Fig. 4

Inhibiting PRC2 component affects cell morphology and increases the mRNA expression of cell differentiation marker ZHFX3. a ZFHX3 mRNA expression increases after RA-treatment in three RA-sensitive cells but not the two RA-insensitive cells. The MYCN amplification status and the RA-sensitive or resistant states of five human NB cell lines are given at the top of this panel. X-axis gives the RA-treatment hours, and Y-axis presents the fold change (2^-ddCt) of relative transcript level using RT-PCR at each time point. Data represent mean values ±95% confidence intervals on the estimates of the means from 2 to 6 biological replicates, each with two technical repeats. The significance for fold-change after RA-treatment without blocking PRC2 using the one-tailed Student t-test is represented as: (0.05 ≤ p < 0.1), * (0.01 ≤ p < 0.05), ** (0.001 ≤ p < 0.01). b Genomic view (hg19) of the gene ZFHX3. Three red arrows point reported somatic mutations in HR-NB. The red box highlights a PRC2 occupancy at ZFHX3 promoter in human ESC cells (ENCODE data). c Cell morphology was examined in the LAN5 cell line after 24 h of treatment with vehicle (A1), DZNep (A2), RA (A3), or DZNep and RA (A4), respectively. Pictures were taken using the 20-fold magnification of a Leica DM IRB light microscope. d The ZFHX3 mRNA levels were increased after 8 h of DZNep treatment. X-axis gives the three HR-NB cell lines, and Y-axis presents the DMSO-normalized relative transcript level (RTL). Real-time PCR was employed to examine quantitative differences in mRNA expression between dimethyl sulfoxide (DMSO) and DZNep-treated cells. These relative expression levels (fold change (2^-ddCt)) were initially normalized to the mean of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data shown are mean relative expression levels ± standard deviation of experiments. The p-value of a two-tailed t-test is given in each scenario, followed by the number of biological replicates