Journal: Cell reports

Article Title: An Aurora kinase A-BOD1L1-PP2A B56 axis promotes chromosome segregation fidelity

doi: 10.1016/j.celrep.2025.115317

Figure Lengend Snippet:

Article Snippet: rabbit anti-PP2A B56 Epsilon , Aviva Systems Biology , Cat# RP56694_P050.

Techniques: Virus, Recombinant, Staining, shRNA, Cloning, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: An Aurora kinase A-BOD1L1-PP2A B56 Axis promotes chromosome segregation fidelity

doi: 10.1101/2023.08.06.552174

Figure Lengend Snippet: A) SW620 cells were first treated for 3 days with UMK57 or DMSO control. They were then re-plated on glass coverslips. After 24 hours, the cells were fixed and stained for Aurora B pT232, ACA and DAPI. A single experiment was performed. The scale bar is 10μm. B) SW620 cells were prepared as in (A) but stained for MCAK pS95 and ACA. A single experiment was performed. The scale bar is 10μm. C) Quantification of the relative kinetochore pT232 Aurora B/ACA intensities from the conditions from panel (A). The condition with the lowest level of pT232 Aurora B/ACA was set to 1 and the other conditions shown as fold-changes. 30 kinetochores were quantified from each of 20 cells from a single experiment. Error bars indicate the mean +/− SEM. Statistical significance was calculated between the indicated conditions using Dunnett’s multiple comparison test. D) As for C but quantifying the relative kinetochore pS95 MCAK/ACA intensities from panel (B). E) Volcano plot analysis of the total protein level proteomic screen results. Proteins of potential interest are highlighted in orange. Statistical significance was calculated using two-tailed t -tests. F) STRING analysis of the statistically significant ( p < 0.1) top-100 positively changed phosphorylated proteins and top-100 negatively changed phospho-proteins showing gene association networks. G) SW620 cells were first treated for 3 days with UMK57 or DMSO control. They were then allowed to remain asynchronous or were synchronized by thymidine-nocodazole. The cells were then harvested and lysed. The proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane and finally blotted as indicated. The shown blots are representative of 3 independent experiments.

Article Snippet: The following antibodies were used for immunofluorescence (IF) and/or immunoblotting (IB): human anti-ACA (Geisel School of Medicine; IF at 1:2000), mouse anti-Hec1 (Santa Cruz C-11; IF,IB at 1:1000), mouse anti-PP2AR1A (Santa Cruz SC-74580; IB at 1:500), mouse anti-PP2AC (Santa Cruz SC-166034; IB at 1:500), rabbit anti-PP2A B56 Alpha ( MyBioSource.com MBS8524809; IB at 1:500), mouse anti-PP2A B56 Beta (Santa Cruz E-6; IB at 1:500), mouse anti-PP2A B56 Gamma (Santa Cruz A-11; IB at 1:500), mouse anti-PP2A B56 Delta (Santa Cruz H-11; IB at 1:500), rabbit anti-PP2A B56 Epsilon (Aviva Systems Biology RP56694_P050; IB at 1:500), rabbit anti-Hec1 pS44 (gift of Jennifer DeLuca; IF at 1:500) , rabbit anti DSN1 pS100 (gift of Iain Cheeseman; IF at 1:250) , rabbit anti-KNL1 pT943/pT1155 (Cell Signaling #40758), mouse anti-α-Tubulin DM1α (Sigma-Aldrich; IF at 1:4000-1:10,000), mouse anti-Aurora A (Cell Signaling Technology 1F8; IF at1:1000), Rabbit anti-Aurora A pT288 (Cell Signaling Technology C39D8; IF at 1:500), Rabbit anti-TACC3 (gift of Jordan Raff; IF at 1:1000) , mouse anti-TPX2 (Cell Signaling Technology D9Y1V; IF at 1:1000), rabbit anti-Aurora B pT232 (Rockland 600-401-677; IF/IB at 1:1000), rabbit anti-MCAK pS95 (Abcam #AB74146; IF/IB at 1:500), rabbit anti-Hec1 pT31 (IF at 1:100,000, WB at 1:1000), rabbit anti-BOD1L1 (IF at 1:1000) (gift of Grant Stewart ), rabbit anti-BOD1L1 (Genetex #GTX119946; WB at 1:500).

