Extended Data Fig. 1: FOXM1 undergoes phase separation.

a, Images of GFP-FOXM1 droplets formation (left), quantification (middle) and phase separation (PS) diagram (right) at room temperature with the indicated NaCl concentrations (25 μM GFP-FOXM1 at pH 7.0); n = 5 fields from 3 independent experiments. b, Images of GFP-FOXM1 droplets formation (left) and quantification (right) at room temperature with the indicated pH (25 μM GFP-FOXM1 in 300 mM NaCl); n = 5 fields from 3 independent experiments. c, GFP-FOXM1 (25 μM) were treated with 5% 1,6-hexanediol (1,6-HD) and then subjected to droplet formation assay in vitro (300 mM NaCl, pH 7.0, room temperature); n = 5 fields from 3 independent experiments. d, Time-lapse micrographs and equivalent diameter (EqDiameter) of AF488-labelled FOXM1 (50 μM) liquid droplets formed at the indicated times with 300 mM NaCl at pH 7.0 at room temperature. e, Representative micrographs (left) and fluorescence recovery after photobleaching (FRAP). Quantification over a 50 s time course at room temperature (right) of GFP-FOXM1 (50 μM) droplets; n = 3 drops from 3 independent experiments. f, Quantified percentages (left) and average number (middle) of nuclear FOXM1 puncta as in Fig. 1g, n = 3 regions from 3 independent experiments; quantified average puncta size per cell (right) as in Fig. 1g, n = 20 cells from 3 independent experiments; cells were fixed. g, Quantified percentages (left) and average number (right) of nuclear FOXM1 puncta as in Fig. 1h, n = 5 regions from 3 independent experiments; cells were fixed. h, Endogenous FOXM1 staining with anti-FOXM1 antibody (green) in MDA-MB-231 cells treated with DMSO or 10% 1,6-HD for 5 min; n = 3 regions from 3 independent experiments; cells were fixed. i, Representative micrographs (left) and quantification (right) of purified GFP-FOXM1 wild-type (WT) and GFP-FOXM1 depleted of its N-terminalautorepressor domain (NRD), forkhead domain (FHD), intrinsically disordered region (IDR)1, IDR2 and transactivation domain (TAD) domain (25 μM); n = 5 fields from 3 independent experiments. j, Related to Fig. 1j: quantified average number of nuclear FOXM1 puncta and the percentages of cells with nuclear FOXM1 puncta were shown; n = 5 regions from 3 independent experiments. k, Images of GFP-FOXM1-IDR1 droplets formation (left) and quantification (right) at room temperature with the indicated GFP-FOXM1-IDR1 concentrations (300 mM NaCl at pH 7.0); n = 5 fields from 3 independent experiments. l, m, Images of unlabeled (l) or AF488-labelled (m) FOXM1-IDR1 droplet formation (left) and quantification (right) at room temperature with the indicated protein concentrations with 300 mM NaCl at pH 7.0; n = 5 fields from 3 independent experiments. n, Iodixanol gradient ultracentrifugation analysis of TCL from HEK293T transfected with Myc-FOXM1 WT or d_IDR1 (top). Aliquots of the fractions were immunoblotted with anti-Myc antibody (bottom left). The band intensities were shown (bottom right). n = 3 independent experiments. o, SDD-AGE analysis of FOXM1 aggregation (top) and SDS-PAGE (bottom) of the TCL derived from HEK293T cells transfected with Myc-FOXM1 WT or d_IDR1. p, MST binding affinity between prokaryotic FOXM1-WT and FOXM1-WT (1 μM); FOXM1-WT and FOXM1-del_IDR1 (1 μM); FOXM1-del_IDR1 and FOXM1-del_IDR1 (1 μM); n = 3 independent experiments. q, MST binding affinity between FOXM1-IDR1 and FOXM1-WT (1 μM) or FOXM1-del_IDR1 (1 μM); n = 3 independent experiments. r, SDD-AGE analysis of endogenous FOXM1 aggregation (top) and SDS-PAGE (bottom) of the TCL derived from MDA-MB-231 cells. s, Purified FOXM1 WT or del_IDR1 proteins were imaged by electron microscopy using negative staining (left). Quantified average fiber length of proteins were shown as indicated (right); n = 5 fields from 3 independent experiments, Scale bar, 200 nm. t, Fluorescence of GFP-FOXM1 (20 μM) and Cy3-FKH DNA (500 nM) mixed at room temperature with 300 mM NaCl at pH 7.0 (top). Quantitative line profile of colocalization along a white arrow of the left image (bottom). u, GFP-FOXM1-d_NRD, FOXM1-d_IDR2, FOXM1-d_TAD (green) and Cy3-FKH DNA (FOXM1 forkhead DNA binding consensus sequence) (magenta) were mixed with the indicated module concentration and were imaged for fluorescence (left). PS diagram of different concentrations of indicated FOXM1 proteins with Cy3-FKH DNA (right). v, Quantitative (left) and representative images (right) of FRAP assay of GFP-FOXM1 WT (20 μM) with Cy3-FKH DNA (500 nM) (top) or GFP-FOXM1 WT (20 μM) alone (bottom); n = 3 drops from 3 independent experiments. w, Fold change of reporter gene activation in HEK293T cells transfected with TSC22D1 promoter- and 6DB promoter- luciferase (luc) constructs plus either empty vector, FOXM1 WT or FOXM1 d_IDR1 (left); quantitative PCR (qPCR) analysis of CDC25B and CyclinA2 mRNA level in HEK293T cells transfected with FOXM1 WT or FOXM1 d_IDR1 expression plasmids (right); n = 3 independent experiments. Data are representative of at least three independent experiments (a-c, e-n, p, q, s, v, w). Scale bar, 5 μm (a-e, h, i, k-m, t-v). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (a-c, f-m, s, w), two-way ANOVA (v). Uncropped gel images are provided in Supplementary Fig. 1.

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