Figure 2: Different temporal requirements for AA and 2i.
From: Collaborative rewiring of the pluripotency network by chromatin and signalling modulating pathways
(a) Top panel: Scheme of experiment: GFP-negative cells (GFP-) were sorted by flow cytometry on the indicated days into wells containing DMSO or AA+2i and analysed for Nanog-GFP-positive cells on day 10 and day 13. Bottom panel: Nanog-GFP-positive cells derived from Nanog-GFP-negative cells sorted on days indicated on the x axis, cultured in DMSO or AA+2i, and analysed on day 10 (solid bars) or day 13 (hatched bars). Individual bars represent independent replicates. (b) Scheme of experiment (left panel) indicating the number of days of AA exposure when 2i was retained for all 10 days and corresponding Nanog-GFP-positive conversion rates obtained (right panel). Error bars represent standard deviation of four replicates. (c) Scheme of experiment (left panel) indicating the number of days of 2i exposure when AA was retained for all 10 days and corresponding Nanog-GFP-positive conversion rates obtained on day 10 (right panel). Error bars represent standard deviation of four replicates. (d) Scheme of experiment (left panel) indicating the number of days of AA exposure alone followed by switch to medium containing 2i alone and corresponding Nanog-GFP-positive conversion rates obtained (right panel). Error bars represent standard deviation of four replicates. (e) Scheme of experiment (left panel) indicating the number of days of 2i exposure alone followed by switch to medium containing AA alone and corresponding Nanog-GFP-positive conversion rates obtained (right panel). Error bars represent standard deviation of four replicates. (f) Nanog-GFP-positive conversion rates obtained when pre-iPSCs were exposed to AA and individual components of 2i—Meki and Gski. Error bars represent standard deviation of three biological replicates. (g) Nanog-GFP-positive conversion rates obtained when pre-iPSCs were exposed AA for initial 2 days and then switched to individual or combined components of 2i for 8 days. Error bars represent standard deviation of three biological replicates.
