human mad2  (Addgene inc)

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Cell viability of K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days after doxycycline (Dox) addition (mean and SD, two-tailed Student’s t test, CENP-C WT cells: n = 6; CENP-C ΔM12BD cells: n = 6; ** p < 0.01). (B) Representative images of DAPI-stained K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Scale bar, 50 μm. (C) Population of normal interphase cells, mitotic cells, and cells with micronuclei in K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Error bars indicate SEM. n = 3 independent experiments; 200 cells from each cell line were quantified in each experiment. (D) The growth curve of K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. K562 WT or CENP-C ΔM12BD cells were treated with or without Dox ( KIF18A OFF or ON). The cell numbers were normalized to those at time 0 of each line. (E) Cell-cycle distribution of conditional knockout of KIF18A in K562 WT cells at each day after Dox addition, based on FACS analysis. (F) Cell-cycle distribution of conditional knockout of KIF18A in K562 CENP-C ΔM12BD cells at each day after Dox addition, based on FACS analysis. (G and H) Quantification of cells with misaligned chromosomes in K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. The experimental scheme is shown. Cells were stained with antibodies against MAD2 (red) to detect misaligned chromosomes and CENP-T (green) as a kinetochore marker. DNA was stained with DAPI (blue). Arrowheads show typical MAD2-positive unaligned chromosomes. Scale bar, 10 μm. The cells with MAD2-positive chromosomes were quantified (H) (mean and SEM, two-tailed Student’s t test; n = 5 independent experiments; n.s., non-significant; ** p < 0.01). (I) Numbers of MAD2 positive kinetochores in each cell in each condition (WT KIF18A ON; WT KIF18A OFF; CENP-C ΔM12BD KIF18A ON; CENP-C ΔM12BD KIF18A OFF) (Mean and SEM, n = 5 independent experiments).

Article Snippet: Mouse anti-human MAD2 , Santa Cruz Biotechnology , Cat#sc-65492; RRID: AB_831526.

Techniques: Knock-Out, Two Tailed Test, Staining, Marker

Journal: EMBO reports

Article Title: The cell cycle protein MAD2 facilitates endocytosis of the serotonin transporter in the neuronal soma.

doi: 10.15252/embr.202153408

Figure Lengend Snippet: Figure 1. The serotonin transporter C-terminus harbors a potential MAD2 interaction motif (MIM). MAD2 protein expression in dorsal raphe neurons.

Article Snippet: Two days after transfection, cells were used for co-IP experiments as described above. siRNA transfection HEK-293 cells in different culture formats were transfected with three target-specific siRNAs against human MAD2 (sc-35837, Santa Cruz Biotechnology) or with siRNA against luciferase (Dharmacon) as a control.

Techniques: Expressing

Journal: Cell death & disease

Article Title: FOXM1 is critical for the fitness recovery of chromosomally unstable cells.

doi: 10.1038/s41419-023-05946-2

Figure Lengend Snippet: Fig. 3 FOXM1 knockdown impairs fitness of MAD2-overexpressing cells. A Representative western blot and quantification of FOXM1 and HA-Mad2 in Kras (K1, K2, K3) and Kras/Mad2 (KM1, KM2, KM3) breast tumor cells. **P = 0.0043; Unpaired t-test. (n = 9 K and 9 KM tumors were analyzed and measured). B Quantitative RT-PCR of mouse Foxm1 expression in mammary tumors from K and KM animals (n = 8 K and n = 11 KM tumors). *P = 0.0338; Unpaired t-test. C Representative pictures (left) and relative cell viability (right) of K and KM tumor cells after siFoxm1 for 6 days (n = 6 K and n = 7 KM tumors with siFoxm1). Cell viability was normalized to untreated cells. *P = 0.0313; Unpaired t-test. D DNA content analysis of K and KM breast tumor cells after siFoxm1 for 6 days represented as the percentage of cells in each cell cycle phase. (n = 4 K and n = 6 KM tumors were analyzed). **P = 0.001, *P = 0.0304; Two-way ANOVA. E Percentage of TUNEL positive cells in K and KM tumor cells 6 days after Foxm1 knockdown (KD). (n = 4 K and 4KM tumors were analyzed). ***P = 0.0006; Two-way ANOVA. F Western blots of FOXM1, HA- Mad2, Cleaved-caspase3 (C-Casp3) and gama-H2AX in K and KM tumor cells after RCM-1 treatment or siFoxm1 for 6 days, performed in two biological replicates. ACTIN was used as a loading control. For original and additional wbs see Supplemental Material.

