Fig. 4: ANLN promotes Pol II CTD phase separation in vitro.

A Representative images and statistical data of in vitro droplet assays with different concentrations of CTD-mEGFP (green) or ANLN-mCherry (red). Proteins were incubated in phase separation buffer with or without 10% 1,6-HD. Scale bar, 20 μm. CTD in order n = 0, 29, 53, 78, 199, 209 and 260, respectively. ANLN in order n = 0, 10, 41, 89, 107, 223 and 172, respectively. The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. B Representative images and quantification of droplet photobleaching recovery at the indicated time points. Scale bars, 5 μm. n = 3 for each condition. The error bars indicate the mean ± SDs. C, D Representative images and statistical data of in vitro droplet assays with 5 μM CTD-mEGFP (green) and different concentrations of ANLN-mCherry (red). Scale bar, 20 μm. n = 114, 100, 84, 130, 169, 76, 122, 145, 105, 162, respectively. The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. E Statistics of droplet area. The droplet size of ANLN or CTD alone and the droplet size formed by both were compared. n = 114, 10, 122, 114, 41, 145, 114, 89, 105, respectively. The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. F Droplet assays of CTD (5 μM, green) and ANLN (0.5 μM, red) in the presence of different concentrations of total RNA isolated from KYSE150 cells. Scale bar, 20 μm. n = 80, 159, 155, 155, 226, 120, 82, respectively. The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. G Droplet assays of CTD (5 μM, green) and ANLN (0.5 μM, red) in the presence of different concentrations of genomic DNA isolated from KYSE150 cells. Scale bar, 20 μm. n = 130, 212, 272, 241, 115, 146, 106, respectively. The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. H Coomassie blue staining of purified proteins. I, J Representative images and statistical data of in vitro droplet assays with 5 μM CTD-mEGFP (green) and different concentrations of ANLN-mCherry WT or PS mutant (red). Scale bar, 20 μm. n = 51, 132, 108, 128, 198, respectively. The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. K–M The distribution characteristics of CTD-mEGFP and ANLN-mCherry WT or PS mutant were analyzed using clock scan. The data are representative of at least three independent experiments with similar results. Source data are provided as a Source Data file.