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.