Techniques: Control, Staining, Comparison, Two Tailed Test, SDS Page, Membrane

Journal: bioRxiv

Article Title: An Aurora kinase A-BOD1L1-PP2A B56 Axis promotes chromosome segregation fidelity

doi: 10.1101/2023.08.06.552174

Figure Lengend Snippet: A) Immunofluorescence images from an asynchronous population of SW620 cells transfected with control shRNA or shRNA against BOD1L1 in prometaphase or metaphase and stained for Hec1, Hec1 pT31 and DAPI. The BOD1L1 and Hec1 channels were adjusted evenly for brightness and contrast for presentation. The DAPI channel of each condition was adjusted independently. Representative images from 2 independent biological experiments are shown. The scale bars of main images are 10 μm, and insets are 1 μm. B) As for panel (A), except for KNL1 pMELT/ACA instead of Hec1 pT31/total Hec1 C) Quantification of the relative kinetochore pT31 Hec1/Hec1 intensities from the conditions from panel (A). The condition with the lowest level of pT31 Hec1/Hec1 was set to 1 and the other conditions shown as fold-changes. 25 kinetochores were quantified from each of 20 cells for each of 2 independent biological repeats. Error bars indicate the mean +/− SEM. Statistical significance was calculated between the indicated conditions using Dunnett’s multiple comparison test. D) As for panel (C), except for Hec1 pS44/total Hec1instead of Hec1 pT31/total Hec1 E) As for panel (C), except for Hec1 pS55/total Hec1 instead of Hec1 pT31/total Hec1 F) As for panel (C), except for DSN1 pS109/ACA instead of Hec1 pT31/total Hec1 G) As for panel (C), except for KNL1 pMELT/ACA instead of Hec1 pT31/total Hec1 H) As for panel (C), except for MCAK pS95/ACA instead of Hec1 pT31/total Hec1 I) As for panel (C), except for Aurora B pT232/ACA instead of Hec1 pT31/total Hec1 J) Quantification of total cellular Aurora A pT288/Total Aurora A. The condition with the lowest level of pT31 Hec1/Hec1 was set to 1 and the other conditions shown as fold-changes. The level of protein was measured from at least 25 cells for each of 2 independent biological repeats. Error bars indicate the mean +/− SEM. Statistical significance was calculated between the indicated conditions using Dunnett’s multiple comparison test.

Article Snippet: The following antibodies were used for immunofluorescence (IF) and/or immunoblotting (IB): human anti-ACA (Geisel School of Medicine; IF at 1:2000), mouse anti-Hec1 (Santa Cruz C-11; IF,IB at 1:1000), mouse anti-PP2AR1A (Santa Cruz SC-74580; IB at 1:500), mouse anti-PP2AC (Santa Cruz SC-166034; IB at 1:500), rabbit anti-PP2A B56 Alpha ( MyBioSource.com MBS8524809; IB at 1:500), mouse anti-PP2A B56 Beta (Santa Cruz E-6; IB at 1:500), mouse anti-PP2A B56 Gamma (Santa Cruz A-11; IB at 1:500), mouse anti-PP2A B56 Delta (Santa Cruz H-11; IB at 1:500), rabbit anti-PP2A B56 Epsilon (Aviva Systems Biology RP56694_P050; IB at 1:500), rabbit anti-Hec1 pS44 (gift of Jennifer DeLuca; IF at 1:500) , rabbit anti DSN1 pS100 (gift of Iain Cheeseman; IF at 1:250) , rabbit anti-KNL1 pT943/pT1155 (Cell Signaling #40758), mouse anti-α-Tubulin DM1α (Sigma-Aldrich; IF at 1:4000-1:10,000), mouse anti-Aurora A (Cell Signaling Technology 1F8; IF at1:1000), Rabbit anti-Aurora A pT288 (Cell Signaling Technology C39D8; IF at 1:500), Rabbit anti-TACC3 (gift of Jordan Raff; IF at 1:1000) , mouse anti-TPX2 (Cell Signaling Technology D9Y1V; IF at 1:1000), rabbit anti-Aurora B pT232 (Rockland 600-401-677; IF/IB at 1:1000), rabbit anti-MCAK pS95 (Abcam #AB74146; IF/IB at 1:500), rabbit anti-Hec1 pT31 (IF at 1:100,000, WB at 1:1000), rabbit anti-BOD1L1 (IF at 1:1000) (gift of Grant Stewart ), rabbit anti-BOD1L1 (Genetex #GTX119946; WB at 1:500).

