Figure 2 from Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation

Abstract

<p>DNA binding by the HOXA9 homeodomain of NHA9 influences puncta morphology and behavior. <b>A,</b> Representative confocal microscopy image of live HEK293T cells expressing G-NHA9–ΔDNA (green). DNA is stained with Hoechst dye (blue). An overlay of the G-NHA9–expressing cell from <a href="#fig1" target="_blank">Fig. 1B</a> is included for comparison (right). <b>B</b> and <b>C,</b> Plots of puncta # (/10<sup>3</sup> μm<sup>3</sup>; <b>B</b>) and <i>V<sub>p</sub></i> (μm<sup>3</sup>; <b>C</b>) versus [G-NHA9 construct] for G-NHA9 (green) and G-NHA9–ΔDNA (red) from data represented in <b>A</b>. Data are plotted on a semi-log (<i>y</i>-axis: log<sub>10</sub>) scale. <b>D,</b> Still images of multiple time points taken from a time-lapse confocal fluorescence microscopy video (Supplementary Video S2) of a fusion event in an HEK293T cell expressing G-NHA9–ΔDNA. <b>E,</b> Confocal micrographs of FRAP of a G-NHA9–ΔDNA punctum in HEK293T cells at different time points after photobleaching (left). Fluorescence recovery curves are shown for bleached (red, right) and unbleached puncta (black, right). The recovery curve for G-NHA9 is also provided for comparison (green). Individual puncta were manually tracked at different times, and the G-NHA9–ΔDNA fluorescence intensity versus recovery time was plotted as the mean ± SD (<i>n</i> = 20). The pairwise <i>P</i> value for the recovery curves between G-NHA9 and G-NHA9–ΔDNA is 2.2 × 10<sup>−16</sup> using the <i>t</i> test. <b>F–H,</b> Plots of the concentration of the NHA9 construct in the nuclear light phase ([LP], μmol/L; <b>F</b>) and within puncta (termed the dense phase; [DP], μmol/L; <b>G</b>), and the <i>K<sub>p</sub></i> (${K_p}{\rm{\ }} = {\rm{\ }}\frac{{[ {{\rm{DP}}} ]}}{{[ {{\rm{LP}}} ]}}$ (<b>H</b>) versus [G-NHA9 construct] for G-NHA9 (green) and G-NHA9–ΔDNA (red). Data are plotted on a semi-log (<i>y</i>-axis: log<sub>10</sub>) scale. <b>I,</b> 1D-density distribution of PCC per cell for G-NHA9 and G-NHA9–ΔDNA to analyze the linear relationship of the signal between mEGFP and Hoechst. Refer to Supplementary Table S2 for mean values ± standard error. The pairwise <i>P</i> values between G-NHA9 and G-NHA9–ΔDNA are shown in each plot (<b>B</b>, <b>C</b>, <b>F–I</b>; see Methods; <i>n</i> = 935 and 780 in <b>B</b>, <b>F</b>, and <b>I</b> including the cells with zero punctum, and <i>n</i> = 378 and 254 in <b>C</b>, <b>G</b>, and <b>H</b> excluding the cells with zero punctum, respectively, for G-NHA9 and G-NHA9–ΔDNA).</p>

Related Publications