Fig. 4

Frrs1b is downstream ofklf3 in the regulation of erythropoiesis. (A) Upper panels, the expression of frrs1b was increased in klf3 morphants at 24 hpf; middle panels, the expression of frrs1b was decreased in klf3 mRNA-injected embryos. The arrowheads indicate the expression of frrs1b in the ICM; lower panels, be1-globin expression was attenuated in frrs1b overexpressed embryos at 36 hpf, similar to the phenotype of klf3 knockdown embryos. The arrowheads mark the expression in the ICM. (B) qPCR results showed that frrs1b or be1-globin expressions were decreased when overexpression of klf3 or frrs1b, respectively (mean±SD, t-test, ***P<0.001, n=3). (C) The o-dianisidine staining was partially rescued when klf3 morphants were injected with frrs1bMO at 2 dpf. (D) The conserved core binding site of Klfs. (E) The schematic structure of the frrs1b promoter that was used in the reporter and ChIP assays. (F) ChIP-PCR. frrs1b-NF/NR are negative control primers that were used to amplify the frrs1b promoter region without the conserved Klf binding site. The frrs1b-F/R primers were used to amplify the promoter region containing the conserved Klf3 binding site. (G) Overexpression of zebrafish Klf3 in HEK293T cells. Actin was used as a loading control. Klf3 protein expression was detected with anti-FLAG antibody. (H and I) The luciferase activity of tested samples was normalized to the control, and the relative luciferase activity was calculated from three independent experiments (mean±SD, t-test, *P<0.05, n=3). HEK293T cells were co-transfected with the Renilla reporter plasmid, the frrs1b promoter construct or a mutant frrs1b promoter construct in which the Klf3 binding site CACC was changed to ACAA, and pCDNA-FLAG-Klf3. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System.