Fig. 5: KLF3 directly regulates AT eosinophil gene expression.

a Metrnl, Penk and Il33 mRNA levels in subcut SVF from a WT and Klf3^−/− mice (n = 4 mice) and b WT, Klf3^−/− (n = 3), WT^WT and WT^Klf3−/− (n = 6) mice. Relative expression was normalised to 18S rRNA and WT values set to 1 for each gene. Levels of secreted c meteorin-like and d IL-33 were measured in supernatants of WT and Klf3^−/− AT explants by ELISA (n = 5 mice). e Expression of thermogenic genes in differentiated preadipocytes co-cultured for 4 h with WT or Klf3^−/− eosinophils (or media alone) was measured by qPCR (n = 4 biological replicates). Relative expression was normalised to 18S and WT eosinophil-treated values set to 1. f Tracks at the promoter and upstream regions of Metrnl, Penk and Il33 were obtained from a KLF3-V5 ChIP-Seq in murine embryonic fibroblasts (GEO Accession No. GSE44748) to assess in vivo KLF3 binding. Scale is shown on the left (KLF3 0-500) and regions are denoted in the top right corner. g CRISPR/Cas9 strategy used to delete KLF3 in EoL-1 cells. h ChIP-qPCR was performed in WT and KLF3^−/− human EoL-1 cells to assess direct binding of KLF3 to Metrnl, Penk and Il33 (n = 3 independent experiments). KLF3 enrichment is normalised to input (non-immunoprecipitated DNA) and a goat IgG control, with human SP1 and VEGFA included as positive and negative control loci respectively. KLF3^−/− EoL-1 cells were used to confirm the affinity of the anti-KLF3 antibody to KLF3 antigen, with no enrichment expected in these cells. For a–d one-sided non-parametric Mann–Whitney U tests were performed where *P < 0.05. For e one-sided non-parametric Mann–Whitney U tests were performed where *P < 0.05 between WT and Klf3^−/− eosinophil co-culture, and ^#P < 0.05 between WT or Klf3^−/− eosinophil co-culture and media alone treatment. For h one-sided non-parametric Mann–Whitney U tests were performed where *P < 0.05 between WT and KLF3^−/− for each region and #P < 0.05 relative to the WT VEGFA negative control. For a–e and g, h, error bars represent means ± SEM. Source data are provided as a Source data file. Eos, eosinophils; kb, kilobase pairs; PAM, protospacer adjacent motif. See also Supplementary Fig. 9.