Journal: PloS one

Article Title: Taxifolin enhances andrographolide-induced mitotic arrest and apoptosis in human prostate cancer cells via spindle assembly checkpoint activation.

doi: 10.1371/journal.pone.0054577

Figure Lengend Snippet: Figure 11. The effect of MAD2 knockdown on the mitotic index of DU145 cells treated with Andro and/or Taxi. Cells were either treated with Andro and/or Taxi for 24 h or transfected with the negative control siRNA or MAD2 specific siRNA for 24 h and later treated with Andro and/or Taxi for additional 24 h. Then cells were fixed and stained with anti-phospho-histone H3 (Ser10) antibody, FITC-conjugated secondary antibody and PI, and analyzed by flow cytometry. Mitotic index is expressed as mean 6 s.d. * indicates significant difference between two groups, *p,0.01. doi:10.1371/journal.pone.0054577.g011

Article Snippet: RNAi depletion of MAD2 DU145 cells were seeded in 60 mm plates to provide a confluency of 50–70% in 24 h. The cells were transfected with either one of the two Mad2L1 siRNA (Origene, MD, USA) or the control siRNA in the presence of siTran transfection reagent (Origene) in Opti-MEM medium (Invitrogen, CA, USA) for 24 h. The transfected cells were rinsed one time with PBS and incubated with DMEM (Invitrogen, CA, USA) medium for 24 h and were further incubated for another 24 h in medium containing 20 mM Andro and 100 mM Taxi.

Techniques: Knockdown, Transfection, Negative Control, Staining, Flow Cytometry

Journal: Cell reports

Article Title: Mad2 Overexpression Uncovers a Critical Role for TRIP13 in Mitotic Exit

doi: 10.1016/j.celrep.2017.05.021

Figure Lengend Snippet: (A) Mad2 versus TRIP13 microarray expression data in three representative datasets from cBioPortal.org. (B) GGCGG CDE/E2F core sites present between -200 and +100 from the transcription start sites of MAD2, BUBR1, and TRIP13. (C) ChIP-Seq data from Encode database (https://genome.ucsc.edu/ENCODE/) for E2F1, E2F4, E2F6 binding near TRIP13 transcription start site in HeLa cells. (D) Mad2 and TRIP13 expression are both elevated by Western blot in Wild Type (WT) and Rb-family (Rb, p107, p130) deficient TKO MEFs. Quantification is shown below corresponding bands.

Article Snippet: Transient human Mad2 overexpressing cells were made by transfecting target cells with Myc-DDK (FLAG) tagged Mad2 (Origene, #RC203273) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Microarray, Expressing, ChIP-sequencing, Binding Assay, Western Blot

Journal: Cell reports

Article Title: Mad2 Overexpression Uncovers a Critical Role for TRIP13 in Mitotic Exit

doi: 10.1016/j.celrep.2017.05.021

Figure Lengend Snippet: (A) Western blot of RPE2 cells transduced with dox-inducible HA-Mad2 construct and/or constitutive TRIP13-GFP construct with or without doxycycline addition for 24 hours. Total quantification (endogenous + exogenous) is shown below corresponding bands. (B) Mitotic timing of RPE-M or T13G cells, +/- 24 hours doxycycline, measured by cell rounding in time-lapse images. n ≥20 cells for each condition. (C) Equal numbers of RPE-M and T13G cells were plated with or without doxycycline on day 0 and were counted and replated on day 2 and day 4, and counted on day 8. Population doublings of RPE-M and T13G cells is plotted over time. n=3 independent experiments (D) Bright field imaging of RPE-M cells without doxycycline treatment and RPE-M and T13G cells with doxycycline treatment for 12 days. (E) Micronuclei were counted in 100 cells in triplicate with DAPI staining in RPE-M or T13G cells treated with doxycycline for 0, 1, 3, or 8 days. * indicates p < 0.05, ** p < 0.01, *** p < 0.001, ns indicates not significant (p > 0.05); Unpaired t test. Data are represented as mean± SD.

Article Snippet: Transient human Mad2 overexpressing cells were made by transfecting target cells with Myc-DDK (FLAG) tagged Mad2 (Origene, #RC203273) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Western Blot, Transduction, Construct, Imaging, Staining

Journal: Cell reports

Article Title: Mad2 Overexpression Uncovers a Critical Role for TRIP13 in Mitotic Exit

doi: 10.1016/j.celrep.2017.05.021

Figure Lengend Snippet: (A) Equal numbers of RPE-M cells transfected with TRIP13 or control siRNAs were plated with or without doxycycline on day 0 and were counted and replated on day 1 and counted on day 3. Population doublings of cells is plotted over time. n=3 independent experiments (B) Western blot of γH2AX and cleaved caspase 3 on RPE-M cells transfected with siRNAs for 6 days +/- Mad2 overexpression for 3 days. (C) Western blot of Mad2 and TRIP13 on parental RPE and Mad2-inducible RPE-M cells transduced with a doxycycline-inducible TRIP13 or control shRNA construct. Total quantification (endogenous + exogenous) is shown below corresponding bands. (D) Tumor volumes over time of RPE and RPE-M cells transduced with TRIP13 or control shRNA injected subcutaneously into nude mice (n=10). Mice were continuously fed with doxycycline-containing feed. Tumor volumes and incidence at week 5 are shown on the right. ** indicates p < 0.01; Unpaired t test. Data are represented as mean± SD.

