Fig 2: Neurospheres of telencephalic embryonic precursor cells express Reg-1a and Extl3. (A) Bright field images of neurospheres at 2 and 48 h of culture on non-adherent and adherent substrate. The graph shows the quantification of neurosphere size (pixel areas). Scale bar, 100 µm. (B) Adherent neurospheres after 48 h of culture immunostained for ß3-Tubulin and Nestin. ß3-Tubulin-positive differentiated cells are predominant outside the neurosphere whereas Nestin-positive cells localize inside and at the edge of the neurosphere. Scale bar, 50 µm. (C) RT-qPCR analysis of Reg-1a and Extl3 mRNA expression in neurospheres grown for 48 h on an adherent substrate (left panel). Values were normalized to those of cyclophilin expression. Western blots of cellular extracts and culture medium from neurospheres grown on an adherent substrate for 48 h (right panel). Reg-1a and Extl3 are expressed in the cells. Reg-1a is secreted in the medium. (D) Immunofluorescence analysis of Reg-1a, Nestin, and ß3-Tubulin expression in neurospheres. Merged images show that Reg-1a localizes to Nestin-positive progenitors and in ß3-Tubulin-positive post-mitotic neurons in both cell bodies and neuritic extensions. Scale bar, 10 µM.

Fig 3: N-terminome analysis of CM of HEK293 cells overexpressing SPPL3. a Immunoblots of TCA-precipitated CM (left) and lysates of carbonate-washed membranes (right) of untransfected (ctrl) and SPPL3-V5 WT-transfected Flp-In™ T-REx™ HEK293 cells. Treatment with doxycycline (Dox) induces SPPL3 overexpression and leads to increased release of EXTL3 and POMK into the CM. b Volcano plots showing peptides detected following HYTANE enrichment of CM from doxycycline-treated and untreated Flp-In™ T-REx™ SPPL3-V5 HEK293 cells (three technical replicates). Samples were analysed in parallel on the indicated LC–MS systems. 2,817 and 1,608 unique N-terminal peptides are plotted, respectively, and labelling/filtering is performed as outlined in Fig. 2e with x = 30 aa. Dashed lines represent threshold values (± 2 SD for log2 difference and p = 0.05). c Schematic overview and mapping of neo-N peptides found to be changed in abundance in CM (see Fig. 2h). Arrow height indicates relative intensities (mean of doxycycline-treated samples) for membrane-proximal peptides changed upon SPPL3 overexpression