Extended Data Fig. 2: Hydrophobic residues in IDR1 are essential for FOXM1 LLPS.
From: Targeting FOXM1 condensates reduces breast tumour growth and metastasis
a, Amino acid sequence analysis of IDR1 region. HM, hydrophobic motif. Pink denotes hydrophobic amino acids. IDR1-I (321-343 aa.), IDR1-II (344-364 aa.), IDR1-III (365-385 aa.). b, Percentage of hydrophobic amino acids in three subdomains of IDR1. c, Hydrophobicity profile of IDR1. The graph shows Kyte & Doolittle scale mean hydrophobicity values (scan window = 13). A scheme on the upper shows the three subdomains in IDR1. d, Domain structure of FOXM1-IDR1 truncations or fragment (left). The numbers above indicate the position of amino acid residues. Coomassie blue staining of bacterially purified GFP-FOXM1-IDR1-WT, D1, D2, D3 and III (right). e, MST analysis of IDR1-WT peptide (1 μM) binding to GFP-IDR1-WT, D1 (deletion aa. 321-344), D2 (deletion aa. 344-364), D3 (deletion aa. 365-385) and III (aa. 365-385); n = 3. f, Coomassie blue staining of GFP-FOXM1-IDR1-WT, HM3-4A (P369A/L370A/L371A/P372A), HM4-4A (L378A/V379A/P380A/I380A), HM5-3A (F383A/P384A/V385A) proteins purified from E. coli. g, MST analysis of prokaryotic purified GFP-IDR1-WT (1 μM) binding to IDR1-WT, HM3-4A, HM4-4A, HM5-3A; n = 3 independent experiments. h, Representative images (left) and quantification (right) of phase separation of GFP-FOXM1-IDR1-WT, HM3-4A, HM4-4A, HM5-3A (25 μM) at room temperature with 300 mM NaCl at pH 7.0; n = 5 fields from 3 independent experiments. i, Immunofluorescence and DAPI staining (left) and immunoblot analysis of the expression (top right) of HeLa cells transfected with GFP-tagged FOXM1 WT or HM3-4A, HM4-4A, HM5-3A (left). Quantified average number of the nuclear FOXM1 puncta and the percentage of cells with nuclear FOXM1 puncta were shown (bottom right); cells were fixed; n = 3 regions from 3 independent experiments. j, Coomassie blue staining of GFP-FOXM1-IDR1-WT or indicated single, double or triple mutations which included L370E, L378E, F383E, L370E/L378E, L370E/F383E, L378E/F383E, L370E/L378E/F383E, V379E and V385E proteins purified from E. coli. k, MST analysis of prokaryotic purified GFP-IDR1-WT (1 μM) binding to IDR1-WT or indicated mutants. Replacement of the hydrophobic residue (s) with negatively charged glutamic acid (E) in HM3-5, including L370E, L378E, F383E, L370E/L378E, L370E/F383E, L378E/F383E, L370E/L378E/F383E, V379E, V385E, could maximally disrupt the hydrophobic environment required for interaction. n = 3 independent experiments. l, Representative images (left) and quantification (right) of phase separation of GFP-FOXM1-IDR1-WT or indicated mutants (25 μM) at room temperature with 300 mM NaCl at pH 7.0; n = 5 fields from 3 independent experiments. m, Immunofluorescence and DAPI staining of HeLa cells transfected with GFP-tagged FOXM1 WT or indicated mutants (left). Quantified average number of the nuclear FOXM1 puncta and the percentage of cells with nuclear FOXM1 puncta were shown (right); cells were fixed; n = 3 regions from 3 independent experiments. Data are representative of at least three independent experiments (e, g, h, i, k-m). Scale bar, 5 μm (h, i, l, m). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (h, i, l, m). Uncropped gel images are provided in Supplementary Fig. 1.
