Fig 1: Defective chromosome segregation in MAD1L1 mutant lymphocytes.(A) Representative immunoblot using anti-MAD1 18322-1-AP in peripheral blood cells. The quantification using three different antibodies is shown to the right. See also fig. S4A. Ab, antibody. (B) Mass spectrometry analysis in blood cells, with separate plots for the total intensity of MAD1 peptide 1 (N terminus; see schematic representation above indicating the relative positions of the Q66* and E628* mutations), MAD1 peptides 2 to 17, and MAD2 peptides. a.u., arbitrary units. (C) Immunofluorescence of BUBR1 and ACAs in lymphocytes treated with taxol. Scale bars, 5 µm. Signals were quantified from mitotic cells from (n = 14), proband (n = 15), mother (n = 12), and father (n = 9) cells. (D) Representative micrographs (top) and fluorescence-activated cell sorting analysis (bottom) of lymphocytes in the presence of taxol. Top panels show immunofluorescence for phosphorylated histone H3 (pH3; red), a-tubulin (green), and DAPI (blue). A minimum of 5000 cells per condition was scored for MPM2 staining (two independent experiments with >300 cells per condition). Scale bars, 5 µm. The percentage of mitotic cells after 6 hours in taxol normalized versus the number of cells that entered mitosis at 2 hours is plotted to the right (n = 2 independent experiments). (E) Representative example (two independent experiments) of interphase cells after treatment with taxol showing aberrant cells (abnormal shape and fragmented lobuli) and micronuclei (arrows), as well as cells large nuclei (asterisks). Scale bars, 10 µm. Plots show the percentage of nuclear aberrations and micronuclei (>300 cells per condition), as well as nuclear volume (>500 cells per condition). In (A), (C), and (E), ns, not significant; **P < 0.01; ***P < 0.001; Student’s t test with Welch’s correction (A), one-way analysis of variance with Bonferroni correction (C and E, nuclear volume), or chi-square, two-coiled Fisher exact test (E, nuclear aberrations and micronuclei).
Fig 2: Simultaneous inhibition of AR and PDEF expression further suppresses tumour cell proliferation compared with the inhibition of AR alone. a AR, PDEF, MAD1 and MYC protein levels in only AR-downregulated (AR KD), simultaneous AR- and PDEF-downregulated (AR KD/PDEF KD) and control MDA-MB-453 cells (NS) were determined by performing western blotting (KD: knockdown; NS: non-specific). b Flow cytometry analysis was performed to detect the proliferation of the above three MDA-MB-453 cell clones; **P < 0.05. c Colony-forming assay was conducted to determine the clone-initiating ability of the above three MDA-MB-453 cell clones; **P < 0.05. d and e Wound-healing (left) and Transwell (right) assays were performed to detect the invasion and migration potential of the above three MDA-MB-453 cell clones (wound-healing assay: original magnification, × 100; Transwell assay: original magnification, × 200); **P < 0.05. f CCK-8 assay was performed to detect the proliferation of the above three MDA-MB-453 cell clones; **P < 0.05. g Images of tumours removed from the nude mice subcutaneously injected with control (NS), stable AR-shRNA-expressing (AR KD) and simultaneous AR-shRNA- and PDEF-shRNA-expressing stable MDA-MB-453 cell clones (AR KD/PDEF KD). h Representative tumour growth curves for the three groups. Data are presented as mean ± SD; **P < 0.05. i H&E staining of tumours removed from the mice in the three groups (magnification, × 400). Results of the IHC staining for detecting Ki67 and MYC expression in tumour samples obtained from the mice in the three groups (Ki67 and MYC; magnification, × 400). j and k Statistics of the percentage of Ki67- and MYC-positive cells in the tumour samples removed from the mice in the three groups; **P < 0.05. l A model showing the role of the AR–PDEF and MAD1–MYC pathways in ER-negative BC cell proliferation
