Fig. 5: TAZ regulates SOX2 and OCT4 expression through phase separation with NANOG.
From: Niche stiffness sustains cancer stemness via TAZ and NANOG phase separation
a The intrinsic disorder tendency of TAZ and NANOG was predicted by IUPred and D2P2 algorithms. A score above 0.5 indicates disorder. b Top, solutions containing the indicated protein at 10 μΜ (ΤΑΖ and NANOG) were examined by phase contrast microscopy. Bottom, the indicated EGFP-TAZ and mCherry-NANOG fusion proteins were mixed at 10 μM, examined by confocal microscopy at room temperature. Representative image from 3 biologically independent experiments. Scale bar, 10 μm. c EGFP-TAZ (10 µM) was mixed with mCherry-NANOG (10 µM) and imaged by confocal microscopy. The fusion of EGFP-TAZ droplets was shown (n = 3 biologically independent experiments). Scale bar, 2 μm. d, e, g, h HEK293T cells were transfected with EGFP-TAZ, mCherry-NANOG, mCherry-NANOG-CAD mutation, or mCherry-NANOG-NAD mutation. d, Representative live cell images of the fluorescence recovery after photobleaching (FRAP) experiment are shown. Scale bar, 2 μm. e, Quantification of FRAP analysis in (d). 0 s is the starting of the bleaching event. Plots were generated from 20 droplets in 4 independent experiments. Mean ± SD. f Confocal images of recombinant EGFP-TAZ (10 µM) in the presence of mCherry-NANOG-NAD or mCherry-NANOG-CAD are shown. Scale bars, 10 μm. The image is representative of three independent experiments. g Confocal microscopy images of HEK293T cells transfected with EGFP-TAZ and mCherry-NANOG/ mCherry-NANOG-CAD/ mCherry-NANOG-NAD. White dashed lines denote the contours of the cell nucleus. Scale bars, 5 μm. See Supplementary Fig. 4b for quantification. h Confocal images of TAZ immunofluorescence and Pou5f1, Sox2, and Fgf4 loci by concurrent nascent RNA-FISH in HEK293T cells. White dashed lines denote the contours of the cell nucleus. Merge (zoom) represents a zoomed-in view of the white box. Line plot analyzed the colocalization of RNA FISH foci and TAZ puncta. Scale bar, 2 μm. See Supplementary Fig. 4c for quantification. i Representative images (maximal projections in z) of ALDH1, TAZ, NANOG and DAPI immunofluorescence staining in paired samples of breast cancer patients with sensitive or resistant responses before and after NAC. White box indicates the areas enlarged in the insets. Scale bar, 10 μm. See Supplementary Fig. 4k for quantification.
