mad2  (Bethyl)

Journal: bioRxiv

Article Title: Ribosome Quality Control Mitigates Proteotoxic Stress in Aneuploid Cells

doi: 10.64898/2026.01.19.700285

Figure Lengend Snippet: A. Representative immunoblot and replicate quantitation of processed-Keima levels in HCT116 RPS3-Keima cells treated for 24 hours with 125nM, 250nM or 500nM Mps1i or DMSO (Ctrl); vinculin was used as loading control. Mean ± SEM, n =4; Kruskal-Wallis test, followed by Dunn’s multiple comparison test: * indicates p =0.0150. B. Representative immunoblot and replicate quantitation of processed-Keima levels in HCT116 RPL28-Keima cells treated for 24 hours with 125nM, 250nM or 500nM Mps1i or DMSO (Ctrl); vinculin was used as loading control. Mean ± SEM, n =4; Kruskal-Wallis test, followed by Dunn’s multiple comparison test: * indicates p =0.0329 (250nM) or p =0.0329 (500nM). C. Representative immunoblots and replicate quantitation of the HCT116 RPS3-Keima cells upon MAD2 or BUB1 siRNA (or non-targeting siRNA); increased levels of processed-Keima indicates ribosome degradation; MAD2 or BUB1 were blotted as knock-down control. Vinculin and tubulin used as loading control. Mean ± SEM, n =4; one sample and Wilcoxon test (each siRNA vs respective non-targeting siRNA=1): * indicates p =0.0257. D. Representative immunoblots and replicate quantitation of the HCT116 RPL28-Keima cells upon MAD2 or BUB1 siRNA (or non-targeting siRNA); increased levels of processed-Keima indicates ribosome degradation; MAD2 or BUB1 have been blotted as knock-down control. Vinculin and tubulin been used as loading control. Mean ± SEM, n =4; one sample and Wilcoxon test (each siRNA vs respective non-targeting siRNA=1): * indicates p =0.0292. E. Representative live-cell images and replicate quantitation of indicated HCT116 Ribo-Keima cells lines treated with Mps1i pulse or DMSO (Ctrl) and collected at 24 hours. Increased Red-Keima intensity indicates ribosome degradation; Hoechst was used to stain DNA; scale bars, 50μm. Upper quartile, lower quartile and median of each violin plot are shown, n =12 fields of view; unpaired Student’s t-test: **** indicates p <0.0001. F. Representative images and replicate quantitation of RFP/GFP ratio in RPE1 RPS3-RFP-GPF cells treated with Mps1i pulse or DMSO (Ctrl) and collected at 24 or 72 hours. RFP/GFP ratio was calculated as explained in Methods and visualised with a rainbow lookup table (calibration bar shown); cyan masks indicate primary nuclei; scale bars 50μm. Upper quartile, lower quartile and median of each violin plot are shown; n=3 biological replicates (30 fields of view each); Kruskal-Wallis test, followed by Dunn’s multiple comparison test: *** indicates p =0.0002, **** indicates p <0.0001.

Article Snippet: The following primary antibodies were used: BUB1 (#ab54893, Abcam), EGFR (in-house polyclonal ab against human EGFR aa 1172–1186, kindly provided by Prof. Di Fiore), GAPDH (#sc-32233, Santa Cruz Biotechnology), HA (#MMS-101P, BioLegend), HSF1 (#4356S, Cell Signaling Technology), Hsp27 (#ADI-SPA-800-D, Enzo Life Science), Hsp70/72 (#ADI-SPA-810-D, Enzo Life Science), Hsp90 (#4877S, Cell Signaling Technology), Keima-red (#M182-3M, MBL), MAD2 (#A300-301A, Bethyl Laboratories), MET (#sc-162, Santa Cruz Biotechnology), mTOR (#2983S, Cell Signaling Technology), PDGFRβ (#3169S, Cell Signaling Technology), Puromycin clone 12D10 (#MABE343, Merck), Tubulin (#T9026, Sigma-Aldrich), Vinculin (#V9131, Sigma-Aldrich), ZNF598 (#ab241092, Abcam).

Techniques: Western Blot, Quantitation Assay, Control, Comparison, Knockdown, Staining

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: Cell Death & Disease

Article Title: BRD4 inhibition suppresses histone H4 UFMylation to increase ferroptosis sensitivity through TXNIP

doi: 10.1038/s41419-025-08166-y

Figure Lengend Snippet: A RT-qPCR analysis of the mRNA levels of CYCLIN A2 , MAD2 , ORC2 , MCM6 , and SSBP1 in Control and TXNIP KO cells treated with DMSO and 2 μM JQ1. B The protein levels of CYCLIN A2, B1, D1, and MAD2 in Control and TXNIP KO cells treated with DMSO and 2 μM JQ1. C GSEA analysis of gene expression in cells with DMSO and 2 μM JQ1 treatment indicated that cMYC target genes were downregulated in JQ1 treated cells. D The enrichment of cMYC at the promoters of CYCLIN A2 , MAD2 , ORC2 , MCM6 , and SSBP1 in Control and TXNIP KO cells with or without JQ1 treatment. E The expression of cMYC in Control and TXNIP KO cells after JQ1 treatment. F The level of protein UFMylation in Control and TXNIP KO cells with 2 μM JQ1 treatment. G The enrichment of UFMylated proteins at the promoters of CYCLIN A2 , MAD2 , ORC2 , MCM6 , and SSBP1 in Control and TXNIP KO cells with or without JQ1 treatment. H The protein levels of CYCLIN A2, MAD2, and total protein UFMylation in cells with 30 μM DKM 2-93 treatment. I The mRNA levels of CYCLIN A2 , MAD2 , ORC2 , MCM6 , and SSBP1 in cells treated with DMSO and 30 μM DKM 2-93. **** p value < 0.001, *** p value < 0.005, ** p value < 0.01, * p value < 0.05 (one-way ANOVA test in A , D , and G ; two-tailed unpaired Student t test in I ); ns not significant.

