Fig. 5

Epigenetic modulation of RSPO2 impairs the proliferation rate of CRC cell lines. A Schema reflecting the genomic position of the RSPO2 gene, the CpG sites analysed in the 450 K methylation platform and the gRNAs designed to modulate the DNA methylation status of its promoter region. B Line plot illustrating the average methylation status of the CpG sites for the indicated categories. Significantly hypermethylated CpG sites observed in the differential methylation comparisons are highlighted in red. C Boxplot showing the gene expression levels of the RSPO2 gene in control or tumour cases obtained from the TCGA-COAD dataset. D Barplots depicting the percentage of DNA methylation observed for the CpG sites included in the RSPO2 promoter region in DLD1 (top) and HCT116 (bottom) cells in the context of control gRNA (grey) or gRNAs targeting this modulated region in cells transduced with dCas9-TET1 (left)- or dCas9-TET1-IM (right)-related chimeras. E Barplots indicating RSPO2 gene expression levels observed upon epigenetic modulation of its promoter region in DLD1 (top) and HCT116 (bottom) cells in the context of dCas9-TET1- or dCas9-TET1-IM-related chimeras, both in control and RSPO2 targeting RNA conditions. F Boxplots displaying the normalized cell proliferation rate observed for the indicated gRNA treatments at two different time points (24 and 96 h) in the context of DLD1 and HCT116 cells transduced with dCas9-TET1 and dCas9-TET1-IM constructs. For D and E, data represent mean ± standard deviation of at least 3 independent experiments, while for F, at least 8 experimental replicas were included. Two-sided Welch’s t-tests were applied for the different statistical comparisons versus each corresponding control condition. ***p value < 0.001; **p value < 0.01; *p value < 0.05; n.s.—nonsignificant