Fig 3.

G9a represses JAK2 transcription. (A) K562 cells were cotransfected with the pGL4.12-JAK2 promoter (1 μg) and pcDNA3-Flag-G9a (0.5 and 1 μg), pEGFP-G9a-ΔSET (0.5 and 1 μg), pLKO.1 (1 μg) as sh-CTL, and G9a shRNAs (0.5 and 1 μg), along with the β-galactosidase expression plasmid. Cell extracts were assayed for luciferase activity. G9a overexpression or knockdown was confirmed by Western blot analysis. (B) K562 cells were transfected with the pGL4.12-JAK2 promoter and treated with 30 μM hemin at different times or with DMSO for 36 h as a control. The cell extracts were assayed for luciferase activity. (C) Restoration of G9a-mediated JAK2 transcriptional repression by BIX 01294. The pGL4.12-JAK2 promoter (1 μg) and pcDNA3-Flag-G9a (1 μg) were cotransfected into K562 cells. Twenty-four hours after transfection, BIX 01294 (1.5, 5, and 10 μM) was treated for 24 h, and the luciferase activity was measured. Luciferase activities were normalized to those of β-galactosidase. The pcDNA3 empty vector was used as a negative control and added to maintain equal amounts of total transfected DNA. All data are representative of at least three independent experiments and presented as means ± SD (n = 4). **, P < 0.01; ***, P < 0.001.