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: Cancer research

Article Title: Polo-like Kinase 1 Is Involved in Invasion through Extracellular Matrix

doi: 10.1158/0008-5472.CAN-07-2348

Figure Lengend Snippet: PLK1 expression is necessary but not sufficient for invasion. A, schematic presentation of the HMT-3522 model of metaplastic breast cancer progression (13). B, RT-PCR analysis for mRNA and Western blot for protein levels of PLK1. S1 cells in two-dimensional or three-dimensional lrECM were grown in the absence of epidermal growth factor (EGF) to completely growth arrest cells as a negative control for PLK1 signal. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C, Western blot for PLK1 in cells transfected with siRNA to PLK1 versus scrambled control siRNA (Scr.); 44% and 46% reduction in T4-2 and S3-C cells, respectively. Invasion assay for T4-2 or S3-C cells (induced by T4-2 conditioned medium) after cells were transfected with PLK1 or scrambled control siRNA; three experiments, duplicate samples. D, the percentage of Ki67-positive T4-2 cells transfected with PLK1 (◆) or scrambled control (■) siRNAs at indicated time points after release from synchrony; invasion assay for these T4-2 cells. E, the percentage of TUNEL-positive T4-2 cells after transfection with the PLK1 or scrambled control siRNAs; two experiments, duplicate samples.

Article Snippet: Tissues were blocked in 1.5% normal horse serum in PBS and incubated with 10 μg/mL of PLK1 antibody [anti-PLK1, human (rabbit); Calbiochem].

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Negative Control, Transfection, Control, Invasion Assay, TUNEL Assay

Journal: Cancer research

Article Title: Polo-like Kinase 1 Is Involved in Invasion through Extracellular Matrix

doi: 10.1158/0008-5472.CAN-07-2348

Figure Lengend Snippet: Vimentin (VIM) and β1 integrin are necessary for PLK1-mediated invasion. A, the mechanism proposed. B, Western blot for vimentin in T4-2 cells transfected with the indicated siRNAs. Vim2 used for subsequent experiments. Invasion assay for T4-2 cells transfected with the indicated siRNAs. Four experiments, duplicate samples. C, cell surface expression of β1 integrin. Four experiments. Values normalized to S1. P < 0.05, between T4-2 and all other cell types. Invasion assay for T4-2 cells treated with the indicated amounts of β1 integrin blocking antibody A2BII. P < 0.05, compared with untreated control, six experiments. D, invasion assay for T4-2 cells treated with siRNAs against PLK1 or vimentin, or β1 blocking antibody, in combinations indicated; four experiments, duplicate samples. P < 0.05, compared with scrambled control siRNA.

Article Snippet: Tissues were blocked in 1.5% normal horse serum in PBS and incubated with 10 μg/mL of PLK1 antibody [anti-PLK1, human (rabbit); Calbiochem].

Techniques: Western Blot, Transfection, Invasion Assay, Expressing, Blocking Assay, Control

Journal: Cancer research

Article Title: Polo-like Kinase 1 Is Involved in Invasion through Extracellular Matrix

doi: 10.1158/0008-5472.CAN-07-2348

Figure Lengend Snippet: PLK1 affects invasion via phosphorylating vimentin and down-regulating cell surface β1 integrin. A, Western blot for Ser82 phosphorylated vimentin in T4-2 cells treated with the indicated siRNAs. B, Western blot for Ser82 phosphorylated vimentin in T4-2 cells infected with lentivirus expressing WT vimentin pVIM (WT) or mutated vimentin pVIM (S82A). Invasion assay for T4-2 samples infected with lentivirus expressing a WT vimentin pVIM (WT) or mutated vimentin pVIM (S82A). Normalized to WT values. P = 0.036, three experiments, triplicate samples. C, cell surface expression of β1 integrin on T4-2 cells treated with the indicated siRNAs. P = 0.0007 (PLK1-scrambled control siRNA) and 0.003 (vimentin-scrambled control siRNA); four experiments. D, cell surface β1 integrin levels (totaland active, as indicated), normalized to T4-2 cells expressing WT vimentin; four experiments.

Article Snippet: Tissues were blocked in 1.5% normal horse serum in PBS and incubated with 10 μg/mL of PLK1 antibody [anti-PLK1, human (rabbit); Calbiochem].

Techniques: Western Blot, Infection, Expressing, Invasion Assay, Control

Journal: Cancer research

Article Title: Polo-like Kinase 1 Is Involved in Invasion through Extracellular Matrix

doi: 10.1158/0008-5472.CAN-07-2348

Figure Lengend Snippet: PLK1 in vivo. A, frequency of tumor formation and mean tumor volumes in fat pad. T4-2 transfected with siRNA against PLK1 (n = 10) versus scrambled control siRNA (n = 5). B, control experiment for immunohistochemical detection of PLK1, comparing mock antibody with PLK1 antibody-treated samples. Bar, 100 µm. C, example; PLK1 immunohistochemical signal in normal, in situ, and invasive samples from the same patient. Bar, 100 µm. D, PLK1 signal intensity. Two normal (N) and two invasive (I) cases as controls, compared with eight cases each containing areas of normal as well as in situ/preinvasive (P) and invasive carcinomas.

Article Snippet: Tissues were blocked in 1.5% normal horse serum in PBS and incubated with 10 μg/mL of PLK1 antibody [anti-PLK1, human (rabbit); Calbiochem].

