Journal: Cell Death & Disease

Article Title: Pre-rRNAs control mitosis by maintaining chromosomal segregation through protecting SMC2 from AURKA-mediated phosphorylation

doi: 10.1038/s41419-025-08169-9

Figure Lengend Snippet: A HeLa cells were transfected with AURKA siRNA-1, AURKA siRNA-2, or control siRNA. These cells were treated with BMH-21 or DMSO, and were synchronized at M phase by nocodazole and harvested by shaking off. Immunoprecipitation was performed with anti-pan-phospho-serine/threonine antibody. The immunoprecipitates were immunoblotted with the indicated antibodies. B HeLa cells were transfected with the indicated doses of Flag-AURKA plasmid or Flag-vector. These cells were treated with BMH-21 or DMSO, and were synchronized at M phase and harvested as described in ( A ). Immunoprecipitation was performed with anti-pan-phospho-serine/threonine antibody. The immunoprecipitates were immunoblotted with the indicated antibodies. C HeLa cells were treated with 0 μM, 5 μM, 10 μM, and 15 μM TCS7010. These cells were treated with BMH-21 or DMSO, and were synchronized at M phase and harvested as described in ( A ). Immunoprecipitation was performed with anti-pan-phospho-serine/threonine antibody. The immunoprecipitates were immunoblotted with the indicated antibodies. D In vitro phosphorylation experiment was performed using purified His-AURKA and GST-SMC2. Proteins were resolved by SDS-PAGE and probed with anti-pan-phospho-lysine antibody. Coomassie blue staining showed the purified proteins. E Sequence alignment of the AURKA phosphorylation consensus within SMC2 and substrates of AURKA. Phosphorylated threonine residues are highlighted in red, arginine at the n − 3 position is highlighted in green, and hydrophobic residues at the n + 1 position are highlighted in blue. F In vitro phosphorylation experiment was performed using purified His-AURKA and GST-SMC2 T574A. Proteins were resolved by SDS-PAGE and probed with the indicated antibodies. Coomassie blue staining showed the purified proteins. G HeLa cells were transfected with the indicated siRNAs and the indicated plasmids (left). A quantitative comparison of multinucleated cells in HeLa cells in the indicated circumstances is shown ( n > 500) (right). ** p < 0.01. *** p < 0.001. **** p < 0.0001. n.s. denotes no significance.

Article Snippet: TCS7010 (S1451) was purchased from Selleck.

Techniques: Transfection, Control, Immunoprecipitation, Plasmid Preparation, In Vitro, Phospho-proteomics, Purification, SDS Page, Staining, Sequencing, Comparison

Journal: Cell Death & Disease

Article Title: Pre-rRNAs control mitosis by maintaining chromosomal segregation through protecting SMC2 from AURKA-mediated phosphorylation

doi: 10.1038/s41419-025-08169-9

Figure Lengend Snippet: A Dot blotting was performed with non-T574 phospho-SMC2 peptide or T574 phospho-SMC2 peptide to detect the specificity of anti-SMC2 T574-P antibody. B HeLa cells were treated with the indicated reagents. These cells were synchronized at M phase by thymidine double blocking and harvested by shaking off. Proteins were resolved by SDS-PAGE and probed with the indicated antibodies. Alpha-tubulin was used as a loading control. C HeLa cells were transfected with the indicated plasmids and treated with BMH-21 or not. These cells were synchronized at M phase by nocodazole and harvested by shaking off. Co-immunoprecipitation was performed with anti-Flag antibody, and the phosphorylation levels of Flag-SMC2 were evaluated by western blot using anti-SMC2 T574-P antibody. Alpha-tubulin was used as a loading control. D HeLa cells were transfected with the indicated siRNAs and treated with BMH-21 or not. These cells were synchronized and harvested as described in ( C ). Proteins were resolved by SDS-PAGE and probed with the indicated antibodies. Alpha-tubulin was used as a loading control. E HeLa cells were transfected with the indicated doses of Flag-AURKA or Flag vector and treated with BMH-21 or not. These cells were synchronized and harvested as described in ( C ). Proteins were resolved by SDS-PAGE and probed with the indicated antibodies. Alpha-tubulin was used as a loading control. F HeLa cells were treated with the indicated doses of TCS7010 or MLN8237, and treated with BMH-21 or not. These cells were synchronized and harvested as described in ( C ). Proteins were resolved by SDS-PAGE and probed with the indicated antibodies. Alpha-tubulin was used as a loading control. G HeLa cells were treated with Act D, BMH-21, or DMSO. These cells were fixed, and indirect immunofluorescent staining was performed using anti-SMC2 T574-P antibody. Chromosomes were stained by DAPI. Scale bar, 8 μm.

