Journal: Scientific Reports

Article Title: Spatiotemporal dynamics of Aurora B-PLK1-MCAK signaling axis orchestrates kinetochore bi-orientation and faithful chromosome segregation

doi: 10.1038/srep12204

Figure Lengend Snippet: ( a ) Nocodazole-arrested HeLa cells were treated with BI2536 or DMSO for another 1 hr, followed by Western blot analysis with antibodies against pSer715-MCAK, MCAK, pThr210-PLK1, PLK1 and α-tubulin, respectively. ( b ) Images of mitotic HeLa cells treated with BI2536 or DMSO. Cell were fixed and stained with anti-pSer715-MCAK antibody (green), anti-pThr210-PLK1 antibody (red), ACA (blue) and DAPI. Insets on the right show single focal planes of the boxed regions. Scale bars, 10 μm and 1 μm (enlarged images). ( c , d ) HeLa cells were synchronized to G 1 /S boundary by a double thymidine block or prometaphase by Nocodazole treatment. Cells were then harvested and analyzed by Western blotting with the indicated antibodies. Tubulin and MCAK served as a loading control respectively in ( c ) and ( d ). ( e ) Double thymidine treated HeLa cells were released into fresh medium at indicated time. The harvested cells were then analyzed by Western blotting with antibodies against pSer715-MCAK, MCAK, PLK1, CyclinB and α-tubulin (loading control). ( f ) Images of HeLa cells stained with anti-pSer715-MCAK antibody (green), ACA (red), DAPI (blue) and anti-α-tubulin antibody. Representative kinetechores are enlarged and shown on the right . Scale bars, 10 μm and 1 μm (enlarged images).

Article Snippet: Other antibodies were obtained from commercial sources: mouse anti-PLK1 monoclonal antibody (Invitrogen); anti-phospho-Thr210 PLK1 antibody (BD Biosciences); anti-Cyclin B antibody (BD Biosciences); mouse anti-EB1 antibody (BD Biosciences); mouse monoclonal anti-GFP antibody (BD Biosciences); anti-α-tubulin antibody DM1A (Sigma-Aldrich).

Techniques: Western Blot, Staining, Blocking Assay

Journal: Scientific Reports

Article Title: Spatiotemporal dynamics of Aurora B-PLK1-MCAK signaling axis orchestrates kinetochore bi-orientation and faithful chromosome segregation

doi: 10.1038/srep12204

Figure Lengend Snippet: ( a ) HeLa cells synchronized to mitosis by Nocodazole were treated respectively with BI2536, VX-680, ZM447439 or DMSO for another 1 hr, followed by Western blot analysis with the indicated antibodies ( b , e ) Synchronized mitotic HeLa cells were treated with BI2536, ZM447439 or DMSO for 1 hr. Cells were then fixed and stained with anti-pSer715-MCAK antibody or anti-pThr210-PLK1 antibody (green), ACA (red) and DAPI (blue). The enlargements show the representative centromeres. ( c , f ). Statistical analysis of pSer715-MCAK and pThr210-PLK1 immunofluorescence intensity at centromeres in ( b ) and ( e ), respectively. The intensity ratio in DMSO-treated group was normalized to 1. Data are shown as means ± SE and derived from at least 10 cells for each condition. ** P < 0.01, *** P < 0.001, Student’s t -test. ( d ) Recombinant GST-PLK1 was phosphorylated by Aurora kinases. In vitro phosphorylation reactions were performed as described in Methods. GST-PLK1 was visualized by Ponceau S staining and phosphorylation was detected by the antibody against pThr210-PLK1 by Western blotting. ( g ) Color-coded images of HeLa cells expressing a centromere-targeted PLK1 FRET sensor in prometaphase of cells arrested by Taxol treatment. PLK1 inhibitor (GW843682x) or Aurora B inhibitor (Hesperadin) was added at the indicated time point during live-cell imaging. Timestamps relative to drug addition is annotated in min. ( h ) Statistical analysis of the FRET/CFP emission ratio on centromeres at the indicated time ( g ). Data are presented as means ± SE derived from over 100 kinetochores of each category from five different cells. Scale bars, 10 μm (all image panels).

