Fig 1: Suppression of endogenous miR-493-3p upregulates Mad2 and induces mitotic anomaliesQuantification of the A. miR-493-3p and B. Mad2 protein levels in HeLa cells 48h after the transfection with miR-control or anti-miR-493-3p. A representative Western blot is shown. C. Representative immunofluorescence images showing Mad2 labelling in prometaphase HeLa cells 48 h post-transfection. Crest served as a kinetochore marker and DNA was stained with DAPI. Scale bar, 10 μm. The graph shows quantification of Mad2 signals in whole cells (n = 40 mitotic cells per a group). D. Quantification of mitotic duration in cells transfected with miR-control or anti-miR-493-3p (n = 300 mitotic cells per a group). E. The graph shows the quantification of anaphase cells with chromatin bridges and lagging chromosomes/chromatin in miR-control and anti-miR-493 transfected HeLa cells (n = 300 anaphase cells per a group). All data is mean +/− s.d. from 3 independent experiments. The asterisks denote statistical significance (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001).
Fig 2: miR-493-3p targets the Mad2-3′UTR and downregulates Mad2 expressionA. Schematic illustration of the predicted targeting site of miR-493-3p in the Mad2 mRNA 3′UTR. B. Quantification of relative firefly luciferase activity, normalized to Renilla luciferase activity, measured from HeLa cells 24 h after co-transfection of Mad2-3′UTR -firefly luciferase reporter construct and miRNA (miR-control or miR-493-3p). C. Quantification of Mad2 mRNA and D. protein levels at 48 h post-transfection in HeLa cells. A representative Western blot is shown. E. Representative immunofluorescence images showing the impact of miRNA overexpression on Mad2 and Bub1 labelling in prometaphase HeLa cells 48 h post-transfection. Crest served as a kinetochore marker and DNA was stained with DAPI. Scale bar, 10 μm. The graph shows quantification of Mad2 and Bub1 at whole cell level (50 mitotic cells were analysed per a group). F. Sites of interference for miR-493-3p and Mad2-target site blocker (TSB-MAD2) in the Mad2-3′UTR (bold letters indicate the seed sequence). G. Quantification of Mad2 protein levels after indicated transfections. A representative Western blot is shown. H. Quantification of miRNA-transfected cells undergoing forced mitotic exit in the presence of taxol or nocodazole (n = 300 cells per group). All data shown is mean +/− s.d. from 3-4 independent experiments. The asterisks denote statistical significance (* = p ≤ 0.05, ** = p ≤ 0.01).
Fig 3: miR-493-3p levels negatively associate with Mad2 gene expression in ovarian cancer cell linesQuantification of A. miR-493-3p expression, B. Mad2 mRNA and C. Mad2 protein in OVCAR-8 and CAOV-3 cell lines. A representative Western blot is shown. D. Quantification of Mad2 mRNA and protein levels 48 h after the transfection of OVCAR-8 and CAOV-3 cells with miR-control or miR-493-3p. Representative Western blots are shown. E. Quantification of mitotic duration (NEBD-to-anaphase) in OVCAR-8 and CAOV-3 cells transfected with miR-control or miR-493-3p (n = 300 cells per group). F. Quantification of miR-493-3p and Mad2 protein levels 48 h after the transfection of OVCAR-8 and CAOV-3 cells with miR-control or anti-miR-493-3p. Representative Western blots are shown. All data is mean +/− s.d. from 3 independent experiments. The asterisks denote statistical significance (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001).
Fig 4: miR-493-3p and Mad2 are inversely expressed in ovarian carcinomasA. Box-plots showing the expression levels of miR-493-3p and Mad2 in ovarian surface epithelium (OSE), high-grade serous ovarian carcinomas (HGSC), and for miR-493-3p also in clear cell ovarian carcinomas (CCC), Oslo cohort. B. Box-plots showing the expression levels of miR-493-3p and Mad2 in normal ovary tissue (normal), low-grade serous ovarian carcinomas (LGSC) and high-grade serous ovarian carcinomas (HGSC), TCGA cohort.
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.
Supplier Page from Abcam for Anti-Mad2L1 antibody [17D10]