Article Snippet: TCS7010 (S1451) was purchased from Selleck.

Techniques: Blocking Assay, SDS Page, Control, Transfection, Immunoprecipitation, Phospho-proteomics, Western Blot, Plasmid Preparation, Staining

Journal: Nature Communications

Article Title: Kinetochore-centrosome feedback linking CENP-E and Aurora kinases controls chromosome congression

doi: 10.1038/s41467-025-64804-1

Figure Lengend Snippet: a Representative RPE-1 cells expressing CENP-A-GFP and centrin1-GFP (gray, centrioles circled) after indicated treatments, showing enlarged insets of polar (P) and aligned (A) kinetochores immunostained for pT232-Aurora B (red). b Ratio of pT232-Aurora B intensity normalized to CENP-A on polar vs. aligned kinetochores per cell under indicated treatments. Colored points represent individual cells; black lines show the mean, with light and dark gray areas marking 95% confidence intervals for the mean and standard deviation, respectively. c Average pT232-Aurora B levels normalized to CENP-A on all kinetochore groups after indicated treatments. Dispersion measures as in ( b ). d Representative cells expressing CENP-A-GFP and centrin1-GFP (cyan, centrioles circled) immunostained for pT232-Aurora B (red) and α-tubulin (gray) after continuous CENP-E inhibition and acute Aurora A inhibition by MLN8054 and TCS7010, with enlarged insets of polar (P) and aligned (A) kinetochores. e – g Quantification of pT232-Aurora B intensity ratio on polar vs. aligned kinetochores e , number of polar chromosomes f , and spindle length g after indicated treatments. Dispersion measures as in ( b) . h RPE-1 cells expressing CENP-A-GFP and centrin-GFP (red, centrioles circled), immunostained for α-tubulin (gray), and imaged by super-resolution Airyscan microscopy showing enlarged insets of congressing (C) and aligned (A) kinetochores. Images are deconvolved projections of 2–5 z-planes. Numbers: b 23, 18, 14, 27 cells averaged from 491 kinetochore pairs, c 83, 71, 61, 57, 47, 41, 52, 79 kinetochore pairs from 91 cells, e 22, 19, 17, 18 cells averaged from 436 kinetochore pairs, f , g 39, 38, 42, 38 cells, all pooled from ≥2 independent biological replicates. Statistics: two-tailed ANOVA with post-hoc Tukey’s HSD test. Symbols indicate: n.s., p > 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; inh., inhibited; chrom., chromosomes; MLN, MLN8237. Source data are provided as a Source Data file.

Article Snippet: Aurora A inhibitor TCS7010 (MedChemExpress, IC 50 value 3.4 nM) at a final concentration of 100 nM, was added 30 minutes before fixation.

Techniques: Expressing, Standard Deviation, Dispersion, Inhibition, Microscopy, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Chromatin-associated α-satellite RNA maintains chromosome stability by reestablishing SAF-A in the mitotic cell cycle

doi: 10.1093/nar/gkaf294

Figure Lengend Snippet: Aurora kinases regulate the expression of α-satellite RNA. ( A ) Representative western blotting demonstrates the inhibition of Aurora kinases in synchronized cells. AURKA inhibitor, TC-S7010 (2.5 μM); AURKB inhibitor, AZD1152 (0.25 μM). H3S10P serves as positive control; ACTB, β-actin, serves as the internal loading control. ( B ) Quantification of H3S10P western blot in (A). ( C ) RT-qPCR analysis showing relative α-satellite RNA levels upon Aurora kinase inhibition. DMSO, no drug control. Results are normalized to Async no drug control. The error bars represent SD, n = 6. Statistical significance is calculated using unpaired t-tests and is reported as P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 ****. ( D ) Representative images of α-satellite RNAs (αSAT) smFISH, CREST, and H3S10P IF in cells treated with Aurora kinase inhibitors. Scale bar, 10 μm.

Article Snippet: To inhibit Aurora kinases, 2.5 μM of TC-S7010 (MedChemExpress) and 0.25 μM of AZD1152 (MedChemExpress) were added to the culture medium 0.5 h before sample collection [ , ].

Techniques: Expressing, Western Blot, Inhibition, Positive Control, Control, Quantitative RT-PCR