Journal: bioRxiv

Article Title: hSpindly’s dynamic controls SAC activity independently of the KBB pathway at unattached kinetochores

doi: 10.1101/2025.07.25.666731

Figure Lengend Snippet: A. Stable inducible RPE-1 GFP-hSpindly cells were treated or not with doxycycline (DOXY). Cell extracts were prepared and immunoblotted with the indicated antibodies. In Wb hSpindly, GFP-hSpindly is the upper band and hSpindly endogenous is the lower band. B. Representative images of hSpindly levels at kinetochores of RPE-1 GFP-hSpindly treated or not with DOXY in the presence of 3.3µM nocodazole (NZ). Endogenous or exogenous hSpindly were visualized by hSpindly antibody or GFP, respectively. Bub1 or CENP-C antibodies were used as kinetochore markers, and DAPI to visualize the DNA. Scale bar: 10μm; insets, 2x zoom. C. Violin plots representing intensity or total area of hSpindly ENDO , hSpindly EXO and Bub1 at kinetochores from the experiment shown in panel B. Lines indicate mean or standard deviation. Statistical analysis was performed with a non-parametric t-test comparing two unpaired groups (***, p<0.001). D. RPE-1 GFP-hSpindly cell extracts after NZ and DOXY treatment for 24 hours were immunoprecipitated with anti-hSpindly (I) or normal mouse serum (PI) as control. Immunoprecipitated materials were analyzed by western blot using the antibodies indicated. E. Stable inducible RPE-1 GFP-hSpindly cells were treated with DOXY and NZ for 24 hours. Before collecting, mitotic cells were incubated with 0.5μM hesperadin or 1μM reversine in the presence of 20μM proteasome inhibitor MG132 for 90 minutes. Cell extracts were used to immunoprecipitate as in D. F, G RPE-1 GFP-hSpindly cell extracts treated with NZ and DOXY for 24 hours were immunoprecipitated using anti-Mad1 or anti-Mad2 antibodies (I), with normal mouse serum used as a control (PI). Anti-Mad2 was used as a control of Mad1 immunoprecipitation. Western blot analysis was performed using the specified antibodies. E: Extracts; Mw: Molecular weight; I: Immune; PI: Preimmune. The upper band marked with asterisk is an unspecific band.

Article Snippet: Proteins were separated by SDS-polyacrilamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with the following antibodies: anti-Bub1 rabbit antibody (1:500, GeneTex GTX30097), anti-BubR1 rabbit antibody (1:1,000, Bethyl Laboratories A300-386A); anti-CyclinB mouse antibody (1:1,000, BD Transduction Lab 610220); anti-β Actin HRP antibody (1:5,000, Santa Cruz sc-47778); anti-GFP rabbit antibody (1:20,000, Immune Systems RGFP-45A); anti-Aurora A kinase rabbit antibody (1:2,000, Novus Biologicals 51843); anti-α tubulin mouse antibody (1:20,000, Sigma T9026); anti-Mad2 rabbit antibody (1:500, Bethyl Laboratories A300-301A); anti-Mad1 mouse antibody (1:500, Santa Cruz sc-47746); anti-hSpindly rabbit antibody (1:1,000, Bethyl Laboratories A301-354A).

Techniques: Standard Deviation, Immunoprecipitation, Control, Western Blot, Incubation, Molecular Weight

Journal: bioRxiv

Article Title: hSpindly’s dynamic controls SAC activity independently of the KBB pathway at unattached kinetochores

doi: 10.1101/2025.07.25.666731

Figure Lengend Snippet: A. RPE-1 GFP-hSpindly cells were washed out after 24 hours of NZ treatment without or with DOXY and collected every 30 minutes. Cell extracts were analyzed by western blot with the antibodies indicated. B. RPE-1 GFP-hSpindly cells were treated with NZ with or without DOXY for 24 hours and collected after 1μm reversine (left panel) or 0.5μm hesperadin (right panel) addition at indicated times. Cell extracts were prepared and immunoblotted with the indicated antibodies. C. Representative images of GFP-hSpindly, Bub1, CENP-C or Mad2 levels at kinetochores in RPE-1 GFP-hSpindly cells treated or not with DOXY in the presence of NZ. DAPI to visualize the DNA. Scale bar: 10μm; insets, 2x zoom. D. Box plots representing intensity of Mad2 at kinetochores from the experiment shown in panel C. Black lines indicate mean. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (***, p<0.001).

