Fig 1: Depletion of KDM4A/B/C increases trimethylation of H3K9 leading to a decrease in HIF-1α in intermittent hypoxia. MCF7 cells were transfected with combined siKDM4A, siKDM4B, and siKDM4C (referred to as siKDM4) or siCONTROL followed by exposure to normoxia, chronic hypoxia (2% v/v), and intermittent hypoxia (5 min/5 min) over 18 h. mRNA expression of (A) KDM4A, (B) KDM4B, (C) KDM4C, (E) HIF1A, (F) HK2, and (G) PLOD2. All values are normalized to normoxia (Log2 scale). Results are the mean ± SD n ≥ 4 independent experiments. ns = not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. D, nuclear extracts of KDM4A, KDM4B, KDM4C, HIF-1α, H3K9me3, and histone H3 (loading control), and cytoplasmic extracts of HK2 and β-actin (loading control).
Fig 2: Chronic hypoxia increases KDM4 expression but limits H3K9me3 demethylation while intermittent hypoxia increases KDM4 expression and enables H3K9me3 demethylation. Cells were exposed to normoxia, chronic hypoxia, and intermittent hypoxia (5 min/5 min). A, KDM4A, KDM4B, and KDM4C in MCF7, HCT116, and MDA-MB-231 cells. Histone H3 is used as a loading control. B, mRNA levels of KDM4A, KDM4B, and KDM4C in HCT116. 500 ng of (C) KDM4A, (D) KDM4B, and (E) KDM4C recombinant proteins were added to microplate wells stably coated with H3K9me3 substrate and exposed to normoxia, chronic hypoxia, or intermittent hypoxia for 4 h. The relative amounts of demethylated H3K9me3 were determined using an HRP-linked antibody (mean ± SD n = 3). F and G, the same enzyme assay was conducted using a range of (F) KDM4B and (G) KDM4C enzyme concentrations. Results are the mean ± SD n = 2. ns = not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Asterisks above the red dots compare normoxia to intermittent hypoxia. Asterisks below the blue square compare chronic hypoxia to intermittent hypoxia. Complete statistical analysis is presented in Table S1. HRP, horseradish peroxidase.
Supplier Page from Abcam for KDM4/JMJD2 Activity Quantification Assay Kit (Colorimetric)