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. 2015 Apr 23;33(5):1390–1404. doi: 10.1002/stem.1926

Figure 4.

Figure 4

Reprogramming mouse embryonic fibroblast cells (MEFs) by 6F is both retinoic acid (RA) receptor ligand‐dependent and ligand‐independent. (A): MEFs were reprogrammed by 4F alone, 4F plus either RARG or LRH1, or 4F plus both RARG and LRH1 (6F) in +VA and −VA medium. Significantly more AP+ colonies are obtained by 6F, even in −VA. (B): Blocking RA signalling by citral (50.0 μM) or CD2665 (1.0 μM) as measured in a luciferase assay. (C): Blocking 4F‐ or 6F‐mediated MEF reprogramming by citral (50.0 μM). Adding ATRA rescues reprogramming. Note that 6F produces substantially more AP+ colonies in the presence of citral compared with the other conditions. (D): The blocking of MEF reprogramming by RARA‐DN. (E): The effects of blocking RA signalling by either citral (50.0 μM) or RARA‐DN on MEF reprogramming. Note that adding citral and expressing RARA‐DN almost completely blocked reprogramming. Adding ATRA at 10.0 nM could partially rescue the block. In the MEF reprogramming experiments, AP+ colonies were scored on day 18 after transfection. *, p < .05; **, p < .01. The experiments were repeated at least three times, and the error bars show standard deviations from the mean of triplicate determinations in one representative experiment. Abbreviations: AP, alkaline phosphatase; ATRA, all‐trans retinoic acid; LRH1, liver receptor homolog‐1; RARA‐DN, dominant‐negative form of RARA; RARG, retinoic acid receptor gamma; VA, vitamin A.