Article Snippet: Proteins were separated by SDS-polyacrilamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with the following antibodies: anti-Bub1 rabbit antibody (1:500, GeneTex GTX30097), anti-BubR1 rabbit antibody (1:1,000, Bethyl Laboratories A300-386A); anti-CyclinB mouse antibody (1:1,000, BD Transduction Lab 610220); anti-β Actin HRP antibody (1:5,000, Santa Cruz sc-47778); anti-GFP rabbit antibody (1:20,000, Immune Systems RGFP-45A); anti-Aurora A kinase rabbit antibody (1:2,000, Novus Biologicals 51843); anti-α tubulin mouse antibody (1:20,000, Sigma T9026); anti-Mad2 rabbit antibody (1:500, Bethyl Laboratories A300-301A); anti-Mad1 mouse antibody (1:500, Santa Cruz sc-47746); anti-hSpindly rabbit antibody (1:1,000, Bethyl Laboratories A301-354A).

Techniques: Western Blot

Journal: bioRxiv

Article Title: hSpindly’s dynamic controls SAC activity independently of the KBB pathway at unattached kinetochores

doi: 10.1101/2025.07.25.666731

Figure Lengend Snippet: A. RPE-1 GFP-hSpindly prometaphase cells induced by DOXY were treated or not with 10μM BAY 1816032 inhibitor for 5 hours in the presence of 20μM MG132. Cell extracts were used to immunoprecipitate hSpindly (I) and normal mouse serum (PI) as a control. Immunoprecipated material was analyzed by western blot. B. RPE-1 GFP-hSpindly were transfected with siRNABub1 for 48 hours. Cells were treated with NZ and DOXY 24 hours before harvesting. Extracts were used to immunoprecipitate Mad1 (I) using normal mouse serum as a control (PI). Complexes were analyzed by immunoblotting with antibodies as indicated. C. Representative images of RPE-1 GFP-hSpindly prometaphase cells in the presence or not of DOXY after Bub1 or KNL1 siRNA treatment for 48 hours. Scale bar: 10μm; insets, 2x zoom. D. Scatter dot boxes representing intensity of Mad2 at kinetochores from the experiment shown in panel C. Black lines indicate mean. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups. (***, p<0.001). E. Representative images of a magnification of kinetochores in RPE-1 GFP-hSpindly prometaphase cells after 48 hours of Bub1 siRNA in the presence of DOXY. Scale bar: 10μm. F. Box plots representing colocalization of Mad2-hSpindly at kinetochores from the experiment shown in panel E. The Manders’ M 1 coefficient is shown. Statistical analysis was performed with a parametric model comparing two unpaired groups. (**, p<0.01). G. Colony assays showing cell viability for RPE-1 GFP-hSpindly cells transfected with siRNABub1 were performed by measuring absorbance 570nm and represented in graphs. Error bars represent the SD (n=3). *p<0.05 (Student’s t-test). E: Extracts; Mw: Molecular weight; I: Immune; PI: Preimmune.

Article Snippet: Proteins were separated by SDS-polyacrilamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with the following antibodies: anti-Bub1 rabbit antibody (1:500, GeneTex GTX30097), anti-BubR1 rabbit antibody (1:1,000, Bethyl Laboratories A300-386A); anti-CyclinB mouse antibody (1:1,000, BD Transduction Lab 610220); anti-β Actin HRP antibody (1:5,000, Santa Cruz sc-47778); anti-GFP rabbit antibody (1:20,000, Immune Systems RGFP-45A); anti-Aurora A kinase rabbit antibody (1:2,000, Novus Biologicals 51843); anti-α tubulin mouse antibody (1:20,000, Sigma T9026); anti-Mad2 rabbit antibody (1:500, Bethyl Laboratories A300-301A); anti-Mad1 mouse antibody (1:500, Santa Cruz sc-47746); anti-hSpindly rabbit antibody (1:1,000, Bethyl Laboratories A301-354A).