Techniques: Immunofluorescence, Transfection, Control, shRNA, Staining, Comparison

Journal: bioRxiv

Article Title: An Aurora kinase A-BOD1L1-PP2A B56 Axis promotes chromosome segregation fidelity

doi: 10.1101/2023.08.06.552174

Figure Lengend Snippet: A) Immunofluorescence images from an asynchronous population of SW620 cells treated either with DMSO control, acute (1 hour) UMK57 or UMK57 for 3 days. The cells were then fixed and stained for total Aurora A and Aurora A pT288. The Aurora A and Aurora A pT288 were adjusted evenly for brightness and contrast for presentation. The DAPI channel of each condition was adjusted independently. Representative images from 2 independent biological experiments are shown. The scale bars are 10 μm. B) Quantification of the relative spindle protein intensities from the conditions from panel (A). The levels of the prometaphase DMSO conditions was set to 1 and the other conditions shown as fold-changes. The fluorescence levels for at least 30 cells for each of 2 independent biological repeats were measured. Error bars indicate the mean +/− SEM. Statistical significance was calculated between the indicated conditions using Dunnett’s multiple comparison test. C) Cells were prepared as for panel (A) but stained for TPX2 and alpha tubulin. D) Cells were prepared as for panel (A) but stained for TACC3 and tubulin. E) Quantification of the relative spindle protein intensities from panels (C) and (D). The tubulin intensities from panels (C) and (D) were combined into a single plot.

Article Snippet: The following antibodies were used for immunofluorescence (IF) and/or immunoblotting (IB): human anti-ACA (Geisel School of Medicine; IF at 1:2000), mouse anti-Hec1 (Santa Cruz C-11; IF,IB at 1:1000), mouse anti-PP2AR1A (Santa Cruz SC-74580; IB at 1:500), mouse anti-PP2AC (Santa Cruz SC-166034; IB at 1:500), rabbit anti-PP2A B56 Alpha ( MyBioSource.com MBS8524809; IB at 1:500), mouse anti-PP2A B56 Beta (Santa Cruz E-6; IB at 1:500), mouse anti-PP2A B56 Gamma (Santa Cruz A-11; IB at 1:500), mouse anti-PP2A B56 Delta (Santa Cruz H-11; IB at 1:500), rabbit anti-PP2A B56 Epsilon (Aviva Systems Biology RP56694_P050; IB at 1:500), rabbit anti-Hec1 pS44 (gift of Jennifer DeLuca; IF at 1:500) , rabbit anti DSN1 pS100 (gift of Iain Cheeseman; IF at 1:250) , rabbit anti-KNL1 pT943/pT1155 (Cell Signaling #40758), mouse anti-α-Tubulin DM1α (Sigma-Aldrich; IF at 1:4000-1:10,000), mouse anti-Aurora A (Cell Signaling Technology 1F8; IF at1:1000), Rabbit anti-Aurora A pT288 (Cell Signaling Technology C39D8; IF at 1:500), Rabbit anti-TACC3 (gift of Jordan Raff; IF at 1:1000) , mouse anti-TPX2 (Cell Signaling Technology D9Y1V; IF at 1:1000), rabbit anti-Aurora B pT232 (Rockland 600-401-677; IF/IB at 1:1000), rabbit anti-MCAK pS95 (Abcam #AB74146; IF/IB at 1:500), rabbit anti-Hec1 pT31 (IF at 1:100,000, WB at 1:1000), rabbit anti-BOD1L1 (IF at 1:1000) (gift of Grant Stewart ), rabbit anti-BOD1L1 (Genetex #GTX119946; WB at 1:500).

