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. 2016 Jan 7;5:e09540. doi: 10.7554/eLife.09540

Figure 2. Nup98-HoxA9 evokes the expression of Hox cluster genes in ES cells.

(A) Differential intranuclear localization of Nup98-HoxA9 in mouse ES, HeLa and NIH3T3 cells. Cells were transfected with the EGFP-Nup98-HoxA9 expressing plasmid for 24 hr, fixed, and stained with DAPI. Samples were analyzed using confocal microscopy. Bar, 10 μm. (B) Subcellular localization of Nup98-HoxA9 and its truncated mutants in ES cells. ES cell clones expressing FLAG-tagged Nup98FG, Nup98-HoxA9, or HoxA9-Ct were fixed and stained with an anti-FLAG (polyclonal) antibody. Nuclei were stained with DAPI. Merged images show FLAG staining (green) and DAPI (red). Bar, 10 μm. (C) Cell morphology of stable ES cell lines expressing FLAG-tagged Nup98-HoxA9, Nup98FG, or HoxA9-Ct. Bar, 100 μm. (D) Differentiation assay of stable ES cell clones. ES cells stably expressing FLAG (control), FLAG-tagged Nup98FG, Nup98-HoxA9, HoxA9-Ct, HoxA9, Nup153-HoxA9, or Nup214-HoxA9 were plated at a density of 103 cells per well in 12-well plates either in the presence or absence of LIF. After 5 d, the plates were fixed and stained with alkaline phosphatase, a marker for undifferentiated stem cells. (E) Gene expression profiling of ES cell lines stably expressing FLAG-tagged Nup98-HoxA9 (#1 and #9; two independent clones) and HoxA9-Ct is compared with that of parental ES cells. A greater than 10-fold upregulation of gene expression was commonly observed in both Nup98-HoxA9 clone #1 and Nup98-HoxA9 clone #9 cells. (F) Hox gene expression profiling. The log2 fold ratios of the normalized signal value of Hox cluster genes from Nup98-HoxA9 or HoxA9-Ct expressing ES cells relative to signals from parental control ES cells are indicated. (G) Upregulation of Hox cluster genes in FLAG-Nup98-HoxA9 expressing ES cell lines was confirmed using semi-quantitative polymerase chain reaction. DAPI, 4',6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; ES, embryonic stem; LIF, leukemia inhibitory factor

DOI: http://dx.doi.org/10.7554/eLife.09540.005

Figure 2.

Figure 2—figure supplement 1. Co-staining of Nup98-HoxA9 with various histone marks.

Figure 2—figure supplement 1.

ES cell lines expressing FLAG (control), FLAG-Nup98FG, or FLAG-Nup98-HoxA9 either cultured in the absence or presence of TSA (50 nM, 24 hr) were fixed and stained with antibodies against FLAG (polyclonal) and indicated histone modifications. DAPI staining was used to visualize nuclei. Merged picture of histone modification (red) and FLAG staining (green) are shown. Bar, 5 μm. DAPI, 4',6-diamidino-2-phenylindole; ES, embryonic stem; TSA, trichostatin A. 
DOI: http://dx.doi.org/10.7554/eLife.09540.006
Figure 2—figure supplement 2. Protein expression levels of Nup98-HoxA9 and Nup98FG.

Figure 2—figure supplement 2.

Immunoblotting was performed with antibodies against FLAG (M2), Nup98, and GAPDH using lysates from ES cell lines (equivalent to 105 cells) expressing FLAG (control), FLAG-Nup98FG, or FLAG-Nup98-HoxA9. Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Osaka, Japan) was used to enhance the signal.
DOI: http://dx.doi.org/10.7554/eLife.09540.007
Figure 2—figure supplement 3. Differentiation assay of stable embryonic stem cell clones.

Figure 2—figure supplement 3.

Alkaline phosphatase assay was performed as in Figure 2D. Data are mean values ± standard error of the mean of three independent experiments.
DOI: http://dx.doi.org/10.7554/eLife.09540.008