mESCs grown under 3% O2 have an impaired LIF signaling, express less ‘pluripotency-involved’ proteins but maintain pluripotency.

Cells were grown with (+LIF) or without LIF for 1 to 3 or 4 days under normoxia or hypoxia as indicated. (A) Protein cell lysates (60 μg) from cells grown under the indicated conditions were analyzed by Western blot. ERK2 was used as a loading control. A representative experiment is shown. Quantification of signals obtained with the different antibodies in the + LIF condition (n = 4) is provided in S3 Fig (B) Gene expression of the indicated genes was analyzed by RT-qPCR. Graphs represent the average level of expression and SEM as depicted in Fig 2 (n = 4). Paired sample t-test was performed to evaluate the significance of the difference in the expression levels observed in normoxia versus hypoxia: *p-value<0.05; **p-value<0.01, ***p-value<0.001; if not stated: not significant. (C) early lineage markers, (D) specific lineage markers in mESCs induced for in vitro differentiation under normoxia (20% O2, N) or hypoxia (3% O2, H). Gene expression was analyzed by RT-qPCR in cells grown for two days under N or H conditions (+LIFd2), in EBs at day 8 of their formation (EBs d8) and seven days after EBs plating. Y axis is in log scale.