Article Snippet: Selection was performed with puromycin (1 μg/ml) or FAC sorting, then infected with inducible Tet-ON lentiviruses carrying human MAD2 and FOXM1 cDNA (MAD2 from Addgene #136347; FOXM1b from Addgene #68811; FOXM1c from Addgene #68810) and selected with hygromycin (300 μg/ml) or puromycin (1 μg/ml).

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, Expressing, TUNEL Assay, Control

Journal: Journal of cell science

Article Title: Changes in subcellular structures and states of pumilio 1 regulate the translation of target Mad2 and cyclin B1 mRNAs.

doi: 10.1242/jcs.249128

Figure Lengend Snippet: Fig. 1. Expression and translational regulation of Mad2 mRNA in mouse oocytes.

Article Snippet: Mouse oocyte extracts were separated by SDS-PAGE with Bolt Bis-Tris Plus Gels (Novex), blotted onto an Immobilon membrane using a Bolt Mini Blot Module (Novex), and probed with an anti-human Pum1 goat antibody (1:1000 dillution) (Bethyl Laboratories, Inc.), an anti-human Cyclin B1 rabbit antibody (1:100 dillution) (Santa Cruz Biotechnology, Inc.), an anti-hamster Cyclin B1 mouse monoclonal antibody (1:1000 dilution) (V152, Abcam), and an anti-human Mad2 rabbit antibody (1:1000 dillution) (Bethyl Laboratories, Inc.).

Techniques: Expressing

Journal: Journal of cell science

Article Title: Changes in subcellular structures and states of pumilio 1 regulate the translation of target Mad2 and cyclin B1 mRNAs.

doi: 10.1242/jcs.249128

Figure Lengend Snippet: Fig. 2. Interaction with Pum1 and cytoplasmic regulation of Mad2 mRNA in mouse

Article Snippet: Mouse oocyte extracts were separated by SDS-PAGE with Bolt Bis-Tris Plus Gels (Novex), blotted onto an Immobilon membrane using a Bolt Mini Blot Module (Novex), and probed with an anti-human Pum1 goat antibody (1:1000 dillution) (Bethyl Laboratories, Inc.), an anti-human Cyclin B1 rabbit antibody (1:100 dillution) (Santa Cruz Biotechnology, Inc.), an anti-hamster Cyclin B1 mouse monoclonal antibody (1:1000 dilution) (V152, Abcam), and an anti-human Mad2 rabbit antibody (1:1000 dillution) (Bethyl Laboratories, Inc.).

Techniques:

Journal: Journal of cell science

Article Title: Changes in subcellular structures and states of pumilio 1 regulate the translation of target Mad2 and cyclin B1 mRNAs.

doi: 10.1242/jcs.249128

Figure Lengend Snippet: Fig. 3. Formation of Pum1 aggregates that surround Cyclin B1 and Mad2 RNA

Article Snippet: Mouse oocyte extracts were separated by SDS-PAGE with Bolt Bis-Tris Plus Gels (Novex), blotted onto an Immobilon membrane using a Bolt Mini Blot Module (Novex), and probed with an anti-human Pum1 goat antibody (1:1000 dillution) (Bethyl Laboratories, Inc.), an anti-human Cyclin B1 rabbit antibody (1:100 dillution) (Santa Cruz Biotechnology, Inc.), an anti-hamster Cyclin B1 mouse monoclonal antibody (1:1000 dilution) (V152, Abcam), and an anti-human Mad2 rabbit antibody (1:1000 dillution) (Bethyl Laboratories, Inc.).