Article Snippet: Antibodies against H3 (17168-1-AP), ubiquitin (10201-2-AP), MAD2 (10337-1-AP), and IgG (30000-0-AP) were purchased from Proteintech.

Techniques: Quantitative RT-PCR, Control, Gene Expression, Expressing, Two Tailed Test

Journal: Cell Death & Disease

Article Title: BRD4 inhibition suppresses histone H4 UFMylation to increase ferroptosis sensitivity through TXNIP

doi: 10.1038/s41419-025-08166-y

Figure Lengend Snippet: A Histone H4 UFMylation in Control and TXNIP KO cells treated with DMSO and 2 μM JQ1. Histone H4 was immunoprecipitated and the precipitated proteins were immunoblotted with UFM1 antibody. B Overepression of TXNIP reduced H4 UFMylation. Empty vector (EV), Flag-TXNIP, and 6His-Flag-H4 were transfected into HEK293T cells. 6His-Flag-H4 proteins and their associated proteins were isolated using Ni-NTA beads and analyzed with indicated antibodies. C The interaction between histone H4 and UFBP1 in Control and TXNIP KO cells with DMSO and 2 μM JQ1 treatment. The protein complex was immunoprecipitated with UFBP1 antibody and probed with histone H4 antibody. D The interaction between histone H4 and cMYC in Control and TXNIP KO cells with and without JQ1 treatment. cMYC was immunoprecipitated and isolated proteins were subjected to Western Blotting analysis with a histone H4 antibody. E Protein UFMylation influenced the association between histone H4 and cMYC. cMYC was immunoprecipitated from Control cells treated with DMSO and JQ1 as well as TXNIP KO cells treated by JQ1 alone and JQ1 combined with DKM 2-93. The precipitates were analyzed using a histone H4 antibody. F Rescued the expression of cMYC target genes by TXNIP KO was depending on UFMylation. Control and TXNIP KO cells were treated by DMSO, 2 μM JQ1, 30 μM DKM 2-93 and a combination of JQ1 (2 μM) with DKM 2-93 (30 μM). The transcription levels of CYCLIN A2 , MAD2 , ORC2 , and MCM6 were determined by RT-qPCR. G The interaction of cMYC with Flag-UFM1-H4 and Flag-H4. HepG2 cells were transfected with empty vector (EV), 6Hist-Flag-UFM1-H4, and 6Hist-Flag-H4 and their associated proteins were pulled down using Ni-NTA beads and probed for cMYC. H UFM1-H4 alleviated the repression of CYCLIN A2 by JQ1. HepG2 cells were transfected with Empty vector (EV), Flag-H4, and Flag-UFM1-H4 and treated with 2 μM JQ1. Total proteins were extracted and CYCLIN A2 expression was analyzed via Western Blotting. **** p value < 0.001, *** p value < 0.005, ** p value < 0.01, * p value < 0.05 (one-way ANOVA test in F ).

Article Snippet: Antibodies against H3 (17168-1-AP), ubiquitin (10201-2-AP), MAD2 (10337-1-AP), and IgG (30000-0-AP) were purchased from Proteintech.

Techniques: Control, Immunoprecipitation, Plasmid Preparation, Transfection, Isolation, Western Blot, Expressing, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: BRD4 inhibition suppresses histone H4 UFMylation to increase ferroptosis sensitivity through TXNIP

doi: 10.1038/s41419-025-08166-y

Figure Lengend Snippet: A Representative images of the HCT116 xenografts treated with control vehicle, JQ1, RSL3, or JQ1 + RSL3. B The growth curve of HCT116 xenografts treated with indicated chemicals in mice. C The tumor weight of HCT116 xenografts after administration of control, JQ1, RSL3, or JQ1 + RSL3. D The expression levels of indicated proteins in HCT116 xenografts treated with control and JQ1. Total proteins were extracted from tumor tissues treated with control and JQ1. E The concentration of malondialdehyde (MDA) in tumor tissues treated with indicated compounds.**** p value < 0.001, *** p value < 0.005, ** p value < 0.01, * p value < 0.05 (one-way ANOVA test in B , C , and E ). F BRD4 inhibition increased TXNIP expression and caused cell cycle arrest, sensitizing cancer cells to ferroptosis. BET inhibitors, such as JQ1, inhibited H3K9Me3 at the TXNIP promoter and increased TXNIP expression. TXNIP stabilized CDK inhibitor P27 by inhibiting its proteasomal degradation. Additionally, TXNIP disrupted the interaction between histone H4 and UFBP1 and reduced H4 UFMylation, inhibiting cMYC binding to its target genes and silencing the transcription of cell cycle regulators, such as CYCLIN A2, MAD2, ORC2, and MCM6. Consequently, cells treated with BET inhibitors entered a dormant state and became sensitive to ferroptosis.

Article Snippet: Antibodies against H3 (17168-1-AP), ubiquitin (10201-2-AP), MAD2 (10337-1-AP), and IgG (30000-0-AP) were purchased from Proteintech.

Techniques: Control, Expressing, Concentration Assay, Inhibition, Binding Assay