Journal: Scientific Reports

Article Title: The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells

doi: 10.1038/s41598-019-43894-0

Figure Lengend Snippet: CMLD-2 effects on MAD2 protein levels in thyroid cancer cells. Panel A. MAD2 is identified as HuR target by RIP assay. Thyroid cancer cells were lysed and immunoprecipitated with anti-HuR or anti-IgG antibodies. HuR–bound mRNAs were amplified using specific MAD2 and MARCH3 (negative control) primers and analyzed by qPCR. All samples were run in triplicate. IgG-immunoprecipitate was arbitrarily set at 1.0 and the enrichment was expressed as relative expression value (Fold Enrichment). Panel B. SW1736, 8505 C, BCPAP and K1 cells were treated with DMSO or CMLD-2 35 µM for 72 h. Cells were collected and MAD2 protein levels were analyzed by Western Blot. Panel C. Densitometric analysis of MAD2 protein levels obtained with Western Blot assay. For each cell line, the results were normalized against Actin and expressed as percentage over control. Results are shown as mean ± SD. ***p < 0.001, ****p < 0.0001 by Student’s t-test.

Article Snippet: To over-express MAD2 protein, we purchased the TrueClone MAD2 cDNA cloned in pCMV6-AC, which contains a full open reading frame of the human Mad2 gene (Origene, Rockville, MD, USA).

Techniques: Immunoprecipitation, Amplification, Negative Control, Expressing, Western Blot

Journal: Scientific Reports

Article Title: The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells

doi: 10.1038/s41598-019-43894-0

Figure Lengend Snippet: Biological effects of MAD2 silencing in thyroid cancer cells. 8505 C and BCPAP cells were transfected with vehicle (mock), non-targeting siRNA (NC, negative control) or three different siRNA (#1, #2 and #3) sequence specific to MAD2 (5 nM) for 72 h and cell viability was analyzed by MTT assay. All experiments were run in sixfold. P < 0.05, **P < 0.001, ***P < 0.0001 by Student’s t-test.

Article Snippet: To over-express MAD2 protein, we purchased the TrueClone MAD2 cDNA cloned in pCMV6-AC, which contains a full open reading frame of the human Mad2 gene (Origene, Rockville, MD, USA).

Techniques: Transfection, Negative Control, Sequencing, MTT Assay

Journal: Scientific Reports

Article Title: The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells

doi: 10.1038/s41598-019-43894-0

Figure Lengend Snippet: MAD2 is involved in CMLD-2 effects in thyroid cancer cell lines. Panel A. 8505 C and BCPAP cells were treated with DMSO, CMLD-2, pCMV empty vector (NC, negative control), pCMV vector specific for MAD2 (1.3 µg), alone or in combinations. Cells were collected after 72 h treatment and MAD2 protein levels were analyzed by Western Blot. Panel B. 8505 C and BCPAP cells were treated with DMSO, CMLD-2, pCMV empty vector (NC, negative control), pCMV vector specific for MAD2 (1.3 µg), alone or in combinations and cell viability was determined by MTT assay after 72 h. NC was arbitrarily set at 1 and cell viability was expressed as relative expression value. All samples were run in sixfold. P < 0.05, **P < 0.001, ***P < 0.0001 by Student’s t-test.

Article Snippet: To over-express MAD2 protein, we purchased the TrueClone MAD2 cDNA cloned in pCMV6-AC, which contains a full open reading frame of the human Mad2 gene (Origene, Rockville, MD, USA).

Techniques: Plasmid Preparation, Negative Control, Western Blot, MTT Assay, Expressing

Journal: Current Biology

Article Title: Mps1 Phosphorylates Its N-Terminal Extension to Relieve Autoinhibition and Activate the Spindle Assembly Checkpoint

doi: 10.1016/j.cub.2018.02.002

Figure Lengend Snippet: Relief of NTE-Mediated Autoinhibition Is Required for Activity (A and B) Stable HeLa T-rex lines expressing MYC-GFP-Mps1-WT and mutants were treated with siMps1, synchronized in mitosis, fixed, and immunostained as indicated. The scale bar represents 5 μm. Quantification of pMELT (A) and Mad2 (B) intensities is shown. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. (C and D) Cells were treated as in (A) but fixed after overnight nocodazole treatment and stained with the indicated antibodies. The mitotic index is shown; n = 3. Cells counted ≥100 per cell lines per experiment. (E) Time-lapse microscopy of cells expressing indicated constructs. Mitotic timing is quantified. ≥50 expressing cells were counted for each condition. n = 2. ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also Figure S6 and , , , and .

