Fig. 4: Zfh1 regulates the transcription of FoxO.

a Scheme depicting mesonotum and metanotum (yellow) used for RNA-seq (illustrated by Miss Yi Wan). b Differentially expressed genes in nota of dsZfh1- and dsGfp-treated fifth-instar nymphs. FoxO is indicated by an arrow. c qRT-PCR analysis of FoxO expression in nota of dsZfh1- and dsGfp-treated fifth-instar nymphs. The experiment was repeated five times with similar results (dots). d Scheme of construction of FoxO::HA mutants using CRISPR/Cas9-mediated homology-directed repair. The target site for Cas9 and protospacer-adjacent motif (PAM) are indicated. An ssDNA donor contains a left homology arm, a HA-tag (HA), and a right homology arm. Ex 1–5, exons 1–5 of FoxO. FoxO-KI-F and FoxO-KI-R, primers used for FoxO::HA verification. e Confirmation of FoxO::HA mutants by western blot using an HA antibody. f Confirmation of FoxO::HA mutants by Sanger sequencing with primers of FoxO-KI-F and FoxO-KI-R. g dsZfh1 decreases FoxO::HA protein level. FoxO::HA mutants were microinjected with dsZfh1 or dsGfp, followed by western blot using an HA antibody. β-Actin is used to show equal protein loading in (e) and (g). The experiments in (e) and (g) were repeated three times independently with similar results. h Scheme of immunoprecipitation assay. Red vertical lines and letters, putative Zfh1 consensus binding sites in the Foxo promoter (ProFoxO). TSS, the transcription start site of FoxO. The HEK293T cells are transfected with the ProFoxO fragment mixed with either pCMV-Zfh1-His or pCMV plasmid. i Relative binding of the Foxo promoter to Zfh1 by immunoprecipitation assays. The ProFoxO fragment was immunoprecipitated with antibodies against His tag. The experiments were repeated three times with similar results (dots). Two-tailed unpaired t-test was used for the statistical analysis (c, ****P = 3.48132E−06) and (i, **P = 0.0075). Data are presented as mean ± SEM in (c) and (i). j Venn diagram and heat map of thoracic genes commonly regulated by Zfh1 and FoxO. Thoraxes treated with dsZfh1, dsFoxO, and dsGfp are denoted as Zfh1-T, FoxO-T, and Gfp-T, respectively. Expression levels are indicated by FPKM (fragments per kilobase of transcript per million mapped reads). Source data are provided as a Source Data file.