Journal: Nature Communications

Article Title: The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation

doi: 10.1038/s41467-024-45683-4

Figure Lengend Snippet: A Volcano plot of EYA4-Myc-BioID interactome. Grey coloured box represents 156 high confidence EYA4 interactors with -log p-value > 1.3 and Log2 fold difference > 2. EYA4, PLK1 and AURKA are indicated on the plot ( n = 4, two-sided Student’s t -test). B EYA4 interactors with known localization to mitotic structures including PLK1. Proteins have been colour coded by Log2 fold difference. C PLK1 tyrosine phosphorylation was assessed by western blots of eluates from immunoprecipitation (IP) of tyrosine phosphorylated proteins using an anti-phosphotyrosine antibody in 293 T or HeLa protein lysates following EYA4 depletion ( n = 3 biological repeats). Asterisk represents non-specific band on the EYA4 blot. D Immunopurified endogenous PLK1 from control or EYA4 depleted 293 T cells that had been arrested in G2 with RO3306 or released into mitosis for 15 or 30 min were probed using an anti-phosphotyrosine antibody (pY, n = 1). E Schematic representation of EYA4 protein with N-terminal transactivation domain and C-terminal tyrosine phosphatase domain as well as putative PDS (AA 123- 129) and known phosphosite (pS128). F Co-immunopurification reactions between immunopurified endogenous PLK1 and an EYA4-Myc construct or EYA4-Myc mutants. Cells have been arrested in G2 with RO3306 or released into early M phase (n = 2 biological repeats). Densitometry of the relative EYA4-Myc or EYA4-Myc mutant in the IP eluate to that in the lysate is shown below the blot. G Immunoflourescence of endogenous PLK1 and EYA4 in G2 arrested HeLa cells treated with control siRNA or siRNA targeting EYA4. Enlarged images show closeups of PLK1 centrosomal foci demonstrating colocalization with EYA4 in the siCon treatment. Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used in the manuscript: The following antibodies were used in the manuscript: PLK F-8 SC17783 Santa Cruz (1:400), Phospho tyrosine 8954 Cell Signaling (1:1000), EYA4 ab93865 Abcam (1:1000), Actin a2066 Sigma (1:1000), Myc 9B11 Cell Signaling (1:1000), Myc 2278S Cell Signaling (1:1000), BUB1 B-3 SC365685 Santa Cruz (1:1000), p-S10 H3 3377 Cell Signaling (1:1000), p-S10 H3 9706 Cell Signaling (1:1000), H3 9715 Cell Signaling (1:1000), p-T210 PLK1 Ab39068 Abcam (1:100 IF, 1:500 WB), p-T210 PLK1 9062 Cell Signaling (1:100), Pericentrin Ab28144 Abcam (1:100), Pericentrin Ab448 Abcam (1:1000), Alpha Tubulin Ab7291 (1:1000) Abcam, Bora 12109 Cell Signaling (1:1000), Cep192 A302324A Bethyl (1:1000), Aurka 12100 Cell Signaling (1:1000), PARP 9542 Cell Signaling (1:1000), Vinculin V9131 Sigma (1:1000), EYA1 22658-1-AP Protein Tech (1:1000), p-S46 TCTP 5251 Cell Signaling (1:200), p-S133 Cyclin B1 4133 Cell Signaling (1:200), p-S198 cdc25C 9529 Cell Signaling (1:200).

