Extended Data Fig. 5: Ser376 phosphorylation abrogates FOXM1 LLPS.
From: Targeting FOXM1 condensates reduces breast tumour growth and metastasis
a, Scheme for the iodixanol gradient ultracentrifugation of HEK293T transfected with Myc-tagged FOXM1 WT or S376E (top). Immunoblot (IB) of the aliquoted fraction with anti-Myc antibodies (bottom left) and quantified band intensities were shown (bottom right). n = 3 independent experiments. b, Iodixanol-gradient ultracentrifugation analysis of TCL from HEK293T cells pre-treated with control, AMPKα2 CA or KD (top). The FOXM1 band intensities were shown (bottom); n = 3 independent experiments. c, SDD-AGE analysis of FOXM1 aggregation (top) and SDS-PAGE (bottom) of the TCL derived from HEK293T cells transfected with Myc-FOXM1 WT or S376E. d, SDD-AGE analysis of FOXM1 aggregation (top) and SDS-PAGE (bottom) of the TCL derived from HEK293T cells transfected with plasmids expressing Myc-FOXM1 WT or S376A and Flag-AMPKα2 CA. e, Immunofluorescence of endogenous FOXM1 and DAPI staining in parental and FOXM1-S376A knock-in HeLa cells treated with control DMSO, metformin (2 mM) or AICAR (0.5 mM) for 16 h (left). Quantified average number of the nuclear FOXM1 puncta and the percentage of cells with nuclear puncta were shown (right); cells were fixed; n = 3 regions from 3 independent experiments. f, Colocalization of active RNA Pol II with FOXM1-GFP in the nuclear puncta of HeLa cells was detected using immunofluorescence staining with antibodies targeting either the active RNA Pol II, which is phosphorylated at Ser 2 (S2P) in its CTD (red), or GFP (green). The images were captured using super-resolution structured illumination microscopy (left). Quantitative line profile of colocalization along a white arrow of the left image (right); cells were fixed. g, Design of guide RNA for CRISPR–Cas9 mutation of both copies of FOXM1 resulting in FOXM1-S376E knock-in (left); sequencing verification of the codon replacement by CRISPR-Cas9 resulting in FOXM1 (Ser376E) was shown (right). h, SDD-AGE analysis of FOXM1 aggregation (top) and SDS-PAGE analysis (below) of TCL derived from parental and FOXM1-S376E knock-in HeLa cells. i, ChIP-qPCR analysis of FOXM1 binding to CENPA and CyclinA2 promoter with control IgG or anti-FOXM1 antibodies (left) and the qPCR analysis of CENPA and CyclinA2 mRNA expression (right) in parental or FOXM1-S376E knock-in HeLa cells; n = 3 independent experiments. j, Fold change in TSC22D1-luc and 6DB-luc activity (left) in HEK293T cells transfected without or with AMPKα2 CA along with control, wild-type FOXM1 (WT) or the catalytically inactive FOXM1 mutant (S376A) expression plasmids; n = 3 independent experiments. k, Iodixanol-gradient ultracentrifugation analysis of TCL from parental or FOXM1-S376E knock-in HeLa cells (top). Quantified FOXM1 band intensities were shown (bottom); n = 3 independent experiments. l, qPCR analysis (right) of CyclinA2 and CDC25B mRNA level in HEK293T cells transfected with or without AMPKα2 CA along with control, FOXM1 (WT) or FOXM1 mutant (S376A) expression plasmids; n = 3 independent experiments. Data are representative of at least three independent experiments (a, b, e, i-l). Scale bar, 5 μm (e, f). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (e, j, k, l). Uncropped gel images are provided in Supplementary Fig. 1.