Article Snippet: Antibodies were used at 1 μg/ml, unless otherwise indicated, as follows: anti-MYC (9E10, Thermo Scientific), anti Knl1 pT875 (1:2000, gift from Dr Yoshinori Watanabe, University of Tokyo), anti-Mps1 (1mg/ml for WB and 5 mg/ml for IF, Sigma), Rabbit polyclonal phosphospecific antibodies against Mps1 T686, T676 and S821 [ ], Rabbit polyclonal phosphospecific antibody against Mps1 T33 (1:500) was generated against phosphopeptide CKNEDL(pT)DELS, anti Mad2 (4μg/ml [ ],) anti-GFP (1:500, Roche), anti-α-tubulin (DM1A, Santa Cruz Biotechnology), CENP-C (1:1000, MBL International), CREST anti-centromere serum (1:1000, ImmunoVision), and Hoechst 33342 (1μg/ml, Sigma).

Techniques: Activity Assay, Expressing, Staining, Time-lapse Microscopy, Construct

Journal: Current Biology

Article Title: Mps1 Phosphorylates Its N-Terminal Extension to Relieve Autoinhibition and Activate the Spindle Assembly Checkpoint

doi: 10.1016/j.cub.2018.02.002

Figure Lengend Snippet:

Article Snippet: Antibodies were used at 1 μg/ml, unless otherwise indicated, as follows: anti-MYC (9E10, Thermo Scientific), anti Knl1 pT875 (1:2000, gift from Dr Yoshinori Watanabe, University of Tokyo), anti-Mps1 (1mg/ml for WB and 5 mg/ml for IF, Sigma), Rabbit polyclonal phosphospecific antibodies against Mps1 T686, T676 and S821 [ ], Rabbit polyclonal phosphospecific antibody against Mps1 T33 (1:500) was generated against phosphopeptide CKNEDL(pT)DELS, anti Mad2 (4μg/ml [ ],) anti-GFP (1:500, Roche), anti-α-tubulin (DM1A, Santa Cruz Biotechnology), CENP-C (1:1000, MBL International), CREST anti-centromere serum (1:1000, ImmunoVision), and Hoechst 33342 (1μg/ml, Sigma).

Techniques: Virus, Recombinant, Phospho-proteomics, Bicinchoninic Acid Protein Assay, Transfection, Luciferase

Journal: Scientific Reports

Article Title: The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells

doi: 10.1038/s41598-019-43894-0

Figure Lengend Snippet: CMLD-2 effects on MAD2 protein levels in thyroid cancer cells. Panel A. MAD2 is identified as HuR target by RIP assay. Thyroid cancer cells were lysed and immunoprecipitated with anti-HuR or anti-IgG antibodies. HuR–bound mRNAs were amplified using specific MAD2 and MARCH3 (negative control) primers and analyzed by qPCR. All samples were run in triplicate. IgG-immunoprecipitate was arbitrarily set at 1.0 and the enrichment was expressed as relative expression value (Fold Enrichment). Panel B. SW1736, 8505 C, BCPAP and K1 cells were treated with DMSO or CMLD-2 35 µM for 72 h. Cells were collected and MAD2 protein levels were analyzed by Western Blot. Panel C. Densitometric analysis of MAD2 protein levels obtained with Western Blot assay. For each cell line, the results were normalized against Actin and expressed as percentage over control. Results are shown as mean ± SD. ***p < 0.001, ****p < 0.0001 by Student’s t-test.

Article Snippet: To over-express MAD2 protein, we purchased the TrueClone MAD2 cDNA cloned in pCMV6-AC, which contains a full open reading frame of the human Mad2 gene (Origene, Rockville, MD, USA).

Techniques: Immunoprecipitation, Amplification, Negative Control, Expressing, Western Blot

Journal: Scientific Reports

Article Title: The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells

doi: 10.1038/s41598-019-43894-0

Figure Lengend Snippet: Biological effects of MAD2 silencing in thyroid cancer cells. 8505 C and BCPAP cells were transfected with vehicle (mock), non-targeting siRNA (NC, negative control) or three different siRNA (#1, #2 and #3) sequence specific to MAD2 (5 nM) for 72 h and cell viability was analyzed by MTT assay. All experiments were run in sixfold. P < 0.05, **P < 0.001, ***P < 0.0001 by Student’s t-test.

Article Snippet: To over-express MAD2 protein, we purchased the TrueClone MAD2 cDNA cloned in pCMV6-AC, which contains a full open reading frame of the human Mad2 gene (Origene, Rockville, MD, USA).

