Fig. 5
Epigenetic memory determination in mESC culture in vitro. a Schematic representation of experimental approach to study the cell-to cell epigenetic memory induced by morphine. Epigenetic changes were measured at 24 h after chronic morphine treatment (P1), and morphine-induced epigenetic memory was evaluated at 48 h (P2) and 96 h (P3) after treatment withdrawal. Representative image of treated and untreated mESCs is also shown in the studied time points. Scale bar = 400 µm. b In vitro dynamic changes of H3K27me3 histone modification induced by 24 h morphine treatment for P1, P2 and P3 after morphine treatment withdrawal in mESCs. β-actin was used as loading control. Sample size n = 5. b Quantification of H3K27me3 levels measured by Image J software.. Statistical significance was determined by Student-T test (*p < 0.05; **p < 0.01; ***p < 0.001), c RT-qPCR analysis for the validation of PRC2 complex subunits expression, Suz12, Ezh2 and Eed were validated at mRNA level (left) and ChIP enrichment level (right). Gapdh and Pcx were used as housekeeping genes for mRNA level analysis and acquired Ct values were normalized respect to the control sample using 2ddCT. d ChIP-RT-qPCR amplification was normalized respect to the 10% of the input sample. Statistical significance was determined by Student-T test (*p < 0.05; **p < 0.01; ***p < 0.001). Sample size n = 5