Techniques: Control, Western Blot, Transfection, Molecular Weight

Journal: bioRxiv

Article Title: hSpindly’s dynamic controls SAC activity independently of the KBB pathway at unattached kinetochores

doi: 10.1101/2025.07.25.666731

Figure Lengend Snippet: A. Stable inducible RPE-1 GFP-hSpindly wild-type (wt) or GFP-hSpindly T552A (T552A) cells were treated or not with doxycycline (DOXY) in the presence of NZ for 24 hours. Cell extracts were prepared and immunoblotted with the indicated antibodies. Endogenous hSpindly (lower band) was used as loading control B. Representative images of hSpindly T552A levels at kinetochores of RPE-1 GFP-hSpindlyT552A prometaphase cells treated or not with DOXY. hSpindlyT552A or Mad2 were visualized by GFP or anti-Mad2, respectively. Bub1 or CENP-C antibodies were used as kinetochore markers, and DAPI to visualize the DNA. Scale bar: 10μm; insets, 2x zoom. C. Violin plots representing intensity or total area of hSpindly T552A compared with hSpindly wild-type (data ). Black dots or lines indicate mean or standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups. (ns, not significant). D. Distribution of hSpindly EXO or hSpindly-T552A EXO populations based on kinetochore signal intensity and area. Each dot represents a kinetochore; lines indicate the mean. E. RPE-1 GFP-hSpindly wt or GFP-hSpindly T552A induced cell extracts after NZ treatment were immunoprecipitated with GFP-Trap beads. Immunoprecipitated materials were analyzed by western blot using the antibodies indicated. E: Extracts; I: Immune.

Article Snippet: Proteins were separated by SDS-polyacrilamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with the following antibodies: anti-Bub1 rabbit antibody (1:500, GeneTex GTX30097), anti-BubR1 rabbit antibody (1:1,000, Bethyl Laboratories A300-386A); anti-CyclinB mouse antibody (1:1,000, BD Transduction Lab 610220); anti-β Actin HRP antibody (1:5,000, Santa Cruz sc-47778); anti-GFP rabbit antibody (1:20,000, Immune Systems RGFP-45A); anti-Aurora A kinase rabbit antibody (1:2,000, Novus Biologicals 51843); anti-α tubulin mouse antibody (1:20,000, Sigma T9026); anti-Mad2 rabbit antibody (1:500, Bethyl Laboratories A300-301A); anti-Mad1 mouse antibody (1:500, Santa Cruz sc-47746); anti-hSpindly rabbit antibody (1:1,000, Bethyl Laboratories A301-354A).

Techniques: Control, Standard Deviation, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: hSpindly’s dynamic controls SAC activity independently of the KBB pathway at unattached kinetochores

doi: 10.1101/2025.07.25.666731

Figure Lengend Snippet: A. Representative images of RPE-1 GFP-hSpindlyT552A prometaphase cells in the presence or not of DOXY after Mock or Bub1 siRNA treatment for 48 hours. GFP-hSpindly T552A, Bub1, CENP-C, Mad2 were visualized with green fluorescence protein or by using the indicated antibodies. Scale bar: 10μm; insets, 2x zoom. B. Box plots representing intensity of Mad2 at kinetochores from the experiment shown in panel A. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups. C. Representative images of a magnification of Kinetochores in RPE-1 GFP-hSpindly T552A prometaphase cells after 48 hours of Mock or Bub1 siRNA in the presence of DOXY. Scale bar: 10μm. D. Box plots representing colocalization of Mad2-hSpindly at kinetochores from the experiment shown in panel C. The Manders’ M 1 coefficient is shown. Black lines indicate mean. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups. (ns, not significant).

