Extended Data Fig. 8: Direct association of FIP4 to aa. 365-385 of FOXM1 in the IDR1 domain (FOXM1365-385) disrupts FOXM1 LLPS and inhibits the transcriptional activity of FOXM1.
From: Targeting FOXM1 condensates reduces breast tumour growth and metastasis
a, Droplet formation (left) and quantification (right) of GFP-FOXM1 (20 μM) mixed with FIPs (5 μM); n = 5 fields from one of three independent experiments. b, MST analysis of prokaryotic purified GFP-FOXM1-WT and del-IDR1 (1 μM) binding to FIP4 or FIP3; n = 3 independent experiments. c, MST analysis of prokaryotic purified GFP-FOXM1-WT and IDR1 (1 μM) binding to FIP3 or FIP4; n = 3 independent experiments. d, e, MST binding affinity between FIP3 (d) or FIP4 (e) and the prokaryotic purified GFP-FOXM1-WT, D1, D2, D3 (1 μM); n = 3 independent experiments. f, g, MST analysis of prokaryotic purified GFP-IDR1-WT (1 μM) binding to FIP3 (f) or FIP4 (g) -WT or indicated mutants. Replacement of the hydrophobic residue (s) with negatively charged glutamic acid (E), including L378E, F383E, L378E/F383E, V379E or V385E, could maximally disrupt the hydrophobic environment required for interaction; n = 3 independent experiments. h, MST binding affinity between FIP3/FIP4 and FITC-labeled IDR1/p-IDR1 as indicated; n = 3 independent experiments. i, A schematic model describing FIP4 that bind to IDR1, causes electrostatic repulsion and thus dissolves the liquid phase separation of FOXM1. j, Comparison of size exclusion chromatography profiles of GFP-FOXM1 alone (Black) and GFP-FOXM1 mixed with FIP4 (0.5 μg/μl) (Red) run under identical buffer conditions on a SuperdexTM 200 increase 10/300 GL column. Approximately 1 mg GFP-FOXM1 was run on a SuperdexTM 200 analytical gel filtration column and UV traces measured at 280 nm were recorded and analyzed (top). Fractions corresponding to each peak were collected and analyzed on 10% denaturing SDS-PAGE followed by Coomassie staining (bottom). k, MST binding affinity between FKH-DNA and prokaryotic purified GFP-FOXM1 (1 μM) pre-incubated without (PBS) or with indicated FIPs (2.5 μM) for 2 h. l, Quantification of the droplet fuse by GFP-FOXM1 and Cy3-FKH DNA and incubated with indicatd FIPs in Fig. 4f; n = 5 fields from one of three independent experiments. m, Quantified average number of nuclear FOXM1 puncta and the percentages of cells with nuclear FOXM1 puncta were shown in Fig. 4g; n = 3 fields from one of three independent experiments. n, FOXM1-GFP condensates in HeLa cells treated with PBS or FIPs (50 μM) for 16 h (left). Quantified average number of nuclear FOXM1 puncta and the percentages of cells with nuclear FOXM1 puncta were shown (right); cells were fixed; n = 3 regions from one of three independent experiments. o, p, FOXM1-GFP condensates in MDA-MB-468 cells (o) or HCC1937 cells (p) treated with PBS or FIPs (50 μM) for 16 h (left). Quantified average number of nuclear FOXM1 puncta and the percentages of cells with nuclear FOXM1 puncta were shown (right); cells were fixed; n = 3 regions from 3 independent experiments. q, Iodixanol gradient ultracentrifugation analysis of TCL derived from MDA-MB-231 cells treaded with FIP4 (50 μM) for 16 h (top). Quantified FOXM1 band intensities were shown (bottom); n = 3 independent experiments. r, SDD-AGE analysis of FOXM1 aggregation (top) and SDS-PAGE of protein expression (bottom) in MDA-MB-231 cells treated with increasing amount of FIP4 (25 μM, 50 μM) for 16 h. s, Immunoblot (IB) analysis of streptavidin biotin-FKH-DNA pull-down derived from HEK293T cells transfected with Myc-FOXM1 and treated without (-) or with indicated FIPs (50 μM) for 16 h. t, Normalized CDC25B and Cyclin A2 mRNA expression (determined by qPCR analysis) in HEK293T cells transfected with FOXM1 expression plasmids and treated without (PBS) or with the indicated FIPs (50 μM) for 12 h (left).Fold change of TSC22D1-luc and 6DB-luc activity in HEK293T cells treated without or with FIPs (50 μM) for 24 h (right); n = 3 independent experiments. u, qPCR analysis of CENPB, CENPF, CDC25B, KRAS, MYC and TGFBR1 mRNA expressions in MDA-MB-231 cells non-treated (PBS) or treated with FIP4 (50 μM) for 48 h; n = 3 independent experiments. v, Representative images (left) and quantification (right) of tumorspheres at day 14. MCF10A-RAS cells treated without (PBS) or with FIP4 (50 μM) were analyzed in mammosphere assay. The number of mammospheres with a diameter > 60 μm was quantified. Scale bar 50 μm; n = 3 wells from one of three independent experiments. Data are representative of at least three independent experiments (a-h, k-q, t-v). Scale bar, 5 μm (a, n-p). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (a, l-p, t-v). Uncropped gel images are provided in Supplementary Fig. 1.
