Fig. 2: DBD is required for PPARγ phase separation.
From: PPARγ phase separates with RXRα at PPREs to regulate target gene expression
a Turbidity assay was used to quantify phase separation of PPARγ truncations (10 μmol/L protein, 150 μmol/L NaCl, 10% PEG-8000). OD600 was normalized to the measurement of mEGFP control (n = 3). Data are shown as means ± SEM. Top, a schematic representation of the PPARγ domains. b Representative fluorescence microscopy images of truncated forms of PPARγ-FL (10 μmol/L protein, 150 μmol/L NaCl, 10% PEG-8000). c Phase diagram of PPARγ-DBD in the presence of different concentrations of NaCl, displaying that phase separation potential of the protein is dependent on salt concentration (10% PEG-8000). d Turbidity assay was used to quantify phase separation of DBD truncated forms of PPARγ-FL (10 μmol/L protein, 150 μmol/L NaCl, 10% PEG-8000). OD600 was normalized to the measurement of mEGFP control (n = 3). Data were shown as means ± SEM. Top, a schematic diagram showing PPARγ with DBD truncated (top) and recombinant mEGFP fusion protein used here (bottom). e Representative fluorescence microscopy images for condensate formation of PPARγ truncations. Cells were transfected with PPARγ truncations for 48 h and imaged, respectively. f The disruption of zinc finger motif impaired the PPARγ phase separation. Representative images of wild-type PPARγ or PPARγ with the disruption of zinc finger motif (PPARγ C>A) fused to mEGFP in droplet formation assay (5 μmol/L protein, 150 μmol/L NaCl, 10% PEG-8000). All cysteines in zinc finger motif were mutated to alanines. g Column scatter charts display average droplet area of each image related to Fig. 2f. Data are shown as means ± SEM (n = 10). h Representative fluorescence microscopy images of a mixture of mEGFP-PPARγ-DBD/mCherry, mEGFP/mCherry-RXRα, mEGFP-PPARγ-DBD/mCherry-RXRα, and mEGFP-PPARγ-LBD/mCherry-RXRα respectively (2 μmol/L protein, 150 μmol/L NaCl, 10% PEG-8000). Scale bar, 5 μm. i Column scatter charts display the droplet diameter in reactions related to panel h. Data are shown as means ± SEM (n = 500). j Phase diagram of PPARγ-DBD in the presence of different concentrations of RXRα (150 μmol/L NaCl, 10% PEG-8000). One-way analysis of variance (ANOVA) for panels a, d, and i. Two-tailed unpaired t-test for panel g. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.