Article Snippet: Proteins were separated by SDS-polyacrilamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with the following antibodies: anti-Bub1 rabbit antibody (1:500, GeneTex GTX30097), anti-BubR1 rabbit antibody (1:1,000, Bethyl Laboratories A300-386A); anti-CyclinB mouse antibody (1:1,000, BD Transduction Lab 610220); anti-β Actin HRP antibody (1:5,000, Santa Cruz sc-47778); anti-GFP rabbit antibody (1:20,000, Immune Systems RGFP-45A); anti-Aurora A kinase rabbit antibody (1:2,000, Novus Biologicals 51843); anti-α tubulin mouse antibody (1:20,000, Sigma T9026); anti-Mad2 rabbit antibody (1:500, Bethyl Laboratories A300-301A); anti-Mad1 mouse antibody (1:500, Santa Cruz sc-47746); anti-hSpindly rabbit antibody (1:1,000, Bethyl Laboratories A301-354A).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: hSpindly’s dynamic controls SAC activity independently of the KBB pathway at unattached kinetochores

doi: 10.1101/2025.07.25.666731

Figure Lengend Snippet: A. Colony assays showing cell viability of RPE-1 GFP-hSpindly or RPE-1 GFP-hSpindly T552A were performed after 24 hours of NZ treatment in the presence of DOXY and in the absence of both endogenous hSpindly and KNL1. Colony forming was quantified by measuring absorbance 570nm and represented in graphs. Error bars represent the SD (n=3). ***, p<0.001 (Student’s t-test). B. Model to illustrate how spindle checkpoint regulation is controlled by the KBB and RZZ pathways at unattached kinetochores. Bub1 kinase activity likely controls the activation of the RZZ pathway by the phosphorylation of hSpindly. hSpindly acts as a recruiter of the Mad1-Mad2 complex controlling the MCC formation with the KBB pathway.

Article Snippet: Proteins were separated by SDS-polyacrilamide gel electrophoresis (SDS-PAGE) and gels were electroblotted onto nitrocellulose membranes and probed with the following antibodies: anti-Bub1 rabbit antibody (1:500, GeneTex GTX30097), anti-BubR1 rabbit antibody (1:1,000, Bethyl Laboratories A300-386A); anti-CyclinB mouse antibody (1:1,000, BD Transduction Lab 610220); anti-β Actin HRP antibody (1:5,000, Santa Cruz sc-47778); anti-GFP rabbit antibody (1:20,000, Immune Systems RGFP-45A); anti-Aurora A kinase rabbit antibody (1:2,000, Novus Biologicals 51843); anti-α tubulin mouse antibody (1:20,000, Sigma T9026); anti-Mad2 rabbit antibody (1:500, Bethyl Laboratories A300-301A); anti-Mad1 mouse antibody (1:500, Santa Cruz sc-47746); anti-hSpindly rabbit antibody (1:1,000, Bethyl Laboratories A301-354A).

Techniques: Activity Assay, Activation Assay, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: p31Comet Splice Variants Induce Distinct Spindle Assembly Checkpoint Dynamics due to Their Unique N-Termini

doi: 10.3390/ijms26073089

Figure Lengend Snippet: Figure 3. Variant 1 exhibits reduced SAC silencing and protein stability in vivo. (A) Western blot of MAD2 immunoprecipitation measuring binding of p31Comet variants. (B) Quantification of immunoprecipitation performed in (A). p31Comet levels were normalized to MAD2. (C) Mitotic index of HeLa cells transiently expressing p31Comet variants/mutants. Mitotic cells were determined by immunofluorescence analysis of phospho-Histone H3 S10 positivity; >100 cells were counted for each replicate, * p < 0.05. The data were analyzed by an Unpaired t-Test. (D) Mitotic timing of HeLa cells expressing p31Comet variants/mutants from 3 separate experiments. Experiments were performed in the presence of 12.5 ng/mL Nocodazole. *** p < 0.001, **** p < 0.0001. The data were analyzed by 2-way ANOVA (E) Western blot of p31Comet variant protein levels over an 8 h time course in the presence of 100 µg/mL CHX to measure differences in stability of p31Comet variants. (F). Quantification of stability assays in (E), N = 3, ** p < 0.01. The data were analyzed by 2-way ANOVA.

Article Snippet: The following antibodies were used in this study: Anti-Cyclin B (mouse), (BD Pharmingen, Franklin Lakes, NJ, USA, 554177); Anti-CMT2 (rabbit) (Abcam, Cambridge, UK, ab150363); Anti-MAD2 (rabbit), Bethyl, Montgomery, TX, USA, A300-301A; Anti-MAD2 (mouse), (BD Transduction Laboratories, Franklin Lakes, NJ, USA 610679); Anti-S-Tag (mouse) (Sigma, St. Louis, MO, USA, SAB2702204); Anti-GST (rabbit), (Santa-Cruz, Dallas, TX, USA, SC-459); Anti-phospho-Histone H3 S10 (rabbit), (Millipore Sigma, St. Louis, MO, USA, 06-570).

