Extended Data Fig. 4: Ser376 phosphorylation abrogates FOXM1 LLPS.

a, Representative micrographs (left) and quantification (right) of GFP-FOXM1 droplet formation. Prokaryotic GFP-FOXM1-WT or S376A (25 μM) was incubated with or without eukaryotic purified AMPK complex (α2/β2/γ2) (1 μg) for 1 h at room temperature in kinase buffer with 5 mM ATP and 10 mM MgCl₂, and then dialyzed into droplet buffer with 300 mM NaCl; n = 5 fields from 3 independent experiments. b, Representative images (left) and quantification (right) of phase separation of GFP-FOXM1 WT, Ser376Asp (S376D) or Ser376Glu (S376E) (25 μM) at room temperature with 300 mM NaCl; n = 5 fields from 3 independent experiments. c, Bacterially purified GFP-FOXM1 WT, S376A, S376D and S376E proteins were analyzed by Coomassie blue staining (left) and immunoblot (right). d, MST binding affinity between prokaryotic expressed GFP-FOXM1 WT or S376E (1 μM) and FKH-DNA. n = 3 independent experiments. e, Representative images (left) and quantification (right) of droplet formation of various concentrations of GFP-FOXM1 WT or S376E with Cy3-FKH DNA (500 nM); n = 5 fields from 3 independent experiments. f, FRAP quantification of the droplet fused by GFP-FOXM1 WT or S376E (10 µM) with Cy3-FKH DNA (500 nM) before and after photobleaching over 80 s (n  =  3 droplets). n = 3 droplets. g, Flow chart of the major steps of generating Ser376-phosphorylated recombinant FOXM1 (left): (1) Step 1: sub-cloning of FOXM1 into pGEX-6p/His vector; (2) Step 2: mutating the codon of Ser376 to TAG to genetically incorporate Phosphoserine into recombinant FOXM1 (see Methods for detail); (3) Step 3: expression and purification of the S376-phosphorylated FOXM1 (FOXM1 S376-Phos). Coomassie blue staining of the indicated proteins purified (right). h, Schematic diagram describing the generation of site-specifically phosphorylated recombinant FOXM1 proteins with a phosphoserine tRNA (see in Methods). Schematic in panel h adapted from ref. ⁴⁶, Cell Press. i, Immunoblot (IB) of the purified proteins with antibody specific to phosphorylated-Ser376 of FOXM1. The anti-Strep blots indicate loading of lanes. j, MST Binding affinity between IDR1 and FITC-IDR1 (1 μM) (IDR1/IDR1), IDR1 and FITC-pSer376-IDR1 (1 μM) (p-IDR1/IDR1), pSer376-IDR1 and FITC- pSer376-IDR1 (1 μM) (p-IDR1/p-IDR1); n = 3 independent experiments. k, FRAP quantification of droplets fused by the non-phosphorylated or Ser376-phosphorylated GFP-FOXM1 (10 µM) mixted with FKH-DNA (500 nM) before and after photobleaching over 50 s (n  =  3 droplets). l, Immunofluorescence and DAPI staining in FOXM1-GFP HeLa stable cells treated with or without metformin (2 mM) or AICAR (0.5 mM) for 16 h (left). Quantified average number of nuclear FOXM1 puncta and the percentages of cells with nuclear puncta were shown (right); cells were fixed; n = 3 regions from 3 independent experiments. m, Immunofluorescence and DAPI staining of MDA-MB-231 cells transfected with GFP-tagged FOXM1 WT or S376E (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. n, Immunofluorescence and DAPI staining of HeLa cells transfected with GFP-tagged FOXM1 WT or S376E (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. o, p, Immunofluorescence and DAPI staining of MDA-MB-468 cells (o) or HCC1937 cells (p) transfected with GFP-tagged FOXM1 WT or S376E (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 (a, b, d-f, j-p). Scale bar, 5 μm (a, b, e, l-p). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (a, b, e, l-p), two-way ANOVA (f, k). Uncropped gel images are provided in Supplementary Fig. 1.

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