Techniques: Western Blot, Immunoprecipitation, Immu-Puri, Construct, Mutagenesis

Journal: Nature Communications

Article Title: The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation

doi: 10.1038/s41467-024-45683-4

Figure Lengend Snippet: A Representative images of mitotic HeLa cells stained for total PLK1 and pT210 PLK1 following knockdowns. B Quantitation of cellular fluorescence intensity of pT210 PLK1 in mitotic cells following knockdowns (independent cells measured from left to right: 80, 102, 96, 89, ** p = 0.0073, **** p ≤ 0.0001, ANOVA with Tukey correction). C Quantitation of cellular fluorescence intensity of pT210/total PLK1 mitotic cells following knockdowns (independent cells measured from left to right: 80, 102, 96, 89, **** p ≤ 0.0001, ANOVA with Tukey correction). D Representative images of individual mitotic HeLa cells stained for PLK1 substrates (green), pS10 H3 (red), and DAPI, with and without depletion of EYA4. E Quantitation of cellular fluorescence intensity of pS46 TCTP in mitotic cells ( n = 43 cells per condition, * p = 0.0105. two-sided Student’s t -test). F Quantitation of cellular fluorescence intensity of pS133 Cyclin B in mitotic cells ( n = 53 cells per condition, p = 0.071, two-sided Student’s t -test). G Quantitation of cellular fluorescence intensity of pS198 CDC25C in mitotic cells ( n = 49 cells per condition, ** p = 0.0031, two-sided Student’s t -test). H Western blots from nocodazole arrested HeLa cells and densitometry of pT210 PLK1/total PLK1 ( n = 3 biological replicates, * p = 0.0495, two-sided Student’s t -test, asterisk indicates a non-specific band). I Representative western blots and pT210/total PLK1 densitometry in nocodazole arrested HeLa cells overexpressing EYA4 or EYA4 mutants. ( n = 3 biological replicates, * p = 0.0417 for comparison between S128A and S128D and 0.0318 for comparison between S128D and Ydef, ANOVA with Tukey correction). J Western blots following treatment with a pan-EYA phosphatase inhibitor in nocodazole arrested HeLa cells and densitometry of pT210 and pT210/total PLK1 ( n = 3 biological replicates, * p = 0.0248, ANOVA with Dunnet correction). “AU” stands for arbitrary units. All quantitative data in this figure are presented as mean values +/− SEM. Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used in the manuscript: The following antibodies were used in the manuscript: PLK F-8 SC17783 Santa Cruz (1:400), Phospho tyrosine 8954 Cell Signaling (1:1000), EYA4 ab93865 Abcam (1:1000), Actin a2066 Sigma (1:1000), Myc 9B11 Cell Signaling (1:1000), Myc 2278S Cell Signaling (1:1000), BUB1 B-3 SC365685 Santa Cruz (1:1000), p-S10 H3 3377 Cell Signaling (1:1000), p-S10 H3 9706 Cell Signaling (1:1000), H3 9715 Cell Signaling (1:1000), p-T210 PLK1 Ab39068 Abcam (1:100 IF, 1:500 WB), p-T210 PLK1 9062 Cell Signaling (1:100), Pericentrin Ab28144 Abcam (1:100), Pericentrin Ab448 Abcam (1:1000), Alpha Tubulin Ab7291 (1:1000) Abcam, Bora 12109 Cell Signaling (1:1000), Cep192 A302324A Bethyl (1:1000), Aurka 12100 Cell Signaling (1:1000), PARP 9542 Cell Signaling (1:1000), Vinculin V9131 Sigma (1:1000), EYA1 22658-1-AP Protein Tech (1:1000), p-S46 TCTP 5251 Cell Signaling (1:200), p-S133 Cyclin B1 4133 Cell Signaling (1:200), p-S198 cdc25C 9529 Cell Signaling (1:200).