Article Snippet: Transient human Mad2 overexpressing cells were made by transfecting target cells with Myc-DDK (FLAG) tagged Mad2 (Origene, #RC203273) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Transfection, Control, Western Blot, Over Expression, Transduction, shRNA, Construct, Injection

Journal: Cell reports

Article Title: Mad2 Overexpression Uncovers a Critical Role for TRIP13 in Mitotic Exit

doi: 10.1016/j.celrep.2017.05.021

Figure Lengend Snippet: (A) Validation of CRISPR-Cas9 knockout of TRIP13. Cell clones were screened for TRIP13 loss by Western blot. DNA from clone 2-19 was cloned and sequenced, with one allele having net deletion of seven base pairs and the second allele having a deletion of two base pairs, both leading to loss of reading frame. The gRNA sequence is indicated in bold. (B) Morphology of TRIP13 knockout cells by bright field microscopy (Left). Quantification of mitotic timing in RPE-M and TRIP13 knockout cells treated with +/- 200 ng/ml nocodazole 4 hours prior to imaging +/- 0.5 uM reversine 1 hour prior to imaging (Right). n ≥20 cells for each condition. (C) Morphology of TRIP13 knockout cells after 3 days Mad2 overexpression by bright field microscopy (Left). Quantification of mitotic timing and fate in RPE-M and TRIP13 knockout cells treated with +/- doxycycline 16 hours prior to imaging (Right). n ≥20 cells for each condition. (D) Mitotic timing of RPE-M or T13G cells treated with doxycycline for 24 hours or a low dose of nocodazole (15 ng/ml) 4 hours prior to imaging. n ≥20 cells for each condition. (E) Mitotic timing of RPE-M cells transfected with control siRNA or TRIP13 siRNA #1 4 days prior to imaging and treated with either doxycycline (24 hours) or low-dose (15 ng/ml) nocodazole (4 hours) prior to imaging. n ≥20 cells for each condition. ** indicates p < 0.01, *** p < 0.001, ns indicates not significant (p > 0.05); Unpaired t test. Data are represented as mean± SD. See also Figures S5 and S6

Article Snippet: Transient human Mad2 overexpressing cells were made by transfecting target cells with Myc-DDK (FLAG) tagged Mad2 (Origene, #RC203273) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Biomarker Discovery, CRISPR, Knock-Out, Clone Assay, Western Blot, Sequencing, Microscopy, Imaging, Over Expression, Transfection, Control

Journal: Cell reports

Article Title: Mad2 Overexpression Uncovers a Critical Role for TRIP13 in Mitotic Exit

doi: 10.1016/j.celrep.2017.05.021

Figure Lengend Snippet: (A) Mitotic timing of TRIP13 wild-type, knockout (Left) or knockdown (Right) cells with or without Mad2-overexpression or reversine (0.5 uM) treatment. Mad2 induced 24 hours prior to imaging with doxycycline, reversine added 1 hour prior to imaging. n ≥20 cells for each condition. (B) Western blot of Cdc20 IPs from mitotic RPE-M and TRIP13 knockout cells +/- Mad2 overexpression +/- reversine. RPE-M and TRIP13 knockout cells were synchronized with 20 hours treatment of Cdk1 inhibitor RO-3306 +/- doxycycline 8 hours after 10 uM RO-3306 addition. Cells were released into fresh media for 30 minutes followed by treatment with 50 ng/ml nocodazole and 10 uM MG132 for 3 hours +/- 1 uM reversine for the last 1.5 hours. Mitotic cells were harvested by mitotic shakeoff. (C) Quantification of Mad2 in Cdc20 IPs in (B) normalized to Cdc20 for each condition (Left). Quantification of the reduction in normalized Mad2 levels in Cdc20 IP after reversine addition for each condition (Right). * indicates p < 0.05, ns indicates not significant (p > 0.05); Unpaired t test. Data are represented as mean± SD. See also Figure S7

Article Snippet: Transient human Mad2 overexpressing cells were made by transfecting target cells with Myc-DDK (FLAG) tagged Mad2 (Origene, #RC203273) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Knock-Out, Knockdown, Over Expression, Imaging, 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: 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