Techniques:

Journal: Molecular medicine reports

Article Title: Deficiency of PTEN leads to aberrant chromosome segregation through downregulation of MAD2.

doi: 10.3892/mmr.2019.10668

Figure Lengend Snippet: Figure 3. MAD2 is downregulated in PTEN‑deficient cells. (A) MAD2 expression was evaluated using cell lysates from control and PTEN‑knockdown cells by western blot analysis. (B) The protein expression levels of MAD2 and cyclin B1 in control and PTEN‑knockdown HeLa cells cultured with or without nocodazole were detected by western analysis. (C) Control and PTEN‑knockdown HeLa cells cultured with or without CHX were harvested at 0, 3 and 6 h after treatment. Then, the protein expression levels of MAD2 were evaluated using western blot analysis. (D) Control and PTEN‑knockdown HeLa cells cultured with or without MG132 were analyzed for MAD2 expression by western blot analysis. (E) Ubiquitin was overexpressed in control and PTEN‑knockdown HeLa cells. Following MG132 treatment, the cells were harvested for analysis via a ubiquitination assay. All data were obtained from 3 independent experi- ments. *P<0.05, **P<0.01 and ***P<0.001. MAD2, mitotic arrest deficient 2; PTEN, phosphatase and tensin homolog; sh, short hairpin RNA; Ub, ubiquitin; CHX, cycloheximide; IP, immunoprecipitates; IB, immunoblot.

Article Snippet: The following primary antibodies were used: rabbit anti-human PTen (1:2,000; cell Signaling Technology, Inc.; cat. no. 9188L), rabbit anti‐human MAD2 (1:1,000) and cyclin B1 (1:4,000; Santa Cruz Biotechnology, Inc.; cat. nos. sc‐28261 and sc‐752, respectively), and mouse anti‐GAPDH (1:20,000; ProteinTech Group, Inc.; cat. no. 60004‐1‐Ig) were used.

Techniques: Expressing, Control, Western Blot, Cell Culture, Ubiquitin Proteomics, shRNA

Journal: Molecular medicine reports

Article Title: Deficiency of PTEN leads to aberrant chromosome segregation through downregulation of MAD2.

doi: 10.3892/mmr.2019.10668

Figure Lengend Snippet: Figure 4. Recovery of MAD2 expression partially ameliorates impaired mitosis. (A) PTEN‑knockdown HeLa cells were transfected with His‑tagged MAD2 expression plasmids prior to the analysis of MAD2 and cyclin B1 expression by western blot analysis. (B) The rate of cell survival for control, PTEN‑knockdown and His‑MAD2‑overexpression PTEN‑knockdown HeLa cells incubated with nocodazole for 36 h was calculated. (C) (Left panel) Representative images indicating the general morphology of mononucleated, binucleated and multinucleated cells at high magnification. (Middle panel) Representative images demonstrating cells of the shControl, shPTEN and shPTEN‑MAD2 groups stained by DAPI. Binucleated cells (arrowhead) and multinucleated cells (arrow) are marked in each image. (Right panel) Statistical analysis of the percentage of binucleated or multinucleated cells in the different groups. A total of 500 cells were counted. Among these cells, binucleated or multinucleated cells were counted. The percentage was calculated as binucleated cell number (or multinucle- ated cell number)/total cell number. All data were obtained from 3 independent experiments, *P<0.05, **P<0.01 and ***P<0.001. BF, bright field; MAD2, mitotic arrest deficient 2; PTEN, phosphatase and tensin homolog; sh, short hairpin RNA; his‑MAD2, His‑tagged MAD2 expression plasmid.

Article Snippet: The following primary antibodies were used: rabbit anti-human PTen (1:2,000; cell Signaling Technology, Inc.; cat. no. 9188L), rabbit anti‐human MAD2 (1:1,000) and cyclin B1 (1:4,000; Santa Cruz Biotechnology, Inc.; cat. nos. sc‐28261 and sc‐752, respectively), and mouse anti‐GAPDH (1:20,000; ProteinTech Group, Inc.; cat. no. 60004‐1‐Ig) were used.

Techniques: Expressing, Transfection, Western Blot, Control, Incubation, Staining, shRNA, Plasmid Preparation