Techniques: Staining, Quantitation Assay, Fluorescence, Western Blot, Comparison

Journal: Nature Communications

Article Title: The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation

doi: 10.1038/s41467-024-45683-4

Figure Lengend Snippet: A Prophase HeLa cells with 1 or 2 centrosome foci (pericentrin staining). B Quantitation of centrosome foci number following knockdowns ( n = 3, ** p = 0.0061, *** p = 0.0003, **** p ≤ 0.0001, ANOVA with Tukey correction). C Integrated intensity of pericentrin foci following knockdowns (Independent cells from left to right: 103, 91, 93, 92, *** p = 0.0001, **** p ≤ 0.0001, ANOVA with Tukey correction). D PLK1 colocalization with pericentrin in G2 cells. E G2 cells identified by gating of EdU and DAPI intensities. F Quantitation of PLK1 integrated intensity at centrosomes in G2 cells (Independent cells measured from left to right: 164, 232, 22, 41, 7, 10, *** p = 0.0004, **** p ≤ 0.0001, ANOVA with Tukey correction). G Examples of spindle defects (Green: pericentrin, Red: alpha-tubulin). H Quantification of spindle defects ( n ≥ 87 mitotic cells across three experiments, Independent cells measured from left to right: 92, 87, 98, 103, 127, 147, * p = 0.0238, *** p = 0.0002, siRNA experiment: ANOVA with Tukey correction, drug treatments: two-sided Student’s t -test). I Mitotic duration following knockdowns or EYA inhibition ( n ≥ 90 across 3 experiments, Total number of independent cells measured from left to right: 99, 101, 100, 97, 100, * p = 0.0122, **** p ≤ 0.0001, ANOVA with Tukey correction). J Mitotic cell death following knockdowns or EYA inhibition ( n ≥ 90 across 3 experiments, *** p = 0.0003, **** p ≤ 0.0001, ANOVA with Tukey correction). K Mitotic duration following overexpression of EYA4 WT or mutants ( n ≥ 100 across 4 experiments, * p = 0.0373 between EV and S128D, p = 0.0274 between S128D and Ydef, *** p = 0.0003, ANOVA with Tukey correction). L Mitotic cell death following overexpression of EYA4 WT or mutants ( n ≥ 100 across 4 experiments, ** p = 0.0059, ANOVA with Tukey correction). All quantitative data in this figure are presented as mean values +/− SEM. Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used in the manuscript: The following antibodies were used in the manuscript: PLK F-8 SC17783 Santa Cruz (1:400), Phospho tyrosine 8954 Cell Signaling (1:1000), EYA4 ab93865 Abcam (1:1000), Actin a2066 Sigma (1:1000), Myc 9B11 Cell Signaling (1:1000), Myc 2278S Cell Signaling (1:1000), BUB1 B-3 SC365685 Santa Cruz (1:1000), p-S10 H3 3377 Cell Signaling (1:1000), p-S10 H3 9706 Cell Signaling (1:1000), H3 9715 Cell Signaling (1:1000), p-T210 PLK1 Ab39068 Abcam (1:100 IF, 1:500 WB), p-T210 PLK1 9062 Cell Signaling (1:100), Pericentrin Ab28144 Abcam (1:100), Pericentrin Ab448 Abcam (1:1000), Alpha Tubulin Ab7291 (1:1000) Abcam, Bora 12109 Cell Signaling (1:1000), Cep192 A302324A Bethyl (1:1000), Aurka 12100 Cell Signaling (1:1000), PARP 9542 Cell Signaling (1:1000), Vinculin V9131 Sigma (1:1000), EYA1 22658-1-AP Protein Tech (1:1000), p-S46 TCTP 5251 Cell Signaling (1:200), p-S133 Cyclin B1 4133 Cell Signaling (1:200), p-S198 cdc25C 9529 Cell Signaling (1:200).

Techniques: Staining, Quantitation Assay, Inhibition, Over Expression

Journal: Nature Communications

Article Title: The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation

doi: 10.1038/s41467-024-45683-4

Figure Lengend Snippet: A Schematic showing an example of immunopurified and Coomassie stained PLK1 which was in-gel digested and used in downstream pilot and PRM mass spec experiments. B PLK1 tyrosine phosphosites detected by mass spec with their positions designated on a PLK1 gene schematic. C The purified phosphatase domain of EYA4 (left, Coomassie-stained SDS-PAGE gel) was used in in-vitro phosphatase assays with synthetic phosphopeptides in duplicate. Peptides dephosphorylated with relatively fast kinetics ( n = 2, pY445, Km 0.46 mM; pY425, Km 0.34 mM; and a positive control peptide from H2AX pY142, 0.51 mM) were fit to Michaelis-Menten curves (middle panel). Peptides with slower kinetics, which could not be fit to a Michaelis-Menten curve at the substrate concentrations tested, were fit using linear regressions (right panel). Individual datapoints represent means. Error bars represent standard deviations. Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used in the manuscript: The following antibodies were used in the manuscript: PLK F-8 SC17783 Santa Cruz (1:400), Phospho tyrosine 8954 Cell Signaling (1:1000), EYA4 ab93865 Abcam (1:1000), Actin a2066 Sigma (1:1000), Myc 9B11 Cell Signaling (1:1000), Myc 2278S Cell Signaling (1:1000), BUB1 B-3 SC365685 Santa Cruz (1:1000), p-S10 H3 3377 Cell Signaling (1:1000), p-S10 H3 9706 Cell Signaling (1:1000), H3 9715 Cell Signaling (1:1000), p-T210 PLK1 Ab39068 Abcam (1:100 IF, 1:500 WB), p-T210 PLK1 9062 Cell Signaling (1:100), Pericentrin Ab28144 Abcam (1:100), Pericentrin Ab448 Abcam (1:1000), Alpha Tubulin Ab7291 (1:1000) Abcam, Bora 12109 Cell Signaling (1:1000), Cep192 A302324A Bethyl (1:1000), Aurka 12100 Cell Signaling (1:1000), PARP 9542 Cell Signaling (1:1000), Vinculin V9131 Sigma (1:1000), EYA1 22658-1-AP Protein Tech (1:1000), p-S46 TCTP 5251 Cell Signaling (1:200), p-S133 Cyclin B1 4133 Cell Signaling (1:200), p-S198 cdc25C 9529 Cell Signaling (1:200).