Article Snippet: Other antibodies were obtained from commercial sources: mouse anti-PLK1 monoclonal antibody (Invitrogen); anti-phospho-Thr210 PLK1 antibody (BD Biosciences); anti-Cyclin B antibody (BD Biosciences); mouse anti-EB1 antibody (BD Biosciences); mouse monoclonal anti-GFP antibody (BD Biosciences); anti-α-tubulin antibody DM1A (Sigma-Aldrich).

Techniques: Western Blot, Staining, Immunofluorescence, Derivative Assay, Recombinant, In Vitro, Expressing, Live Cell Imaging

Journal: Scientific Reports

Article Title: Spatiotemporal dynamics of Aurora B-PLK1-MCAK signaling axis orchestrates kinetochore bi-orientation and faithful chromosome segregation

doi: 10.1038/srep12204

Figure Lengend Snippet: ( a ) Color-coded images of HeLa cells expressing PLK1 or Aurora B sensor show chromosome alignment after Syntelin washout. Cells were treated with 1 μM Syntelin for 30 min followed by three washes for subsequent real-time imaging analyses. Timestamps are in minutes. ( b ) Quantitative analysis of FRET/CFP emission ratio on centromeres relative to the metaphase plate. Schematic illustration of centromere position calculation (see also ). ( c ) HeLa cells were treated with MG132 for 1 hr to allow for metaphase plate formation. Parallel samples were then treated with 1 μM Taxol or DMSO for 40 min before staining for PLK1 (green), Aurora B (red) and ACA (blue). Insets show individual kinetochore pairs used for line scans. ( d ) Live-cell imaging of chromosome segregation in GFP-MCAK constructs-addback cells. Cells were treated according to the protocol outlined in the upper panel. Arrows indicate lagging chromosomes during segregation. Timestamps in hr:min. ( e ) Quantitative analysis of the defective anaphases seen in ( d ). Data are presented as means ± SD from three independent experiments. * P < 0.05, Student’s t -test. ( f ) Cold-stable KT-MT attachment in mCherry-MCAK mutants-addback cells. Cells treated with Monastrol were released into MG132-containing medium for another 1 hr, and then fixed and stained for MTs (green), ACA (red) and DAPI (blue). Arrows indicate erroneous kinetochore attachments. ( g ) Quantitative analysis of cells exhibiting one or more aberrant attachments in various mutant MCAK-expressing cells. Data are presented as means ± SD from three independent experiments. * P < 0.05, Student’s t -test. ( h ) Model of accurate regulation of MCAK by Aurora B-PLK1 axis at kinetochore. Aurora B locates closely to PLK1 at prophase, enabling PLK1 to be activated by Aurora B at inner-centromeres (upper row). During the prometaphase-to-metaphase transition, stretch on kinetochore pairs may separate PLK1 and MCAK from Aurora B to outer-kinetochores. The stimulated MCAK activity ensures error correction in KT-MT attachment (middle row). Once the amphitelic attachments are generated, both activities of PLK1 and MCAK are down-regulated, and anaphase begins with chromosomes segregating towards the opposite poles (lower row). Scale bars, 10 μm (all image panels).

Article Snippet: Other antibodies were obtained from commercial sources: mouse anti-PLK1 monoclonal antibody (Invitrogen); anti-phospho-Thr210 PLK1 antibody (BD Biosciences); anti-Cyclin B antibody (BD Biosciences); mouse anti-EB1 antibody (BD Biosciences); mouse monoclonal anti-GFP antibody (BD Biosciences); anti-α-tubulin antibody DM1A (Sigma-Aldrich).

