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Fig. 6
The primitive hematopoietic deficiency caused by eaf1 depletion can be partially rescued by ectopic expression of the dominant negative form of foxo3b (dn-foxo3b). (A) The dominant negative form of foxo3b (dn-foxo3b) behaved similarly to the dominant negative form of human FOXO3a (dn-FOXO3a) as predicted. Overexpression of HA-tagged foxo3b (HA-foxo3b) as well as its active form, HA-tagged foxo3b-TM (HA-foxo3b-TM), activated the FOXO reporter (pGL2-DBE) (lanes 4 and 5) significantly, but overexpression of the HA-tagged dominant negative form of foxo3b (HA-dn-foxo3b) failed to do so (lane 6). The dosages of injected plasmids were pGL2-promoter, pGl2-DBE, 12.5 pg/embryo; HA-empty/foxo3b/foxo3b-TM/dn-foxo3b, 62.5pg/embryo; pRL-SV40, 25.0 pg/embryo. (B) The dominant negative form of foxo3b (dn-foxo3b) suppressed the ability of wild type foxo3b to transactivate the foxo3b reporter (pGL2-DBE) HA-empty/foxo3b/dn-foxo3b, 37.5pg/embryo. (C) Dn-foxo3b interacted with Gata1 in 293 T cells. (D) Dn-foxo3b interacted with Spi1 in 293 T cells. Co-immunoprecipitation assays were performed using plasmids as indicated. (E) Dn-foxo3b restored the suppressive activity of wildtype foxo3b on gata1 reporter. (F) Dn-foxo3b could restore the suppressive activity of wildtype foxo3b on spi1 reporter. (G) O-dianisidine staining indicated that co-injection of dn-foxo3b mRNA partially restored hemoglobin levels in eaf1 morphants (c) compared to co-injection of GFP mRNA control (b) at 52 hpf. Expression of multiple markers hbae3 (f), hbbe1 (i), mpo (l), and l-plastin (o) could also be restored by co-injection of dn-foxo3b mRNA in eaf1 morphants, but not by co-injection of GFP mRNA in eaf1 morphants (e, h, k and n). Eaf1–MO3, 4 ng/embryo; dn-foxo3b mRNA, 820 pg/embryo. (H) Semi-quantitative RT-PCR confirmed the restoration of hbbe1, hbae3, mpo and l-plastin by co-injection of dn-foxo3b in eaf1 morphants (*p<0.05). (I) The expression of injected dn-foxo3b mRNA at 52 hpf embryos was confirmed by Western blot using anti-Myc antibody.
Reprinted from Developmental Biology, 388(1), Hu, B., Zhang, W., Feng, X., Ji, W., Xie, X., and Xiao, W., Zebrafish eaf1 suppresses foxo3b expression to modulate transcriptional activity of gata1 and spi1 in primitive hematopoiesis, 81-93, Copyright (2014) with permission from Elsevier. Full text @ Dev. Biol.