Figure 7: Convergence of AA and 2i effectors to regulate Esrrb level.

(a) Top panel—Scheme of the experiment: siRNA transfections targeting Esrrb or control (anti-luciferase) were performed on days −1, +1, +4 and +7, after addition of AA on day 0. On day 2, media were switched to that containing 2i alone. Bottom panel—Quantification of Nanog-GFP-positive cells obtained on day 10 of the experiment upon knockdown of Esrrb. Error bars represent standard deviation from four replicates. Asterisk indicates significance *P<0.05 assessed by t-test. (b) Top panel—Scheme of the experiment: siRNA transfections targeting Nanog or control (anti-luciferase) were performed on days −1 and +1 of simultaneous exposure to AA and 2i, which began on day 0. Bottom panel—Quantification of Esrrb and Nanog expression by quantitative reverse transcription—PCR (qRT–PCR) following 48 h of AA+2i treatment. (c) Top panel—Scheme of the experiment: siRNA transfections targeting Egfr or control (anti-luciferase) were performed on days −1, and +1 of exposure to AA, which began on day 0. Bottom panel—Quantification of Esrrb and Egfr expression by qRT–PCR following 48 h of AA treatment. Data from three independent siRNAs indicated as #1,#2 and #3 and five, three or one biological replicates, respectively, are presented. (d) Left panel—Kdm3b expression after siRNA transfections targeting control (anti-luciferase), Kdm3b, Egfr and Esrrb were performed on days −1, and +1 of exposure to AA alone (day 2 levels) or AA+2i (day 2 levels) or exposure to AA for 2 days followed by switch to 2i for 2 days (day 4 levels). Middle panel—same as above for Egfr. Right panel—same as above but for Esrrb. Error bars represent standard deviation of two biological replicates. (e) Model for action of AA and 2i during reprogramming. Treatment with AA functions through Kdm3b early and Tet-mediated 5hmC increases late in the reprogramming process. In parallel, AA and 2i activate separate key pluripotency genes. 2i also suppresses signalling effectors like Egfr, which in combination with AA can directly regulate a unique cohort of pluripotency genes such as Esrrb. This results in the complete activation of the pluripotency network (dotted line).