Figure 3. Nuclear accumulation of Jmjd3 is required for effective demethylation of H3K27me3. SV40NLS was fused with C-terminal domain of Jmjd3 (Jmjd3-C+SV40NLS). HEK cells were transfected with Flag-tagged Jmjd3-C+SV40NLS or Jmjd3-C. (A) Anti-Flag western blot confirms the expression of these proteins. Pan-histone H3 levels were used a protein loading control. (B) Localization of Jmjd3-C or Jmjd3-C+SV40NLS and level of H3K27me3 were determined by immunofluorescence using anti-Flag antibodies (green) and anti-H3K27me3 antibody (red). Cell nuclei were stained with DAPI (blue). Arrowheads show nuclear H3K27me3 levels in the Jmjd3-C transfectants, whereas arrows show H3K27me3 levels in the Jmjd3-C+SV40NLS transfectants. (C) The signal intensity of H3K27me3 in Jmjd3-C or Jmjd3-C+SV40NLS transfected cells was quantified. Values represent the mean of individual transfectants. n = total numbers of counted cells. The error bars represent one standard error. Statistics were performed using the Student’s t test analysis. (D) Expression of Flag-UTX-C and Flag-UTX-C+SV40NLS was confirmed by anti-Flag western blot. Pan-histone H3 levels were used as a protein loading control. (E) Localization of UTX-C or UTX-C+SV40NLS and level of H3K27me3 were determined by immunofluorescence using anti-Flag antibodies (green) and anti-H3K27me3 antibodies (red). Cell nuclei were stained with DAPI (blue). Arrowheads show nuclear H3K27me3 levels in the UTX-C transfectants, whereas arrows show H3K27me3 levels in the UTX-C+SV40NLS transfectants. (F) The signal intensity of H3K27me3 in UTX-C or UTX-C+SV40NLS transfected cells was quantified. The values represent the mean of the individual transfectants. n = total number of counted cells. Error bars represent one standard error from mean. Statistics were performed using the Student’s t test analysis. (G) Heterogeneous subcellular localization of Jmjd3 in MEFs. MEFs were stained with anti-Jmjd3 antibodies (green) and DAPI (blue). Arrow and arrowhead show cells in which Jmjd3 accumulated in the nucleus and was distributed between cytoplasm and nucleus, respectively.