Supplementary Data

• Supplementary Materials and Methods - Supplementary Materials and Methods (PDF 197 KB)
• Supplementary Table 1 - RT-qPCR primer list (PDF 155 KB)
• Supplementary Figure 1 - Amino acid sequence alignment (by ClustalX1.8) of Foxo3 homologues in the orange-spotted grouper, zebrafish, and human. The identical, highly conserved, and less conserved amino acid residues were indicated at the bottom by *, :, and ., respectively. The conserved structural motifs were also shown and indicated by arrowheads at the top. The nuclear localization signal (NLS) and the nuclear export signal (NES) were gray shaded and indicated at the bottom. The Akt consensus phosphorylation sites were marked by white letters against a black background, and the motifs for Akt phosphorylation, R-X-R-X-X-S/T-Hyd, were indicated by underlines, where 'X' is any amino acid and ‘Hyd’ is a bulky hydrophobic residue. The protein sequences were downloaded from Entrez (NCBI). For details, please refer to Figure 1. (PDF 48 KB)
• Supplementary Figure 2 - The schematic diagram showing the genomic structures of foxo3 homologues in the orange-spotted grouper, zebrafish, and human. The exons are boxed and indicated by Roman numerals at the bottom, and the introns are depicted by lines. The translated exons are shaded. The Arabic numeral above the parentheses of the foxo3 gene structures represented the nucleotides cut off. The orange-spotted grouper foxo3a and foxo3b genomic structures were determined by comparing the corresponding cDNA sequence cloned and genomic DNA sequence (from the draft genome sequence of the orange-spotted grouper, and provided with courtesy by Dr Yong Zhang). The genomic structures of foxo3 homologues in zebrafish and human were determined by sequences downloaded from Entrez (NCBI) with following accession numbers: NW_001877750 (zebrafish foxo3a), NC_007131 (zebrafish foxo3b), and NC_000006 (human Foxo3). (PDF 231 KB)
• Supplementary Figure 3 - Specificity of anti-grouper Foxo3a or Foxo3b antiserum as determined by Western blot analysis. The proteins were separated on 12% SDS-PAGE gels, transferred to polyvinylidene fluoride membranes, and then immunoreacted with: A, the mouse anti-grouper Foxo3a antiserum (1:1000); B, anti-Foxo3a antiserum pre-absorbed by excessive EcFoxo3a; C, the mouse anti-grouper Foxo3b antiserum (1:1000); or D, anti-Foxo3b antiserum pre-absorbed antiserum by excessive EcFoxo3b. EcFoxo3a antigen, the recombinant Foxo3a fusion polypeptide used to immunize mouse; EcFoxo3b antigen, the recombinant Foxo3b fusion polypeptide used to immunize mouse; EcFoxo3a and EcFoxo3b, the nuclear protein extracts of COS-7 cells transfected with the pcDNA3.0-derived expression constructs for the full length Foxo3a and Foxo3b polypeptides, respectively; pcDNA3.0, the protein extracts of COS-7 cells transfected with the empty pcDNA3.0 vector; The secondary antibodies were 1:5000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) (catalog number: 115-035-003, Jackson ImmunoResearch Laboratories, Inc., PE, USA). The blots were visualized using BeyoECL Plus kit (Beyotime). The molecular standards identified with relative molecular mass (Mr×10⁻³) were shown to the left of images. (PDF 1302 KB)
• Supplementary Figure 4 - Negative controls for Foxo3a (A~E) and Foxo3b (F~J) immunoreactivities in the ovary of the orange-spotted grouper. The sections from the same ovarian tissues as shown in Figure 4 were immunoreacted with the anti-Foxo3a (1:300) and anti-Foxo3b antiserum (1:200) pre-absorbed by excessive EcFoxo3a and EcFoxo3b proteins respectively, and visualized by DAB chromogen. Scale bar is 25 μm. (PDF 990 KB)
• Supplementary Figure 5 - Western blot analysis of the orange-spotted grouper Foxo3a (A) and Foxo3b (B) expression in nuclear extracts of COS-7 cells. 1, un-transfected cells; 2, transfected with the pcDNA3.0 empty expression vector; 3, transfected with the Foxo3a- or Foxo3b-expression vector. The nuclear protein extracts were separated on 12% SDS-PAGE gels, transferred to polyvinylidene fluoride membranes. The antiserum against Foxo3a (1:1000), Foxo3b (1:1000), or mouse anti-ACTB monoclonal antibody (1:500; catalog number: 60009-1-Ig; ProteinTech Group, Inc.) was used as the primary antibody. The secondary antibody was 1:5000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) (catalog number: 115-035-003; Jackson ImmunoResearch Laboratories, Inc.). The blots were visualized using BeyoECL Plus kit (Beyotime). The molecular standards identified with relative molecular mass (Mr×10⁻³) were shown to the left of images. (PDF 137 KB)
• Supplementary Figure 6 - The competition test in gel-shift analysis of Foxo3a binding to the putative Foxo site in the orange-spotted grouper cyp19a1a promoter. The assay was set up essentially the same as Figure 8 except that the cold probe was included in the reaction with 50-, 100-, 200-, and 500-fold excess respectively as indicate by a triangle at the top. The specific band became fainter with increasing amounts of excessive cold probe and almost disappeared at 500-fold excess. W, wild-type nucleotide probe; M, mutated nucleotide probe; C7, the nuclear extracts of COS-7 cells; P3, the nuclear extracts of COS-7 cells transfected with pcDNA3.0 plasmid; EcFox3a, the nuclear extracts of COS-7 cells transfected with Foxo3a expression vector; Np, without addition of COS-7 nuclear proteins; Nu, without addition of unlabeled probes. (PDF 280 KB)
• Supplementary Figure 7 - ChIP analysis of Foxo3a and Foxo3b binding to the upstream promoter regions (-2226/-1921 bp and -1024/-687 bp) of the orange-spotted grouper cyp19a1a in the ovary at different developmental stages. The DNA fragments were immunoprecipitated using specific antibodies against Foxo3a, Foxo3b, RNA polymerase II, or normal mouse IgG, and analyzed with PCR amplification of 38 cycles. The PCR products were separated on 2.0% agarose gels and visualized by staining with ethidium bromide. M, DL1000 bp DNA marker (Takara, Bio Inc., Shiga, Japan); EVO, early vitellogenic stage ovary; MVO, mid-vitellogenic stage ovary; MO, mature ovary; Li, liver. The experiments were repeated at least twice, and similar results were obtained. (PDF 440 KB)