Figure 1. Transient low dose treatment of 5-AZA-CdR induces durable and DNA demethylation-independent activation of a gene-set enriched for interferon responsive genes and RIG-1 pathway.

HCT116 cells were transiently treated with 0.3uM of 5-AZA-CdR for 24 hours (1 population doubling time). RNA was extracted before treatment and at day 5, 14, 24 and 42 after drug withdrawal and subjected to cDNA microarray. A) Expression profile for each of the four identified clusters. The solid line represents the median value for each group, while the dotted lines are the lower quartile (lower dotted line) and the upper quartile (upper dotted line) for each group. B) Kernel density plots showing the Pearson correlation (r) between gene expression and DNA methylation for each probe across the 42 days time-course experiment. The y-axis indicates the density of probes. C) Selected examples of genes from Group 1, Group 2, Group 3, and Group 4 are shown. Left y-axis represents the average expression level, and right y-axis represents the average DNA methylation level. The x-axis denotes time (in days) after 5-AZA-CdR withdrawal. Error bars represent the standard deviation D) Three most significant Canonical Pathways enriched at group 4 genes using Ingenuity Pathway Analysis. The significance value for each canonical pathway was calculated by Fisher’s exact test right-tailed. Left y-axis displays the -log of p-value. The orange points represent the ratio between the number of genes in Group 4 that belongs to that specific pathway by the total number of genes annotated to that specific pathway. E) Diagram of the canonical pathway entitled “Role of RIG1-like Receptors in Antiviral Innate Immunity”. Genes highlighted in pink belongs to Group 4. IPS-1 (MAVS) is a central node of the pathway, linking RIG-1/MDA5 receptors to IRF7. See also Figure S1.