Techniques: Variant Assay, In Vivo, Western Blot, Immunoprecipitation, Binding Assay, Expressing, Immunofluorescence

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Scores of the signature genes resulted from the differential expression analysis using as input the EGA RNA-seq data compared to the three lung samples.

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Expressing

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: A The blot of siRNA BAG2 and MDK, C the blot of siRNA MAD2L1. β-Tubulin was used as a loading control and GAPDH was the positive control of the siRNA transfection. As shown in the blots and also in graphs ( B ) and ( D ) there was a significant reduction of the protein level in the silenced cells, as compared to the negative CTRL. E Graph showing the comparison of protein expression in MeT-5A and MSTO cell lines of BAG2, MAD2L1, and MDK (Mann–Whitney test; p value < 0.05 were considered significant, p value = 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****)).

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Control, Positive Control, Transfection, Comparison, Expressing, MANN-WHITNEY

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: A AOC of MeT-5A and MSTO at 72 h after treatment with siRNA-negative-control (siRNA CTRL−), siRNA BAG2, siRNA MAD2L1, siRNA MDK. The AOC is normalized to the time of transfection ( t 0 ). B Overall, FI of MeT-5A and MSTO was measured at 72 h after silencing; the graphs show the comparison between MeT-5A and MSTO for the three-siRNA transfection. ANOVA test; p value = 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****). C – E End point of apoptosis analysis in MSTO and MeT-5A cells in the presence of 5 µM caspase 3/7 Green Dye and siRNA BAG2, siRNA MAD2L1, and siRNA MDK, respectively. Mann–Whitney test; p value 0.0332(*), 0.0021(**), 0.0002(***), <0.0001(****).

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Negative Control, Transfection, Comparison, MANN-WHITNEY

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: A – D Representative BAG2 immunostainings in RMP, EMM, BMM, and SMM, respectively. Moderate to strong expression in the three types of MPM, while no expression is visible in the hyperplastic mesothelium in RMP (red arrow). E – H Representative MAD2L1 immunostainings in RMP, EMM, BMM, and SMM, respectively. Strong expression is present in the three types of MPM, but there are areas of discernable expression in the mesothelial cells on the pleural surface in RMP as well (blue arrow positive mesothelial area, red arrow negative area). I – L Representative MDK immunostainings in RMP, EMM, BMM, and SMM, respectively. Strong expression is seen in the three types of MPM; however, some focal expression is also detectable in the mesothelial cells on the pleural surface in RMP (blue arrow focal staining, red arrow negative staining). Magnification: RMPs ×400, MPMs ×200.

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Expressing, Staining, Negative Staining

Journal: Cancer Gene Therapy

Article Title: BAG2 , MAD2L1 , and MDK are cancer-driver genes and candidate targets for novel therapies in malignant pleural mesothelioma

doi: 10.1038/s41417-024-00805-4

Figure Lengend Snippet: Graphs for BAG2 immunostaining in RMP vs MPM regardless of subtype ( A ) and RMP vs the different subtypes of MPM ( B ), respectively. Graphs for MAD2L1 immunostaining in RMP vs MPM regardless of subtype ( C ) and RMP vs the different subtypes of MPM ( D ), respectively. Graphs for MDK immunostaining in RMP vs MPM regardless of subtype ( E ) and RMP vs the different subtypes of MPM ( F ), respectively. Kruskal–Wallis test followed by a post hoc Dunn’s test with Bonferroni correction was used to evaluate the statistical difference between the median of each group, p value <0.001(***), <0.01(**), <0.05(*).

Article Snippet: IHC was carried out on 3.5 μm paraffin sections by using recombinant Anti-BAG2 Rabbit mAb [EPR3567] (1:400), Anti-Midkine Rabbit mAb [EP1143Y] (1:100; cod ab79406, ab52637; Abcam, Cambridge, MA, USA), and Anti-MAD2 (MAD2L1) Rabbit pAb (1:250; cod TA308923; OriGene, Rockville, MD, USA).

Techniques: Immunostaining