Fig 3: Upregulation of MAD1 expression suppresses PDEF-mediated growth of ER-negative BC cell lines. a PDEF, MAD1 and MYC protein levels in PDEF-overexpressing, simultaneous PDEF- and MAD1-expressing and control SKBR-3 cell clones were determined by performing western blotting. b Transwell assay was performed to detect the migration of the above three SKBR-3 cell clones. (Transwell assay: original magnification, × 200); **P < 0.05. c Colony-forming assay was performed to determine the clone-initiating ability of the above three SKBR-3 cell clones; **P < 0.05. d Results of the flow cytometry analysis showed that MAD1 overexpression inhibited PDEF overexpression-induced increase in the number of S-phase SKBR-3 cells; **P < 0.05. e Images of tumours removed from the nude mice subcutaneously injected with control SKBR-3 cells (control), stable PDEF-overexpressing SKBR-3 cell clones (PDEF) and stable simultaneous PDEF- and MAD1-overexpressing SKBR-3 cell clones (PDEF/MAD1). f Representative tumour growth curves for the three groups. Data are presented as mean ± SD; **P < 0.05. g H&E staining of tumours removed from the mice in the three groups; magnification, × 400. IHC staining of Ki67 and MYC in tumour samples obtained from the mice in the three groups (Ki67 and MYC) (magnification, × 400). Representative images of H&E staining of metastatic nodules in the lung tissues of nude mice (lungs) (magnification, × 200). h and i Statistics of the percentage of Ki67- and MYC-positive cells in the three groups; **P < 0.05
Fig 4: PDEF upregulates MYC-mediated gene transcription by promoting MAD1 degradation. a Western blotting was performed to detect the expression levels of intracellular signalling components, including MEK/ERK, PI3K/AKT, MYC/MAD1, AR and EGFR, in PDEF-overexpressing SKBR-3 cells. b Western blotting was performed to detect the expression levels of MEK/ERK, PI3K/AKT, MYC/MAD1, AR and EGFR in PDEF-downregulated MDA-MB-453 cells (NS: non-specific; KD: knockdown). c Co-IP assay was performed with the anti-PDEF antibody in PDEF-overexpressing SKBR-3 cells and control vector-infected cells. The interaction between precipitated MYC and PDEF was detected using the anti-PDEF antibody. d Co-IP assay was performed with the anti-PDEF antibody in PDEF-downregulated MDA-MB-453 cells and control vector-infected cells. The interaction between precipitated MAD1 and PDEF was detected using the anti-PDEF antibody. e PDEF and MYC mRNA levels in PDEF-overexpressing SKBR-3 cells or PDEF-downregulated MDA-MB-453 cells were determined by performing RT-qPCR (left panels). PDEF and MAD1 mRNA levels in PDEF-overexpressing SKBR-3 cells or PDEF-downregulated MDA-MB-453 cells were determined by performing RT-qPCR (right panels); **P < 0.05. f Top panel: Schematic diagram of the PDEF-binding regions within the MAD1 locus. Lower left panel: Results of the direct PDEF ChIP assay followed by RT-qPCR of PDEF-downregulated MDA-MB-453 cells (black bars) or control vector-infected (white bars) cells; data are presented as mean ± SD. Lower right panel: Semi-quantitative PCR of a negative control (IgG) sample
Fig 5: Biallelic loss-of-function mutations in MAD1L1.(A) Chromograms showing the two heterozygous mutations in the MAD1L1 gene in the proband. (B) Schematic representation of the MAD1 protein and the mutations found in the proband. NTD, N-terminal domain; MIM, MAD2 interaction motif. (C) Predicted structure of MAD1 (AlphaFold) showing critical residues limiting the different domains and the position of the Q66 and E628 residues mutated. (D) Pedigree of the family with the status of MAD1L1 gene shown on the right superior angle of each individual. AMI, acute myocardial infarction; DMC, diabetes mellitus complications; Inf, infection; RI, renal insufficiency; SB, stillbirth; t2, trisomy chromosome 2.
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