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. 2021 Sep 22;12:5579. doi: 10.1038/s41467-021-25761-7

Fig. 5. DNA affinity influences KLF4-mediated liquid condensation.

Fig. 5

a Fluorescence microscopy images of 3 µM duplex DNA in TS buffer and 100 nM YOYO-1 with (bottom) or without (top) 10 µM DBD. Similar results were obtained for 2 replicates. b Fluorescence microscopy images of DBD:DNA mixtures at 3:1 and 3:1.5 ratios show that adding cognate DNA can reverse LLPS. Similar results were obtained for 2 replicates. c Mixing DBD and DNAs at different concentrations produces condensates (black circles) or homogeneous solutions (open circles) that define LLPS phase diagrams. Images were scored as condensate if the coefficient of variation was >0.2 and the mean fluorescent intensity was >0.4 arbitrary units. Values were determined using ImageJ from 2–3 independent replicates. Fluorescence images at right are taken at 3 µM DBD and 250 nM DNA, conditions boxed in red in the phase diagrams. d LLPS phase diagrams determined as in (c) but for double mutant DBDE476D/R501A with DNAs. e Fluorescence images of HEK 293T cells expressing KLF4-mTurq (left), KLF4R501A-mTurq (middle) and KLF4E476D/R501A-mTurq (right). Bottom row shows close-ups of single cells with the same mean fluorescence (~5000 AU). Similar results were obtained for 2 biological replicates. f HEK 293T cells expressing KLF4-mTurq variants classified as diffuse (gray, left) or punctate (red) are plotted by mean fluorescence intensity; the long horizontal bars mark the median, and the top and bottom bars on the vertical lines denote the 90-10 percentiles. Wildtype “diffuse” cells show lower fluorescence than those cells expressing KLF4R501A-mTurq or KLF4E476D/R501A-mTurq (n = 112 diffuse cells for each fusion across 2 biological replicates). Cells with at least 5 distinct puncta (0.5 µm spots with intensity center >1500 arbitrary units, determined using Imaris software) were classified as punctate. Statistical tests were performed using a two-sided Student’s paired t-test. Y-axis limit was set to 30,000 for visualization purposes. Fields that yielded 112 diffuse cells also gave punctate cell counts of 209 (WT), 39 (KLF4R501A-mTurq) and 64 (KLF4E476D/R501A-mTurq) across 2 biological replicates.