Fig. 9.

Both Cdx2 and Elf5 are down-regulated in response to inhibiting the ERK1/2 pathway in trophoblast stem cells (TS cells). (A) TS cells were grown in complete TS cell medium with or without the potent MEK inhibitor PD184352 for 6, 12 or 24 h; whole-cell extracts were then fractionated by SDS-PAGE and subjected to Western blotting with antibodies to Cdx2, Elf5, P-ERK1/2 and ERK1. Cdx2 and Elf5 protein levels decreased markedly in a time-dependent manner when ERK1/2 activity was inhibited by PD184352, and the down-regulation of Cdx2 was clearly preceding that of Elf5. Blots shown are from a single experiment which was performed twice yielding identical results. (B and C) Normalised real-time (Q)RT-PCR analyses of Cdx2 and Elf5 mRNA levels from two independent sets of experiments (black and gray bars) performed in parallel with the protein studies in (A). (B) When TS cells were maintained in complete TS cell medium, the expression levels of Cdx2 were not or only moderately affected over a time-course of 24 h. When ERK1/2 pathway activity was inhibited by PD184352, Cdx2 mRNA levels decreased drastically compared to their untreated controls at all time points (reduction ~90%). (C) Similarly, Elf5 mRNA levels remained constant in complete growth medium but decreased progressively upon PD184352 treatment. This down-regulation was lagging behind the decrease of Cdx2 levels, starting at 12 h and reaching a maximal reduction of ~85% only after 24 h.