Techniques: Transfection, Negative Control, Sequencing, MTT Assay

Journal: Scientific Reports

Article Title: The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells

doi: 10.1038/s41598-019-43894-0

Figure Lengend Snippet: MAD2 is involved in CMLD-2 effects in thyroid cancer cell lines. Panel A. 8505 C and BCPAP cells were treated with DMSO, CMLD-2, pCMV empty vector (NC, negative control), pCMV vector specific for MAD2 (1.3 µg), alone or in combinations. Cells were collected after 72 h treatment and MAD2 protein levels were analyzed by Western Blot. Panel B. 8505 C and BCPAP cells were treated with DMSO, CMLD-2, pCMV empty vector (NC, negative control), pCMV vector specific for MAD2 (1.3 µg), alone or in combinations and cell viability was determined by MTT assay after 72 h. NC was arbitrarily set at 1 and cell viability was expressed as relative expression value. All samples were run in sixfold. P < 0.05, **P < 0.001, ***P < 0.0001 by Student’s t-test.

Article Snippet: To over-express MAD2 protein, we purchased the TrueClone MAD2 cDNA cloned in pCMV6-AC, which contains a full open reading frame of the human Mad2 gene (Origene, Rockville, MD, USA).

Techniques: Plasmid Preparation, Negative Control, Western Blot, MTT Assay, Expressing

Journal: The Journal of Pathology

Article Title: Low MAD2 expression levels associate with reduced progression-free survival in patients with high-grade serous epithelial ovarian cancer

doi: 10.1002/path.3035

Figure Lengend Snippet: Nuclear MAD2 IHC staining intensity is associated with progression-free survival (PFS) in high-grade serous EOC. (a) Full-face FFPE sections demonstrating IHC staining intensity for MAD2, low (2+), intermediate (3+), high (4+). (b) Multivariate Cox's regression hazard analysis: correlation of low MAD2 IHC staining intensity expression with progression-free survival (PFS) in patients with high-grade serous EOC. Multivariate Cox's regression hazard analysis (adjusted for stage, tumour grade and optimum surgical debulking < 1 cm) showed a significant correlation between low MAD2 IHC staining intensity and PFS ( p = 0.0003; HR 4.689)

Article Snippet: Proteins were then electrophoretically transferred to a nitrocellulose membrane (100 V for 60 min) and analysed with antibodies raised against MAD2 (BD Transduction Laboratories), cyclin B1, poly(ADP–ribose) polymerase (PARP), phospho (ser10)-Histone H3, cleaved caspase 3 (Cell Signaling) and β-actin (Sigma).

Techniques: Immunohistochemistry, Expressing

Journal: The Journal of Pathology

Article Title: Low MAD2 expression levels associate with reduced progression-free survival in patients with high-grade serous epithelial ovarian cancer

doi: 10.1002/path.3035

Figure Lengend Snippet: Ovarian cancer cells with low MAD2 protein expression are less sensitive to paclitaxel. (a) Bar graph of qRT–PCR for MAD2 mRNA expression. Relative expression levels compared to A2780 cells were determined using the standard curve method (ABI Prism 7700 Sequence Detection System, User Bulletin no. 2) and quantified using the TaqMan® comparative CT method of analysis. (b) Western blot analyses for MAD2 protein expression and western blot for β-actin loading control. (c) Line graph representing a dose–response curve for cell viability assessed in a MTT assay in ovarian cancer cells treated with 1–500 n m paclitaxel for 48 h. The data presented are representative of at least three independent experiments. Student's t -test was used for comparison of means (* p < 0.05, ** p < 0.01 and *** p < 0.001)

Article Snippet: Proteins were then electrophoretically transferred to a nitrocellulose membrane (100 V for 60 min) and analysed with antibodies raised against MAD2 (BD Transduction Laboratories), cyclin B1, poly(ADP–ribose) polymerase (PARP), phospho (ser10)-Histone H3, cleaved caspase 3 (Cell Signaling) and β-actin (Sigma).

