Extended Data Fig. 3: AMPK directly phosphorylates FOXM1 at S376 in IDR1.

a, Fold change of reporter gene activity in HEK293T cells transfected with TSC22D1 promoter- and 6DB promoter-luc constructs non-treated or treated with metformin (2 mM) or AICAR (0.5 mM) for 16 h; n = 3 ndependent experiments. b, qPCR analysis of CDC25B and CyclinA2 mRNA level in HEK293T cells non-treated or treated with metformin (2 mM) or AICAR (0.5 mM) for 16 h; n = 3 independent experiments. c, Immunofluorescence and DAPI staining in FOXM1-GFP stable HeLa cells treated with or without 2-DG (10 mM) 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. d, Immunofluorescence and DAPI staining in FOXM1-GFP stable HeLa cells released from 16 h of glucose starvation (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. e-g, Endogenous FOXM1 staining with anti-FOXM1 antibody (green) in MDA-MB-231 cells (e), MDA-MB-468 cells (f) and HCC1937 cells (g) treated with with metformin (2 mM) or AICAR (0.5 mM) for 16 h (left). Quantified average number of 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. h, Diagram showing intracellular crosslinking mass spectrometric strategies targeting FOXM1 (Methods) (left). MS analysis of interacting proteins captured by the resultant covalent linkage in situ (right). i, Immunoblot (IB) of total cell lysate (TCL) and IP derived from HEK293T cells transfected with Myc-FOXM1-WT and Flag-tagged AMPKα1 or AMPKα2 plasmids. j, Immunoblot (IB) of input and immunoprecipitates (IP) of control IgG or anti-FOXM1 antibodies derived from HEK293T cells. k, AMPK kinase complex (α2/β2/γ2) was purified with Flag-beads and eluted by Flag peptide. Flag-AMPKα2 or its kinase-dead mutant (K47R) was co-expressed with HA-AMPKβ2 and HA-AMPKγ2 plasmid in HEK293T cells for 48 h. Illustration in panel k adapted from ref. ²⁸, EMBO Press. l, Images of GFP-FOXM1 droplets formation pre-incubated with AMPK complex (α2/β2/γ2) or its kinase dead form (α2^K47R/β2/γ2) purified from HEK293T cells 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 (25 μM GFP-FOXM1, at pH 7.0, at room temperature). n = 3 fields from 3 independent experiments. m, Fold change in TSC22D1-luc and 6DB-luc activity in HEK293T cells transfected with Flag-AMPKα2 constitutively active form (CA) or its kinase-dead (KD) mutant (T172A) along with FOXM1 expression plasmids; n = 3 independent experiments. n, qPCR analysis of CDC25B and CyclinA2 mRNA level in HEK293T cells transfected with AMPKα2-CA or AMPKα2-KD along with FOXM1 expression plasmids; n = 3 independent experiments. o, Immunoblot (IB) of TCL and anti-Flag IP derived from HEK293T cells transfected with Myc-FOXM1 and the indicated Flag-AMPKα constructs. p, Immunoblot (IB) analysis of the TCL and anti-Myc IP derived from HEK293T cells transfected with Myc-FOXM1 and Flag-AMPKα2 constitutively active (CA) form or the Flag-active AMPK truncation kinase-dead (KD) mutant (T172A), as indicated. q, Immunoblot (IB) of TCL and IP using control IgG or anti-FOXM1 antibodies derived from control Ampkα WT and Ampkα1^−/−α2^−/− double knock-out (DKO) MEFs treated with metformin (2 mM) for 16 h. r, Immunoblot (IB) analysis of the TCL derived from AMPKα WT and Ampkα1^−/−α2^−/− double knock-out (DKO) MEFs treated with metformin (2 mM) for 16 h. s, SDS-PAGE of anti-Flag (FOXM1) immunoprecipitants from HEK293T cells with Coomassie brilliant blue staining. FOXM1 band at the corresponding location was indicated. t, Immunoblot (IB) of TCL and anti-Myc IP derived from HEK293T cells transfected with Myc-FOXM1 WT/S376A and Flag-AMPKα2-CA (left), or Myc-FOXM1 and Flag-AMPKα2-CA/KD (right). u, Immunoblot (IB) analysis of the TCL and IP of control IgG or anti-FOXM1 antibodies derived from AMPKα WT and Ampkα1^−/−α2^−/− double knock-out (DKO) MEFs treated with AICAR (0.5 mM) for 16 h. v, Left: design of guide RNA for CRISPR-Cas9 mutation of both copies of FOXM1 resulting in FOXM1 (Ser376A) in HeLa cells: upward arrow indicates the position of the codon in exon 7 that encodes Ser376; red underline (WT allele) indicates the codon (TCA) encoding Ser376; PAM (blue underline, WT allele) indicates the protospacer-adjacent motif; red font (in single-stranded oligodeoxynucleotide [SS ODN] and protein sequence) indicates the replacement codon (GCT) and amino acid (alanine [A]). Right: sequencing verification of the codon replacement by CRISPR-Cas9 resulting in FOXM1 (Ser376A) was shown. w, Immunoblot (IB) the TCL and anti-FOXM1 IP derived from the homozygous S376A knock-in HeLa cells treated with or without metformin (2 mM) for 16 h. Data are representative of at least three independent experiments (a-g, l-n). Scale bar, 5 μm (c-g, l). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (a-g, l-n). Uncropped gel images are provided in Supplementary Fig. 1.

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