Techniques: Expressing, Imaging, Staining, Live Cell Imaging, Construct, Mutagenesis, Activity Assay, Generated

Journal: bioRxiv

Article Title: Mitotic phosphorylation of ADAR1 regulates its centromeric localization and is required for faithful mitotic progression

doi: 10.1101/2025.05.28.656747

Figure Lengend Snippet: (A) Flow cytometry analysis showing DNA content (DRAQ5) on the x-axis and phospho-Histone H3 (Ser10) on the y-axis. HeLa cells were transfected with control siRNA (siControl, upper panels) or siRNA targeting ADAR1 (siADAR1, lower panels), and collected at 48, 60, and 72 h post-transfection (left to right). Percentages of cells in each cell cycle phase (G1, S, G2/M) and mitotic population (M, boxed area) are indicated. Green dots represent cells positive for phospho-Histone H3 (Ser10), indicating mitotic cells, while purple dots represent non-mitotic populations. (B) Western blotting analysis of mitotic and DNA damage markers following ADAR1 knockdown. HCT116 (left) and HeLa (right) cells were transfected with control siRNA (siControl) or two independent siRNAs targeting ADAR1 (siADAR1-1 and siADAR1-2). Protein lysates were collected and subjected to immunoblotting for ADAR1, cleaved PARP, phospho-DNA-PKcs, phospho-RPA32, RPA32, γH2AX, Cyclin B1, phospho-histone H3 (Ser10), phospho-PLK1 (T210), PLK1, phospho-Aurora A (T288), Aurora A, phospho-CDC2 (T15), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control). Markers were selected to assess cell cycle status during mitosis and the presence of DNA damage.

Article Snippet: The following primary antibodies were used for western blotting and IP: anti-ADAR1 (Santa Cruz, sc-73408, 1:1000), anti-GAPDH (Cell Signaling Technology [CST], #5174, 1:4000), anti-γH2AX (S139) (CST, #9718, 1:500), anti-phospho-Histone H3 (S10) (CST, #53348, 1:1000), anti-phospho-Aurora A/B/C (Thr288/232/198) (CST, #14475, 1:1000), anti-Aurora A (CST, #2914, 1:1000), anti-Aurora B (Abcam, ab2254, 1:1000), anti-phospho-PLK1 (T210) (CST, #9062, 1:1000), anti-PLK1 (Santa Cruz, sc-17783, 1:500), anti-CDC25C (CST, #4688, 1:1000), anti-α-tubulin (CST, #2125, 1:1000), anti-SMC1 (CST, #6892, 1:1000), anti-SMC2 (CST, #5392, 1:1000), anti-SMC3 (CST, #5696, 1:1000), anti-RAD21 (CST, #4321, 1:1000), and anti-DHX9 (Abcam, ab26271, 1:1000).

Techniques: Flow Cytometry, Transfection, Control, Western Blot, Knockdown

Journal: bioRxiv

Article Title: Mitotic phosphorylation of ADAR1 regulates its centromeric localization and is required for faithful mitotic progression

doi: 10.1101/2025.05.28.656747

Figure Lengend Snippet: (A) Identification of mitotic regulators interacting with ADAR1 by co-immunoprecipitation (co-IP). HeLa cells expressing Flag-tagged ADAR1p110 or wild-type (WT) controls were harvested under asynchronous (Async) or mitotically synchronized (Msync) conditions. Cell lysates were subjected to anti-Flag IP, and both input, flow-through (FT), and IP fractions were analyzed by western blotting. Immunoblots were probed for candidate interacting proteins, including DExD/H-box helicase (DHX9), Aurora kinases (Aurora A, B, C), PLK1, CDC25C, SMC3, SMC2, and phospho-specific forms of Aurora kinases and PLK1. GAPDH served as a negative control for nonspecific binding. (B) Lysates from Flag-ADAR1p110-expressing cells were subjected to IP using an anti-Flag antibody, and eluted protein complexes were re-immunoprecipitated using either anti-SMC3 or control IgG antibodies.

Article Snippet: The following primary antibodies were used for western blotting and IP: anti-ADAR1 (Santa Cruz, sc-73408, 1:1000), anti-GAPDH (Cell Signaling Technology [CST], #5174, 1:4000), anti-γH2AX (S139) (CST, #9718, 1:500), anti-phospho-Histone H3 (S10) (CST, #53348, 1:1000), anti-phospho-Aurora A/B/C (Thr288/232/198) (CST, #14475, 1:1000), anti-Aurora A (CST, #2914, 1:1000), anti-Aurora B (Abcam, ab2254, 1:1000), anti-phospho-PLK1 (T210) (CST, #9062, 1:1000), anti-PLK1 (Santa Cruz, sc-17783, 1:500), anti-CDC25C (CST, #4688, 1:1000), anti-α-tubulin (CST, #2125, 1:1000), anti-SMC1 (CST, #6892, 1:1000), anti-SMC2 (CST, #5392, 1:1000), anti-SMC3 (CST, #5696, 1:1000), anti-RAD21 (CST, #4321, 1:1000), and anti-DHX9 (Abcam, ab26271, 1:1000).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Western Blot, Negative Control, Binding Assay, Control

