Porcine KLF9 is a direct target gene of TH. (A) The volcano plot revealed that 78 genes were dysregulated, including KLF9, which was predominantly downregulated in the thymuses of hypothyroid mutants. (B) KLF9 and THRB mRNA expression were notably decreased in the thymuses and spleens of hypothyroid pigs compared with those of their WT littermates (WT, n = 15; D409G, n = 10; **P < .01). (C) K562 cells were treated with 100 nM T3 for 1 to 24 hours, and KLF9 expression was measured by qRT-PCR. The data revealed that T3 significantly upregulated KLF9, as well as THRA and THRB. The data are presented as the means ± SD of 3 replications (*P < .05, **P < .01). (D) Schematic representation of the porcine KLF9-truncated promoter. Seven putative TREs located in 2 regions (R1 and R2) were predicted. (E) Luciferase assay of the reporters containing R1 or R2 of the truncated KLF9 promotor in 293T cells. Compared with the cells cotransfected with R2 and empty vector (pcDNA3.1), both THRA and THRB markedly enhanced the luciferase activity of R2, whereas R1 had no luciferase expression activity (P > .05). (F) The luciferase activity assay revealed that only mTRE7 showed significantly decreased luciferase activity after cotransfection with THRA, compared with the other mTRE2-6. (G) The luciferase activity of TRE7 was significantly elevated in response to any TR isoform, whereas decreased expression of the mutated TRE7 was observed in the presence of both TR isoforms. The data are presented as the means ± SD of 3 replications (**P < 0.01). FDR, false discovery rate; m, mutation; Td, TH deficiency.