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.