Skip to main content
. 2015 Sep 22;290(47):28107–28119. doi: 10.1074/jbc.M115.662197

FIGURE 4.

FIGURE 4.

Transactivation of Etv2 by the Vegf/Flk1-Calcineurin-NFAT3 cascade. A, the Etv2 minireporter is responsive to Vegf stimulation in 293T cells. Mutation of the E#1–3, Gata, or Smad motifs has no effect on Vegf stimulation. However, mutation of the CRE#1–3 motifs or Ets#1–4 motifs results in loss of response to Vegf stimulation. *, p < 0.05. Ctrl, control; Luc, luciferase. B, mutation of the Ets#2 or Ets#3 motif has no effect on Vegf stimulation in 293T cells. However, mutation of the Ets#1 or #4 motif attenuates the transactivation of Etv2 minireporter stimulation by Vegf. The reduction is further enhanced when all Ets#1–4 motifs were mutated. *, p < 0.05. C, the Etv2 minireporter was transactivated by CnA* ∼7-fold in 293T cells. Mutation of the E#1–3, Gata, or Smad motifs does not affect the transactivation by CnA*. Mutation of the CRE#1–3 motifs or Ets#1–4 motifs results in a reduction of transactivation (∼2-fold) or complete loss of transactivation, respectively. The myoglobin promoter (Mb pmt) reporter serves as a positive control for the transcriptional activation of CnA*. *, p < 0.05. D, mutation of Ets#1 resulted in a severe reduction of transactivation by CnA* (∼1.5-fold), and mutation of Ets#4 impaired the activity (∼4-fold) in 293T cells. However, mutation of the Ets#2 or #3 motif has no significant effect on transactivation by CnA*. Mutation of all Ets #1–#4 motifs resulted in complete loss of transactivation. *, p < 0.05. E, Nfat3 transactivates the Etv2 minireporter ∼6-fold. Mutation of the Ets#1 motif results in complete loss of activity, and mutation of the Ets#4 motif attenuates the activity (2-fold) in 293T cells. Mutation of Ets#2 or Ets#3 has no impact on transactivation. Mutation of all Ets#1–#4 motifs completely abolishes transcription activation. *, p < 0.05. F, ChIP assays using Nfat3 reveal that Nfat3 binds to CRII, whereas the interaction with CRI is minimal in wild-type EBs on day 4. *, p < 0.05. G, experimental schematic in which the cell line harboring the Etv2 minireporter is differentiated and treated with vehicle (DMSO), CSA, or FK506 from EB D2–4. EBs were harvested on D4 to capture peak Etv2 expression. H, Etv2 expression is decreased significantly relative to Gapdh with treatment of either CSA or FK506 compared with control DMSO treatment. ***, p < 0.001. I, ZsGreen reporter expression is decreased significantly relative to Gapdh with treatment of either CSA or FK506 compared with control DMSO treatment. **, p < 0.01.