Journal: bioRxiv

Article Title: Mitotic phosphorylation of ADAR1 regulates its centromeric localization and is required for faithful mitotic progression

doi: 10.1101/2025.05.28.656747

Figure Lengend Snippet: (A) HeLa cells were collected under asynchronous (Async), mitotically arrested (M; nocodazole-treated), or S phase-arrested (S; thymidine-treated) conditions. Cell lysates were analyzed by SDS-PAGE with (+) or without (–) Phos-tag acrylamide to detect phosphorylated ADAR1p110. λ-Phosphatase treatment was used to confirm phosphorylation dependency. In Phos-tag gels (top panel), ADAR1p110 exhibited a mobility shift that was strongly enhanced in mitotic samples, appearing as multiple slower-migrating bands. This shift was abolished by phosphatase treatment, indicating that the observed shift is phosphorylation-dependent. Conventional SDS-PAGE (bottom panel) was performed to assess total ADAR1p110 and ADAR1p150 protein levels as loading controls. (B) Mass spectrometry-based phosphopeptide mapping was performed on 3×Flag-tagged ADAR1p110 purified from 293T cells under mitotically synchronized conditions. The amino acid sequence starting from residue 514 is shown. Orange marks indicate phosphorylation sites. Below the sequence, a schematic representation of ADAR1p110 is provided, including the Z-DNA binding domain (green), the dsRBDs (blue), and the deaminase domain (red). (C) HeLa cells were transfected with siRNA targeting the 3′-untranslated region (3′-UTR) of ADAR1, followed by transfection with mCherry-tagged ADAR1p110 constructs. The constructs included WT, phospho-mimetic mutants (3×D and S614D), and phospho-deficient mutants (3×A and S614A). Cells were harvested 48 h after transfection, and total lysates were analyzed by western blotting using antibodies against mCherry (exogenous ADAR1p110), endogenous ADAR1p110, phospho-histone H3 (Ser10), and GAPDH. Phospho-histone H3 (S10) band intensity was used as a readout for mitotic accumulation under each condition. (D) HeLa cells were treated with kinase inhibitors under Async or Msync conditions. Cells were exposed to PLK1 inhibitors BI2536 and GSK461364, and CDK12/13 inhibitors SR-4835 and THZ531, across indicated concentrations. Whole-cell lysates were analyzed by Phos-tag SDS-PAGE followed by immunoblotting to detect phosphorylated ADAR1p110. Phosphorylated forms were visualized as slower-migrating bands. A decrease or disappearance of these bands indicates a loss of phosphorylation upon kinase inhibition. (E) HeLa cells were synchronized in Msync using nocodazole and transfected with either control siRNA (siNC1) or CDK13-targeting siRNA (siCDK13). Asynchronous cells were included for reference. Whole-cell lysates were subjected to Phos-tag SDS-PAGE followed by western blotting to assess the phosphorylation status of ADAR1p110. A reduction in the slower-migrating phosphorylated form of ADAR1p110 was observed upon CDK13 knockdown, confirming its role in mitotic phosphorylation.

