Figure 5: AA+2i increases connectivity of upregulated genes.

(a) Networks generated from overlaying upregulated gene expression data onto STRING database. Nodes, representing genes, are identified by circles and their connections, or edges, by the lines joining them. Commonly regulated nodes cluster together, whereas loosely related nodes are at a greater distance. Size of the nodes is proportional to connectivity. A zoomed-in view of the Nanog subnetwork and its edges is presented. Orange=shared between the AA+2i and AA network, blue=shared between the AA+2i and 2i networks, green=unique to the AA+2i network. (b) Bar graph of upregulated nodes shared by AA (orange) and AA+2i (green). The number of edges for each node is indicated on the y axis. (c) Quantitative reverse transcription–PCR (qRT–PCR) data for expression of Nanog and Kdm3b following 2 days of exposure to AA combined with siRNA targeted against luciferase (si Ctrl) or Kdm3b. Error bars are standard deviation of three biological replicates. Asterisk indicates significance *P<0.05, by t-test. (d) Counts of Nanog-positive colonies obtained from Nanog-inducible pre-iPSC line in the presence of 2i alone as compared with control AA+2i condition. Data from two independent replicates are presented. (e) Venn diagrams of overlap between upregulated DE genes associated with maintenance of pluripotency. Genes in bold represent DE genes with greater than twofold expression change over DMSO. (f) qRT–PCR data over a 48-h time course of pluripotency genes responsive to AA or 2i alone following exposure to AA (orange), 2i (blue) or AA+2i (green). (g) qRT–PCR data for Esrrb expression analysed during a time-course exposure to AA (orange), 2i (blue) or AA+2i (green) or switch to 2i after either 48 h of DMSO or AA.