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. 2021 Oct 11;125(12):1647–1656. doi: 10.1038/s41416-021-01571-y

Fig. 4. Effects of the combination of 5-aza-dC and TBT on neuroblastoma differentiation.

Fig. 4

a Schedule of the combined treatment of 5-aza-dC (0.1, 0.3 or 1 µM) and TBT (1 or 10 µM). b Representative immunofluorescence staining data. Expression of ßIII-tubulin was enhanced in NB-1 cells by pre-treatment with 1 µM of 5-aza-dC for three days prior to exposure to 1 µM of TBT. Scale bar = 100 µm. c Quantification of neurite length and number in NB-1 cells. The number of neurites per cell increased by 5-aza-dC treatment prior to TBT treatment. d Induction of gene expression by 5-aza-dC, TBT and combined treatment. Expression levels (log2 values) of 50,739 probes were obtained by an expression microarray, and drug-treated cells (y-axis) were compared with non-treated cells (x-axis). Genes with no change were plotted on the black dashed line, and genes upregulated two-fold or more and with expression > −1.0 after a drug treatment are surrounded by a trapezoid with red dashed lines. The numbers of upregulated genes for 5-aza-dC, TBT and combined treatment were 373, 184 and 511, respectively. e Numbers of genes overlappingly upregulated by the treatment of 5-aza-dC, TBT and their combination. f Expression of neuronal markers in the seven cell lines. Additional or synergistic effects were observed by the combination of 5-aza-dC and TBT (1.0 µM 5-aza-dC and 10 µM TBT for NB-1; 0.3 µM and 1.0 µM for NB-39nu; 0.3 µM and 10 µM for IMR-32, SH-SY5Y, BE(2)-C and TGW; 0.1 µM and 1.0 µM for KELLY). Each RT-qPCR analysis was performed in triplicate, and the results are presented as mean ± SE. n.s. non-significant. g Expression of the genes involved in RA signaling in seven cell lines. The expression of CYP26B1, S100A6, HIC1 and CDH11 were additively or synergistically upregulated by the combination of 5-aza-dC and TBT treatment (CYP26B1 expression: 0.3 µM 5-aza-dC and 1.0 µM TBT for NB-1, NB-39nu, IMR-32 and TGW; 0.3 µM and 10 µM for SH-SY5Y and BE(2)-C; 0.1 µM and 1.0 µM for KELLY) (Other genes: 1.0 µM 5-aza-dC and 10 µM TBT for NB-1; 0.3 µM and 1.0 µM for NB-39nu and SH-SY5Y; 0.3 µM and 10 µM for IMR-32 and TGW; 0.1 µM and 1.0 µM for KELLY). Values represent the mean ± SE of three experiments. n.s. non-significant.