Techniques: In Vivo, Transfection, Control, Immunohistochemical staining, In Situ

Journal: BMC Cancer

Article Title: MicroRNA-100 is a potential molecular marker of non-small cell lung cancer and functions as a tumor suppressor by targeting polo-like kinase 1

doi: 10.1186/1471-2407-12-519

Figure Lengend Snippet: PLK1 is a direct target of miR-100. ( A ) Alignment between the predicted miR-100 target sites and miR-100 is shown. ( B ) RT-PCR and Western Blot assays were performed to detect the expression of PLK1 mRNA and protein expression in A549 cells transfected with miR-100 mimics (miR-NC mimics) or anti-miR-100 (anti-miR-NC). ( C ) A549 cells were co-transfected with miR-100 mimics or anti-miR-100 and pLUC vector with PLK1 3’-UTR-wild or mut. After 24 hours, the luciferase activity was measured. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. * and ** indicate P < 0.05 and P < 0.01, respectively. The data are expressed as the mean value ± SEM of the results obtained from three independent experiments. Corresponding P values analyzed by t -tests are indicated.

Article Snippet: The blots were incubated with primary antibodies against human PLK1 (Santa Cruz, CA, USA) at 1:500 overnight at 4°C and then with anti-rabbit IgG (horseradish peroxidase-conjugated secondary antibody) for 1 h at room temperature.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: BMC Cancer

Article Title: MicroRNA-100 is a potential molecular marker of non-small cell lung cancer and functions as a tumor suppressor by targeting polo-like kinase 1

doi: 10.1186/1471-2407-12-519

Figure Lengend Snippet: Effects of siRNA targeting PLK1 on growth, apoptosis and cell cycle of NSCLC cells. ( A ) RT-PCR and Western Blot analysis of PLK1 mRNA and protein expression in A549-siRNA/PLK1 or A549-siRNA/control cells. ( B ) MTT analysis of growth in A549 cells at different time points (0, 24, 48, 72 or 96h) after transfection with siRNA/PLK1 or siRNA/control. ( C ) Hoechst staining analysis of apoptosis in A549/miR-100 or A549/miR-NC cells. The percentage of Hoechst-positive nuclei per optical field (at least 50 fields) was counted. ( D ) Flow cytometric analysis of cell cycle distribution in A549-siRNA/PLK1 or A549-siRNA/control cells. * and ** indicate P < 0.05 and P < 0.01, respectively. Each experiment was performed at least in triplicate. Corresponding P values analyzed by t -tests are indicated.

Article Snippet: The blots were incubated with primary antibodies against human PLK1 (Santa Cruz, CA, USA) at 1:500 overnight at 4°C and then with anti-rabbit IgG (horseradish peroxidase-conjugated secondary antibody) for 1 h at room temperature.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Control, Transfection, Staining

Journal: BMC Cancer

Article Title: MicroRNA-100 is a potential molecular marker of non-small cell lung cancer and functions as a tumor suppressor by targeting polo-like kinase 1

doi: 10.1186/1471-2407-12-519

Figure Lengend Snippet: DNA vector-mediated PLK1 overexpression could partially rescue the effects of miR-100 mimics on malignant phenotypes of A549 cells. ( A ) RT-PCR and Western Blot analysis of PTEN mRNA and protein expression in A549/miR-NC, A549/miR-100 or A549/miR-100 co-transfected with pcDNA/control or pcDNA/PLK1. ( B ) MTT analysis of growth in A549/mi-NC, A549/miR-100 or A549/miR-100 co-transfected with pcDNA/control or pcDNA/PLK1. ( C ) Hoechst staining analysis of apoptosis in A549/miR-NC, A549/miR-100 or A549/miR-100 co-transfected with pcDNA/control or pcDNA/PLK1. The percentage of Hoechst-positive nuclei per optical field (at least 50 fields) was counted. ( D ) Flow cytometric analysis of cell cycle distribution in A549/miR-NC, A549/miR-100 or A549/miR-100 co-transfected with pcDNA/control or pcDNA/PLK1. * indicates P < 0.05. The data are expressed as the mean value ± SEM of the results obtained from three independent experiments. Corresponding P values analyzed by ANNOVA tests are indicated.

Article Snippet: The blots were incubated with primary antibodies against human PLK1 (Santa Cruz, CA, USA) at 1:500 overnight at 4°C and then with anti-rabbit IgG (horseradish peroxidase-conjugated secondary antibody) for 1 h at room temperature.

Techniques: Plasmid Preparation, Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Control, Staining

Journal: BMC Cancer

Article Title: MicroRNA-100 is a potential molecular marker of non-small cell lung cancer and functions as a tumor suppressor by targeting polo-like kinase 1

doi: 10.1186/1471-2407-12-519

Figure Lengend Snippet: PLK1 was significantly upregulated in NSCLC tissues and inversely correlated with miR-100 expression. ( A ) The averaged mRNA level of PLK1 in NSCLC tissues (0.85 ± 0.15) was significantly higher than that in corresponding nontumor tissues (0.23 ± 0.06). GADPH was used as an internal control. ( B ) A statistically significant inverse correlation between miR-100 and PLK1 mRNA levels in 20 cases of NSCLC tissues (Spearman’s correlation analysis, r = −0.543; P < 0.001). Corresponding P values analyzed by a t -test or Spearman correlation test are indicated.

Article Snippet: The blots were incubated with primary antibodies against human PLK1 (Santa Cruz, CA, USA) at 1:500 overnight at 4°C and then with anti-rabbit IgG (horseradish peroxidase-conjugated secondary antibody) for 1 h at room temperature.

Techniques: Expressing, Control