Techniques: Staining, Mass Spectrometry, Purification, SDS Page, In Vitro, Positive Control

Journal: Nature Communications

Article Title: The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation

doi: 10.1038/s41467-024-45683-4

Figure Lengend Snippet: A Representative western blots of pT210 and total PLK1 in PLK1-myc WT and PLK1-myc Y445F expressing HeLa cells that have been mitotically synchronized with nocodazole (* represents endogenous pT210 or total PLK1). B Densitometry pertaining to ( A ) ( n = 3 biological replicates, *** p = 0.0006, two-sided Student’s t -test). C Representative western blots of endogenous PLK1 depletion rescue by PLK1-myc WT and PLK1-myc Y445F (* represents endogenous PLK1). D Densitometry of cleaved PARP/PARP pertaining to ( A ) ( n = 4, ** p = 0.0013, *** p = 0.0005, **** p ≤ 0.0001, ANOVA with Tukey correction for multiple comparisons). E , F Quantification of mitotic cell death ( E ) or mitotic duration ( F ) from live cell microscopy experiments involving endogenous PLK1 depletion and rescue with PLK1-myc WT or PLK1-myc Y445F overexpression ( n = 75 mitotic entry events across 3 experiments, pertaining to ( E ): ** p = 0.0011, *** p = 0.0001, **** p ≤ 0.0001, pertaining to ( F ): * p = 0.0326, ** p = 0.0080, *** p = 0.0004, **** p ≤ 0.0001, both panels used ANOVA with Tukey correction for multiple comparisons). G – J Quantification of mitotic cell death ( G , I ) or mitotic duration ( H , J ) from live cell microscopy experiments involving depletion of EYA4, and overexpression of PLK1-myc WT or PLK1-myc Y445F overexpression ( n = 75 mitotic entry events across 3 experiments, * p = 0.0139, ** p = 0.0075). K Alamar blue viability dose-response curves following 72 h treatment with benzarone across a range of concentrations in cells stably overexpressing an EV construct, PLK1-myc WT, or PLK1-myc mutants ( n = 4 replicates, mean IC50 values presented, * p ≤ 0.05, **** p ≤ 0.0001, two-sided Student’s t -tests). “AU” stands for arbitrary units. All quantitative data in this figure are presented as mean values +/− SEM. Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used in the manuscript: The following antibodies were used in the manuscript: PLK F-8 SC17783 Santa Cruz (1:400), Phospho tyrosine 8954 Cell Signaling (1:1000), EYA4 ab93865 Abcam (1:1000), Actin a2066 Sigma (1:1000), Myc 9B11 Cell Signaling (1:1000), Myc 2278S Cell Signaling (1:1000), BUB1 B-3 SC365685 Santa Cruz (1:1000), p-S10 H3 3377 Cell Signaling (1:1000), p-S10 H3 9706 Cell Signaling (1:1000), H3 9715 Cell Signaling (1:1000), p-T210 PLK1 Ab39068 Abcam (1:100 IF, 1:500 WB), p-T210 PLK1 9062 Cell Signaling (1:100), Pericentrin Ab28144 Abcam (1:100), Pericentrin Ab448 Abcam (1:1000), Alpha Tubulin Ab7291 (1:1000) Abcam, Bora 12109 Cell Signaling (1:1000), Cep192 A302324A Bethyl (1:1000), Aurka 12100 Cell Signaling (1:1000), PARP 9542 Cell Signaling (1:1000), Vinculin V9131 Sigma (1:1000), EYA1 22658-1-AP Protein Tech (1:1000), p-S46 TCTP 5251 Cell Signaling (1:200), p-S133 Cyclin B1 4133 Cell Signaling (1:200), p-S198 cdc25C 9529 Cell Signaling (1:200).

Techniques: Western Blot, Expressing, Microscopy, Over Expression, Stable Transfection, Construct

