Extended Data Fig. 7: The chromatin landscapes and enhancer remodeling mediated by S376E knock-in reduce malignancy and improve immunogenicity of tumor cells.

a, Scatter plot (left) and enriched motifs (right) for FOXM1 CUT&Tag in parental HeLa cells. b, Normalized read distribution profiles of CUT&Tag signal across typical enhancers (TEs) (left) and super enhancers (SEs) (right) in parental and FOXM1-S376E KI HeLa cells. c, Venn diagram analysis shows the overlap of FOXM1-bound peaks and H3K27ac-bound peaks in parental and FOXM1-S376E-KI HeLa cells (left) as determined by CUT&Tag; The FOXM1-bound peak number (middle) and the percentage of H3K27ac-bound peaks co-occupied with FOXM1 (right) were shown. d, Venn diagram showing the overlap of increased binding genes occupied by FOXM1 and H3K27ac in parental cells (left) and FOXM1-S376E-KI cells (right) as determined by CUT&Tag. e, Gene ontology (GO) functional clustering of increased binding genes co-occupied by FOXM1 and H3K27ac in parental and FOXM1-S376E-KI cells in d. f, KEGG pathway analysis of different expressed genes between the parental and FOXM1-S376E-KI cells by RNA-seq. g, Pathway enrichment map for significantly enriched gene sets in FOXM1-S376E-KI cells; shown by pre-ranked gene-set enrichment analysis (GSEA). h, Immunofluorescence and quantification of endogenous MAGEB2 (left) and SPA17 (right) staining in parental and FOXM1-S376E-KI HeLa cells; cells were fixed; n = 3 regions from 3 independent experiments. i, Immunoblot (IB) analysis of parental and FOXM1-S376E-KI HeLa cells. j, Quantification of endogenous dsDNA in parental and FOXM1-S376E-KI HeLa cells in Fig. 3h (n = 3 regions from 3 independent experiments). k, Immunofluorescence (left) and quantification (right) of endogenous dsRNA and DAPI staining in parental and FOXM1-S376E-KI HeLa cells transfected without or with polyinosinic-polycytidylic acid (poly I:C) as indicated; cells were fixed; n = 3 regions from 3 independent experiments. l, cGAMP concentrations in cytoplasmic fractions of WT or cGAS^−/− HT1080 transfected without or with cytoplasmic DNA isolated from FOXM1-S376E-KI HeLa cells, measured by cGAMP ELISA. n = 3 independent experiments. m, qPCR analysis of IFNB1, CCL5 and CXCL10 mRNA in parental and FOXM1-S376E-KI HeLa cells; n = 3 independent experiments. n, Quantification of endogenous γH2AX in parental and FOXM1-S376E-KI HeLa cells in Fig. 3k (n = 3 regions from 3 independent experiments). o, Immunofluorescence (left) and quantification (right) of endogenous dsDNA and DAPI staining of FOXM1 knock-out MDA-MB-231 cells stably expressing Myc-FOXM1 WT or S376E; cells were fixed; n = 3 regions from 3 independent experiments. p, Immunofluorescence (left) and quantification (right) of endogenous dsDNA and DAPI staining of control and FOXM1 knock-out MDA-MB-231 cells stably expressing Myc-FOXM1 WT or S376E and transfected without or with poly (I:C) as indicated; cells were fixed; n = 3 regions from 3 independent experiments. q, qPCR analysis of IFNB1, CCL5 and CXCL10 mRNA in FOXM1 knock-out MDA-MB-231 cells stably expressing Myc-FOXM1 WT or S376E; n = 3 independent experiments. r, Immunoblot (IB) analysis of the indicated proteins in FOXM1 knock-out MDA-MB-231 cells stable expressing Myc-FOXM1 WT or S376E. s, Immunofluorescence (left) and quantification (right) of endogenous γH2AX and DAPI staining in FOXM1 knock-out MDA-MB-231 cells stable expressing Myc-FOXM1 WT and S376E; cells were fixed; n = 3 regions from 3 independent experiments. t, Transwell migration assay (left) and transwell invasion assay (right) using FOXM1 knock-out MDA-MB-231 cells stable expressing Myc-FOXM1-WT or S376E. Representative figures from three independent experiments are shown. The quantification of migrated cells per field is shown in the bar graphs; Scale bar, 50 μm. u, T cell‐mediated cancer cell killing assay: FOXM1 knock-out MDA-MB-231 cells stable expressing Myc-FOXM1-WT or S376E co‐cultured with or without activated human peripheral blood mononuclear cells (PBMC) (see in Methods for detail) for 72 h were subjected to crystal violet staining (left). MDA‐MB‐231‐to‐T cell ratio = 1:3. Quantification of cytotoxicity of T cells (right). n = 3 wells from 3 independent experiments. Data are representative of at least three independent experiments (h, j-q, s, u). Scale bar, 10 μm (h, k, o, p, s). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (h, j-q, s-u) or Weighted Kolmogorov–Smirnov statistic test (g). Uncropped gel images are provided in Supplementary Fig. 1.

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