Article Snippet: The following primary antibodies were used for western blotting and IP: anti-ADAR1 (Santa Cruz, sc-73408, 1:1000), anti-GAPDH (Cell Signaling Technology [CST], #5174, 1:4000), anti-γH2AX (S139) (CST, #9718, 1:500), anti-phospho-Histone H3 (S10) (CST, #53348, 1:1000), anti-phospho-Aurora A/B/C (Thr288/232/198) (CST, #14475, 1:1000), anti-Aurora A (CST, #2914, 1:1000), anti-Aurora B (Abcam, ab2254, 1:1000), anti-phospho-PLK1 (T210) (CST, #9062, 1:1000), anti-PLK1 (Santa Cruz, sc-17783, 1:500), anti-CDC25C (CST, #4688, 1:1000), anti-α-tubulin (CST, #2125, 1:1000), anti-SMC1 (CST, #6892, 1:1000), anti-SMC2 (CST, #5392, 1:1000), anti-SMC3 (CST, #5696, 1:1000), anti-RAD21 (CST, #4321, 1:1000), and anti-DHX9 (Abcam, ab26271, 1:1000).

Techniques: SDS Page, Phospho-proteomics, Mobility Shift, Mass Spectrometry, Purification, Sequencing, Residue, Binding Assay, Transfection, Construct, Western Blot, Inhibition, Control, Knockdown

Journal: Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology

Article Title: Phosphorylation of the histone H3.3 variant in mitosis and meiosis of the urochordate Oikopleura dioica.

doi: 10.1007/s10577-006-1112-z

Figure Lengend Snippet: Figure 4. H3.3S31P during different stages of mitosis in Oikopleura dioica. H3.3S31P, red; polo-like kinase, green; DNA, blue. In prophase, centrosomes, labelled with polo-like kinase , had migrated to the spindle poles and chromosomes began to condense. H3.3S31P was weak in prophase, increased with increasing chromosome condensation through prometaphase and peaked in metaphase with some chromosomal regions showing enrichment. No H3.3S31P was detected during anaphase and telophase. Scale bars in mm.

Article Snippet: Rabbit polyclonal anti-histone H3.3 phosphorylated at serine 31 (H3.3S31P, Abcam, ab2889), rat anti-histone H3 phosphorylated at S28 (H3S28P Abcam, ab10543) and rabbit anti-polo-like kinase antibody (Rockland, 600-401-446) were used at final dilutions of 1/100.

Techniques:

Journal: Diagnostics

Article Title: The Role of Aurora Kinase A in HBV-Associated Hepatocellular Carcinomas: A Molecular and Immunohistochemical Study

doi: 10.3390/diagnostics16010160

Figure Lengend Snippet: Immunohistochemical evaluation across study groups. ( 1a ) Control liver tissue demonstrating no AURKA expression. In some cases, weak cytoplasmic staining was observed in pericentral hepatocytes (arrowhead), consistent with localized cellular stress in this region. Arrows indicate areas with focal cytoplasmic positivity. AURKA, ×100. ( 1b , c ) HBV-HCC and Cr-HCC tissues showing moderate-to-strong AURKA positivity with predominant nuclear staining and variable cytoplasmic staining. Cytoplasmic staining is consistent with AURKA involvement in microtubule dynamics during cellular stress and mitotic regulation. AURKA, ×100. ( 2a ) Control tissue negative for p53 Ser315 , ×100. ( 2b ) HBV-HCC demonstrating moderate and diffuse nuclear p53 Ser315 positivity; intratumoral staining heterogeneity observed (arrows), ×100. ( 2c ) Cr-HCC showing strong and diffuse nuclear p53 Ser315 staining, ×100. ( 3a , b ) Control and HBV-HCC tissues negative for PLK1 Thr210 , ×100. ( 3c ) Cr-HCC demonstrating strong and diffuse nuclear PLK1 Thr210 staining, ×100. ( 4a ) Control tissue negative for BRCA1, ×100. ( 4b ) HBV-HCC with prominent nuclear BRCA1 staining, ×100. ( 4c ) Cr-HCC with weak and focal nuclear BRCA1 staining, ×100.

Article Snippet: Primary antibodies included AURKA (1:100, GeneTex, Hsinchu City, Taiwan), Phospho-p53 Ser315 (1:100, GeneTex, Taiwan), and BRCA1 (1:200, Elabscience, Houston, TX, USA), incubated for 30 min. For Phospho-PLK1 Thr210 (1:100, Affinity Biosciences, Changzhou, China), low-pH antigen retrieval was applied, followed by a 60 min incubation.

Techniques: Immunohistochemical staining, Control, Expressing, Staining