a) CSC sphere formation assays assessing the effect of vehicle or 5 ng/mL NTF3 treatment (7 days) on GFP-expressing (black) or SLITRK3 overexpressing (red) H226 cells. Lines, SEM. (b) Impact of SLITRK3 overexpression and NTF3 treatment on the fraction of CSCs relative to total cell count based on FACS analysis of CD133+ status. Lines, SEM. (c) In situ immunofluorescent detection (red) of phosphorylated NTRK3 (p-NTRK3) in H226 cells expressing GFP or overexpressing SLITRK3, with or without NTF3 treatment. Nuclei (blue) were stained with DAPI. (d) Western blot analysis of p-NTRK3 levels in HARAB cells following stable knockdown of SLITRK3 by shRNA vectors (shRNA1 and shRNA2) relative to a nonsilencing control vector. (e) Result of immunoprecipitation performed using whole cell lysates from HARAB cells. A SLITRK3 antibody was used for immunoprecipitation and a NTRK3 antibody for Western blot detection. The bracket indicates the 2 bands that correspond to NTRK3. Rabbit IgG was used as the negative control. The band at 50 kDa corresponds to the antibody heavy chain subunit. Results are representative of 3 experiments