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. 2017 Jan 4;12(4):1011–1019. doi: 10.1021/acschembio.6b00958

Figure 3.

Figure 3

Cellular demethylase activity of KDM4A demonstrates a graded response to oxygen concentration in U2OS cells. (a–d). Representative images from immunofluorescence analysis of the oxygen sensitivity of cellular KDM4A. Doxycycline (Dox)-inducible U2OS stable cells were induced to overexpress FLAG-KDM4A WT and incubated at (a) 0.1%, (b) 1.0%, (c) 5.0%, or (d) 21% O2 for 24 h. H3K9me3 signal in induced cells (indicated by arrows) was normalized against signal in uninduced cells and correlated to enzyme activity (Figure S4). (e) Cellular KDM4A H3K9me3 demethylase activity in O2 concentrations from 0.1 to 5.0% relative to 21%. Quantification is based on three biological repeats; error bars denote sd. One-way ANOVA with Dunnett’s multiple comparison test against the result for 21% O2 was performed in GraphPad Prism. N > 50 cells were used for each experiment; cells incubated in each oxygen concentration were processed, imaged, and analyzed simultaneously. (f)(i)Western blot analysis of cell lysates from induced and uninduced U2OS F-KDM4A and parental U2OS cells (P) reveals stabilization of HIF-1α in cells incubated at 0.1–5.0% O2 compared to cells incubated at 21% O2. β-actin was used as a loading control. (ii) Multiplexed fluorescent Western blot (red, anti-KDM4A; green, anti- FLAG and anti-actin) and (g) quantitative graph showing levels of FLAG-KDM4A protein in U2OS F-KDM4A cells treated ± dox and incubated at 0.1–21% O2 for 24 h. The membrane was stained simultaneously for KDM4A, FLAG, and β-actin to enable direct quantification and comparison. P denotes parental U2OS cells, which were used to control for dox-dependent effects. All signals were normalized to the β-actin loading control. Levels of overexpressed FLAG-KDM4A were quantified relative to those in normoxic cells treated with dox. Quantitation is based on three biological repeats; error bars denote sd and two-way ANOVA with Dunnett’s multiple comparison test was performed using GraphPad Prism. (h) Hypoxia does not affect expression levels of KDM4A mRNA. Relative mRNA levels of KDM4A U2OS F-KDM4A cells ± dox incubated for 24 h in 0.1% −21% oxygen, and U2OS parental cells (P) incubated at 1.0 and 21% O2, were analyzed by RT-qPCR. A two-way ANOVA with Dunnett’s multiple comparison was performed using GraphPad Prism; error bars denote sd; N = 2; control= −dox; and 21% O2.