Fig. 3: ANLN enhances initiation related Pol II clustering.

A Representative super-resolution and wide field images showing Pol II-pS5 (green) in HeLa cells after 48 h of ANLN knockdown. Scale bar, 3 μm. B The relative number of different types of Pol II-pS5 clusters after ANLN knockdown. n = 45 (si-NC); n = 45 (si-ANLN). The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. C Relative fluorescence intensity of Pol II-pS5 signals per cell per focal plane. n = 45 (si-NC); n = 45 (si-ANLN). The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. D Representative super-resolution images showing Pol II-pS5 (green) in ANLN rapid depletion KYSE150 cells introduced with ANLN WT or NLS mutant. Scale bar, 3 μm. E The relative number of different types of Pol II-pS5 clusters. n = 84 (Control); n = 66 (ANLN KD); n = 81 (ANLN KD + WT); n = 48 (ANLN KD + NLS mut). The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. F Relative fluorescence intensity of Pol II-pS5 signals per cell per focal plane. n = 84 (Control); n = 66 (ANLN KD); n = 81 (ANLN KD + WT); n = 48 (ANLN KD + NLS mut). The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. G Western blot showing the efficiency of ANLN depletion and rescue in KYSE150 cells. H Prediction of intrinsically disordered residues in ANLN and POLR2A protein by PONDR program. I, J The key amino acid regions that mediate the phase separation ability of ANLN were predicted by PSPHunter. K The indicated plasmids were transfected into HEK293T cells for 48 h, the cells were lysed and subjected to immunoprecipitation with HA magnetic beads. L Representative super-resolution images showing Pol II-pS5 (green) in ANLN rapid depletion KYSE150 cells introduced with ANLN WT or PS mutant. Scale bar, 3 μm. M The relative number of different types of Pol II-pS5 clusters. n = 42 (Control); n = 45 (ANLN KD); n = 39 (ANLN KD + WT); n = 51 (ANLN KD + PS mut). The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. N Relative fluorescence intensity of Pol II-pS5 signals per cell per focal plane. n = 42 (Control); n = 45 (ANLN KD); n = 39 (ANLN KD + WT); n = 51 (ANLN KD + PS mut). The error bars indicate the mean ± SDs. Two-tailed Student’s t tests were performed to determine the significance. O Western blot showing the efficiency of ANLN depletion and rescue in KYSE150 cells. The data are representative of at least three independent experiments with similar results. Source data are provided as a Source Data file.