Journal: Nature Communications

Article Title: The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation

doi: 10.1038/s41467-024-45683-4

Figure Lengend Snippet: A Co-immunoprecipitation reactions with overexpressed PLK1-myc WT or PLK1-myc Y445F in RO3306 arrested G2 cells and nocodazole arrested M-phase cells. Western blots from protein lysates (left panel), and western blots from G2 arrested eluates (middle panel) and M-phase arrested cells (right panel). B Representative simulation snapshot of the backside of the phosphopeptide binding pocket of PLK1s PBD (bound phosphopeptide in yellow). Y445 is indicated in a phosphorylated state (pY445). The image highlights the interaction of pY445 with R507 (green sticks), as well as hydrogen bonds formed by D429 and N446, and increased rigidity within the connecting loop (AA 502-506) highlighted in orange. C , D Plots of the root mean square fluctuation (RMSF) of Cα atoms in the WT PLK1 PBD (black line) and Y445-phosphorylated PLK1 PBD (red line). The decreased RMSF within the connecting loop (AA 502-506) is indicative of decreased flexibility either when the PBD is bound to a model phosphopeptide ( C ), or in the unbound state ( D ) ( n = 5 simulations). Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used in the manuscript: The following antibodies were used in the manuscript: PLK F-8 SC17783 Santa Cruz (1:400), Phospho tyrosine 8954 Cell Signaling (1:1000), EYA4 ab93865 Abcam (1:1000), Actin a2066 Sigma (1:1000), Myc 9B11 Cell Signaling (1:1000), Myc 2278S Cell Signaling (1:1000), BUB1 B-3 SC365685 Santa Cruz (1:1000), p-S10 H3 3377 Cell Signaling (1:1000), p-S10 H3 9706 Cell Signaling (1:1000), H3 9715 Cell Signaling (1:1000), p-T210 PLK1 Ab39068 Abcam (1:100 IF, 1:500 WB), p-T210 PLK1 9062 Cell Signaling (1:100), Pericentrin Ab28144 Abcam (1:100), Pericentrin Ab448 Abcam (1:1000), Alpha Tubulin Ab7291 (1:1000) Abcam, Bora 12109 Cell Signaling (1:1000), Cep192 A302324A Bethyl (1:1000), Aurka 12100 Cell Signaling (1:1000), PARP 9542 Cell Signaling (1:1000), Vinculin V9131 Sigma (1:1000), EYA1 22658-1-AP Protein Tech (1:1000), p-S46 TCTP 5251 Cell Signaling (1:200), p-S133 Cyclin B1 4133 Cell Signaling (1:200), p-S198 cdc25C 9529 Cell Signaling (1:200).

Techniques: Immunoprecipitation, Western Blot, Binding Assay

Journal: Nature Communications

Article Title: The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation

doi: 10.1038/s41467-024-45683-4

Figure Lengend Snippet: G2, Phosphorylation of the putative PDS on EYA4/EYA1 (S128 on EYA4) allows for interaction with PLK1 and dephosphorylation of pY445 within the PBD. G2→Prophase, Dephosphorylation of pY445 changes the structure of the PLK1 PBD, favouring the interaction between PLK1 and PLK1 activation complex members, and permitting greater levels of pT210 phosphorylation by AURKA. Prophase, Active PLK1 supports centrosome maturation and separation in preparation for mitotic spindle formation. Prophase→Successful Mitosis, Accurate and timely completion of mitosis. Alternatively, EYA4/EYA1 loss or EYA inhibition causes mitotic aberrations, increased mitotic duration, and mitotic cell death. Created with biorender.com.

Article Snippet: The following primary antibodies were used in the manuscript: The following antibodies were used in the manuscript: PLK F-8 SC17783 Santa Cruz (1:400), Phospho tyrosine 8954 Cell Signaling (1:1000), EYA4 ab93865 Abcam (1:1000), Actin a2066 Sigma (1:1000), Myc 9B11 Cell Signaling (1:1000), Myc 2278S Cell Signaling (1:1000), BUB1 B-3 SC365685 Santa Cruz (1:1000), p-S10 H3 3377 Cell Signaling (1:1000), p-S10 H3 9706 Cell Signaling (1:1000), H3 9715 Cell Signaling (1:1000), p-T210 PLK1 Ab39068 Abcam (1:100 IF, 1:500 WB), p-T210 PLK1 9062 Cell Signaling (1:100), Pericentrin Ab28144 Abcam (1:100), Pericentrin Ab448 Abcam (1:1000), Alpha Tubulin Ab7291 (1:1000) Abcam, Bora 12109 Cell Signaling (1:1000), Cep192 A302324A Bethyl (1:1000), Aurka 12100 Cell Signaling (1:1000), PARP 9542 Cell Signaling (1:1000), Vinculin V9131 Sigma (1:1000), EYA1 22658-1-AP Protein Tech (1:1000), p-S46 TCTP 5251 Cell Signaling (1:200), p-S133 Cyclin B1 4133 Cell Signaling (1:200), p-S198 cdc25C 9529 Cell Signaling (1:200).

Techniques: De-Phosphorylation Assay, Activation Assay, Inhibition

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