Fig 2: Change of susceptibility to arabinofuranosyl cytidine (Ara-C) for SNAP25-expressing Daoy cells. The half-maximal inhibitory concentration of Ara-C was determined under a dose-dependent manner (from 0.1 to 10 mM) of Ara-C following incubation for 48 h. The surviving cells were counted by CCK-8 assay. Data are the means ± SD of triplicate independent experiments. Ara-C, arabinofuranosyl cytidine; CCK-8, cell counting kit-8. SD, standard derivation.

Fig 3: Morphological changes of cells with synaptosomal-associated protein 25 (SNAP25) expression. (A) The dendrites of Daoy cells with SNAP25 expression. (B) The dendrites of VGH-Med cells with SNAP25 expression. The cells transfected with pEGFP C3 vector emitted a green fluorescence throughout the cell and the SNAP25-overexpressing cells (pEGFP C3-SNAP25) formed dendrites that emitted green fluorescence. DAPI (blue) indicated the nucleus. (C) SNAP25 expression in normal neural cells. White arrowheads indicate the dendrites. GFP, green fluorescent protein; GFAP, glial fibrillary acidic protein; TAU, Tau protein; PNM, pan neuronal marker; DAPI, 4',6-diamidino-2-phenylindole; IBMX, 3-isobutyl-1-methylxanthine.

Fig 4: Validation of overexpressed synaptosomal-associated protein 25 (SNAP25) in Daoy cells. (A) High mRNA level of SNAP25 in cells transfected with pEGFP C3-SNAP25. The increasing SNAP25 mRNA levels in the stable clones were quantified by RT-qPCR. The difference in mRNA levels was compared using the Student's t-test. Data are representative of at least 3 experiments with similar results, and a value of p<0.05 was considered to indicate significance. (B) Expression of SNAP25 protein in cells transfecdted with pEGFP C3-SNAP25. The increasing SNAP25 protein expression was immunoblotted by western blot analysis. GFP, green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig 5: Expression differences of synaptosomal-associated protein 25 (SNAP25) between medulloblastoma and normal brain cells. (A) Comparative mRNA levels of total SNAP25 and variants by RT-qPCR. SNAP25 mRNA levels were quantified in three medulloblastoma (MB) cell lines (Daoy cells: sonic hedgehog subgroup; VGH-Med; D341 Med: group 3 subgroup) and normal brain cells. Each SNAP25 mRNA level in MB cell lines was normalized with their endogenous glyceraldehyde-3-phosphate dehydrogenenase (GAPDH) mRNA level and relative to normal brain cells. All data were then log2 transformed [log2(total SNAP25 of normal brains), 0]. (B) Map of primers for quantifications of total SNAP25 and variants. -, indicates the conserved sequences. (C) Endogenous SNAP25 protein in MB cell lines. Protein levels of SNAP25 (25 kDa) and GAPDH (36 kDa) were detected by western blot analysis. Lanes 1 and 2, normal medulla oblongata from BioChain and Abcam, respectively; lanes 3 and 4, left and right cerebellum from BioChain; lanes 5 and 6, normal brains from Abcam and ProSci; lanes 7 to 9, Daoy, VGH-Med and D341 Med MB cell lines. T, V1, and V2 indicated the amplified regions. T, total SNAP25; V1 or var 1, variant 1; V2 or var 2, variant 2. CDS, coding sequence; 5'-UTR, 5'-untranslated region; 3'-UTR, 3'-untranslated region; ATG, start codon; TAA, stop codon.