Techniques: Expressing, Quantitative RT-PCR, Sequencing, Western Blot, MTT Assay

Journal: The Journal of Pathology

Article Title: Low MAD2 expression levels associate with reduced progression-free survival in patients with high-grade serous epithelial ovarian cancer

doi: 10.1002/path.3035

Figure Lengend Snippet: Cellular responses to paclitaxel are compromised in ovarian cells that express low levels of MAD2. (a) Western blot analyses for cyclin B1, phospho(Ser10)-Histone H3 and PARP cleavage in the ovarian cancer cell lines treated with 10 and 50 n m paclitaxel for 24 h. (b) Western blot analyses for PARP and cleaved caspase 3, in the ovarian cancer cell lines treated with 10 and 50 n m paclitaxel for 48 h. A western blot for β-actin was used as a loading control. The data presented are representative of at least three independent experiments. (c) MTT viability assay of A2780 cells transiently transfected with MAD2 siRNA and scrambled controls and treated with paclitaxel for 24 h. Western blot analyses of A2780 cells transiently transfected with MAD2 siRNA and scrambled controls and treated with paclitaxel for 24 h. Data presented are representative of at least three independent experiments (* p < 0.05, ** p < 0.01)

Article Snippet: Proteins were then electrophoretically transferred to a nitrocellulose membrane (100 V for 60 min) and analysed with antibodies raised against MAD2 (BD Transduction Laboratories), cyclin B1, poly(ADP–ribose) polymerase (PARP), phospho (ser10)-Histone H3, cleaved caspase 3 (Cell Signaling) and β-actin (Sigma).

Techniques: Western Blot, MTT Viability Assay, Transfection

Journal: The Journal of Pathology

Article Title: Low MAD2 expression levels associate with reduced progression-free survival in patients with high-grade serous epithelial ovarian cancer

doi: 10.1002/path.3035

Figure Lengend Snippet: Binding of miR-433 to the 3′ UTR of MAD2 mRNA down-regulates MAD2 protein expression. (a) Schematic representation of the miR-433 binding region in the MAD2 3′ UTR and the sequence of the MAD2 morpholino oligonucleotide complimentary to the miR-433-MAD2 binding domain. (b) Bar graph of relative miR-433 gene expression in the ovarian cancer cell lines. Relative expression levels compared to A2780 cells were determined using the standard curve method (ABI Prism 7700 Sequence Detection System, User Bulletin No. 2) and quantified using the TaqMan® comparative CT method of analysis. (c) MAD2 and β-actin-loading control western blot analyses of A2780 cell lines transfected with pre-miR-433 and scrambled controls or MAD2 morpholino and morpholino scrambled controls. Bar graphs represent MAD2 expression as a percentage of control measured by densitometry using ImageJ. The data presented are representative of at least three independent experiments. (d) Comparison of normalized luciferase activity in A2780 cells transfected with MAD2–pMIR–REPORT and pre-miR-scr or pre-miR-433. Student's t -test was used for comparison of means (* p < 0.05, ** p < 0.01 and *** p < 0.001)

Article Snippet: Proteins were then electrophoretically transferred to a nitrocellulose membrane (100 V for 60 min) and analysed with antibodies raised against MAD2 (BD Transduction Laboratories), cyclin B1, poly(ADP–ribose) polymerase (PARP), phospho (ser10)-Histone H3, cleaved caspase 3 (Cell Signaling) and β-actin (Sigma).

Techniques: Binding Assay, Expressing, Sequencing, Western Blot, Transfection, Luciferase, Activity Assay

Journal: The Journal of Pathology

Article Title: Low MAD2 expression levels associate with reduced progression-free survival in patients with high-grade serous epithelial ovarian cancer

doi: 10.1002/path.3035

Figure Lengend Snippet: Transient transfection of pre-miR-433 compromises paclitaxel responses in A2780 cells. (a) Bar graph representing the relative viability assessed by MTT assay in A2780 cells transiently transfected with pre-miR-433 and treated with 10 and 50 n m paclitaxel compared to scrambled control. (b) Western blot analyses for MAD2, cyclin B1, phospho(Ser10)–Histone H3, PARP cleavage and cleaved caspase 3 in A2780 cells transiently transfected with scrambled control microRNAs and pre-miR-433. The cell lines were treated with 10 and 50 n m paclitaxel for 24 h. A western blot for β-actin was used to demonstrate loading. The data presented are representative of at least three independent experiments. Mean differences were calculated by Student's t -test, where statistical significance was assessed as * p < 0.05

Article Snippet: Proteins were then electrophoretically transferred to a nitrocellulose membrane (100 V for 60 min) and analysed with antibodies raised against MAD2 (BD Transduction Laboratories), cyclin B1, poly(ADP–ribose) polymerase (PARP), phospho (ser10)-Histone H3, cleaved caspase 3 (Cell Signaling) and β-actin (Sigma).

Techniques: Transfection, MTT Assay, Western Blot