Fig. 2: Evidence that KDM3A catalyses H3K9ac hydroxylation in cells.
From: KDM3A catalyses the oxidation of acetyl-lysine to hydroxyacetyl-lysine on histone H3K9
a, Western blots of calf thymus histones incubated with different concentrations of recombinant KDM3ACD and of purified HEK293T histones incubated with equimolar KDM3ACD, KDM3AFL or KDM3BFL, under standard assay conditions. Histones were analysed using PTM-specific antibodies, and anti-histone H4 was used as a loading control. b, Western blot of whole-cell lysate from HEK293T cells transiently transfected with KDM active (WT) or inactive (Mut) constructs: HA-tagged C-terminal regions (including C2HC4 and JmjC domain) of KDM3A, KDM3B and JMJD1C, and Flag-tagged full-length KDM3A. Cell lysates were analysed using histone PTM-specific antibodies, and anti-histone H4 and Ponceau S staining were used as loading controls. c–e, Immunofluorescence of HEK293T cells transfected with KDM active (WT) or inactive (Mut) HA-KDM3ACD-WT/Mut. Fixed HEK293T cells were incubated with HA and either H3K9acOH (c), H3K9ac (d) or H3K9me2 (e) antibodies, and co-stained with DAPI (nucleic acid). Scale bars are shown on the DAPI images. White arrowsheads indicate transfected HA-positive cells. Consistent with the western blot analysis, the HA signal of KDM3A Mut was weaker and more diffuse in the nucleus of transfected HEK293T cells, compared to KDM3A WT, an observation suggestive of reduced exogenous KDM3A Mut stability. f, Summary of immunofluorescence data (c–e) between transfected HA-KDM3ACD WT or Mut for PTMs H3K9acOH, H3K9ac and H3K9me2. Data are presented as mean values ± s.d. of three biological replicates (n > 2,000 cells per replicate). Statistical significance was determined using a two-tailed t-test: H3K9acOH (P < 0.001), H3K9ac (P = 0.022), H3K9me2 (P = 0.001): *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