Techniques: Immunofluorescence, Control, Staining, Fluorescence, Comparison

Journal: bioRxiv

Article Title: An Aurora kinase A-BOD1L1-PP2A B56 Axis promotes chromosome segregation fidelity

doi: 10.1101/2023.08.06.552174

Figure Lengend Snippet: A) Immunofluorescence images from an asynchronous population of SW620 cells transfected with control shRNA or shRNA against BOD1L1 in prometaphase or metaphase and stained for Hec1, Hec1 pT31 and DAPI. The BOD1L1 and Hec1 channels were adjusted evenly for brightness and contrast for presentation. The DAPI channel of each condition was adjusted independently. Representative images from 2 independent biological experiments are shown. The scale bars of main images are 10 μm, and insets are 1 μm. B) As for panel (A), except for KNL1 pMELT/ACA instead of Hec1 pT31/total Hec1 C) Quantification of the relative kinetochore pT31 Hec1/Hec1 intensities from the conditions from panel (A). The condition with the lowest level of pT31 Hec1/Hec1 was set to 1 and the other conditions shown as fold-changes. 25 kinetochores were quantified from each of 20 cells for each of 2 independent biological repeats. Error bars indicate the mean +/− SEM. Statistical significance was calculated between the indicated conditions using Dunnett’s multiple comparison test. D) As for panel (C), except for Hec1 pS44/total Hec1instead of Hec1 pT31/total Hec1 E) As for panel (C), except for Hec1 pS55/total Hec1 instead of Hec1 pT31/total Hec1 F) As for panel (C), except for DSN1 pS109/ACA instead of Hec1 pT31/total Hec1 G) As for panel (C), except for KNL1 pMELT/ACA instead of Hec1 pT31/total Hec1 H) As for panel (C), except for MCAK pS95/ACA instead of Hec1 pT31/total Hec1 I) As for panel (C), except for Aurora B pT232/ACA instead of Hec1 pT31/total Hec1 J) Quantification of total cellular Aurora A pT288/Total Aurora A. The condition with the lowest level of pT31 Hec1/Hec1 was set to 1 and the other conditions shown as fold-changes. The level of protein was measured from at least 25 cells for each of 2 independent biological repeats. Error bars indicate the mean +/− SEM. Statistical significance was calculated between the indicated conditions using Dunnett’s multiple comparison test.

Article Snippet: The following antibodies were used for immunofluorescence (IF) and/or immunoblotting (IB): human anti-ACA (Geisel School of Medicine; IF at 1:2000), mouse anti-Hec1 (Santa Cruz C-11; IF,IB at 1:1000), mouse anti-PP2AR1A (Santa Cruz SC-74580; IB at 1:500), mouse anti-PP2AC (Santa Cruz SC-166034; IB at 1:500), rabbit anti-PP2A B56 Alpha ( MyBioSource.com MBS8524809; IB at 1:500), mouse anti-PP2A B56 Beta (Santa Cruz E-6; IB at 1:500), mouse anti-PP2A B56 Gamma (Santa Cruz A-11; IB at 1:500), mouse anti-PP2A B56 Delta (Santa Cruz H-11; IB at 1:500), rabbit anti-PP2A B56 Epsilon (Aviva Systems Biology RP56694_P050; IB at 1:500), rabbit anti-Hec1 pS44 (gift of Jennifer DeLuca; IF at 1:500) , rabbit anti DSN1 pS100 (gift of Iain Cheeseman; IF at 1:250) , rabbit anti-KNL1 pT943/pT1155 (Cell Signaling #40758), mouse anti-α-Tubulin DM1α (Sigma-Aldrich; IF at 1:4000-1:10,000), mouse anti-Aurora A (Cell Signaling Technology 1F8; IF at1:1000), Rabbit anti-Aurora A pT288 (Cell Signaling Technology C39D8; IF at 1:500), Rabbit anti-TACC3 (gift of Jordan Raff; IF at 1:1000) , mouse anti-TPX2 (Cell Signaling Technology D9Y1V; IF at 1:1000), rabbit anti-Aurora B pT232 (Rockland 600-401-677; IF/IB at 1:1000), rabbit anti-MCAK pS95 (Abcam #AB74146; IF/IB at 1:500), rabbit anti-Hec1 pT31 (IF at 1:100,000, WB at 1:1000), rabbit anti-BOD1L1 (IF at 1:1000) (gift of Grant Stewart ), rabbit anti-BOD1L1 (Genetex #GTX119946; WB at 1:500).

Techniques: Immunofluorescence, Transfection, Control, shRNA, Staining, Comparison