Thermal stability of FGF affects their ability to stimulate ERK phosphorylation. (A): ERK phosphorylation correlates with the activation of FGF receptors in human embryonic stem cells (ESCs). H1 cells were incubated in E8 media (100 ng/ml FGF2 and 2 ng/ml TGFβ) for 30 minutes with drug treatments (10 μM SU5402—FGF receptor [FGFR] inhibitor or 10 μM SB43542—TGFβ inhibitor, or both). Proteins were harvested to analyze ERK1/2 phosphorylation (pERK1/2) by Western blot. (B): Inhibition of ERK phosphorylation suppresses the expression of pluripotency marker NANOG. H1 ESCs were incubated with 10 μM U0126 (MEK inhibitor), and NANOG expression was measured after 3 days of incubation. (C): Screening for FGFs supporting sustained ERK phosphorylation. H1 ESCs were plated into basic media (E8 media without TGFβ) with different FGFs (100 ng/ml) for 24 hours, and proteins were then collected to detect ERK phosphorylation. (D): Screening for FGFs supporting pluripotency. H1 cells were maintained in the same media as (C) for 3 days, cells were harvested to measure the expression of pluripotency marker NANOG by RT-qPCR. GAPDH was used as control. (E): Screening for FGFs that stimulate ERK phosphorylation in short exposure. Media used in (C) were applied for 15 minutes on FGF-starved ESCs, before proteins were collected to analyze for ERK phosphorylation. FGF18 was also tested for its activity in 15-minute incubation (Supporting Information Fig. S1A). (F): Exam thermal stability of FGF in activating ERK phosporylation. Media used in (C) were incubated at 37°C for 6 hours, and then applied for 15 minutes on FGF-starved ESCs, before proteins were collected for ERK phosphorylation analysis. Abbreviations: ERK, extracellular-signal-regulated kinase; FGF, fibroblast growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MEK, map-erk kinase; RT-qPCR, reverse transcription – quantitative polymerase chain reaction; TGF, transforming growth factor.