Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A Left: Renilla luciferase activity analyses in HEK 293 T cells co-transfected with psiCHECK2-PLK1-CDS, psiCHECK2-PLK1-3′UTR, or psiCHECK-PLK1-3′UTR-MUT + miR-183-5p or negative control miRNA (NC) (Ambion). Results are 48 h post transfection. Right: Renilla luciferase activity analyses in HEK 293 T cells co-transfected with psiCHECK2-PLK1-CDS or psiCHECK2-PLK1-3′UTR + miR-18a-3p, miR-100-5p, or negative control miRNA (NC). Data ( n = 3/group) are presented as mean + % SD. B Left: Immunoblots showing PLK1 expression following no treatment (NT), transfection with negative control (NC) or miR-183-5p. Right: RT-qPCR demonstrating expression levels of miR-183-5p following transfection (results are x10 3 ). C Left: Immunoblots showing PLK1 expression following no treatment (NT) or miR-183-5p, following transfection with PLK1 CDS with 3′UTR (CDS + 3′UTR) or PLK1 CDS only (CDS only). Right: RT-qPCR demonstrating expression levels of miR-183-5p following transfection (results are x10 3 for MDA-MB-231, BT549 and ZR-75-1, x10 for T47D). D Western blots show results of the rescue experiments (see material and methods) performed on the MDA-MB-231 (left) and T47D (right) cell lines. Bar graphs are densitometry results of PLK1-fold expression. Vinculin was used as internal control. Data are the mean of at least 3 independent experiments ( N = 3) + SD. ** p -value <0.01 compared to negative control by Student’s unpaired t test. E TCGA, IBCD analysis. Left: Violin plots represent miR-183-5p expression in Cancerous versus Normal breast tissue ( p -value = 3.35 × 10 −21 ) and in Luminal A versus TNBC ( p -value = 0.25). Right: Violin plots depicting PLK1 expression in Cancerous versus Normal breast tissue ( p -value = 4.22 × 10 −20 ) and in Luminal A versus TNBC. ( p -value = 1.01 × 10 −6 ). N = 46 paired luminal A, 15 paired TNBC, 61 cancer vs normal.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Luciferase, Activity Assay, Transfection, Negative Control, Western Blot, Expressing, Quantitative RT-PCR, Control

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A WST8 assay demonstrating the effects of NMS-P937 treatment: 0, 10, 25, 50, 75, 100, and 150 nM after transfection with negative control (NC) or miR-183-5p or si-PLK1. Left hand graphs. TNBC cell lines MDA-MB-231 (top left, p -value = 3.26e-14) and BT549 (top right, p -value = 0.00112). Luminal A cell lines T47D (lower left, p -value < 2e-16) and ZR-75-1 (lower right p -value = 1.93e-6). Right hand graphs. Assay was repeated using si-PLK1. Data ( N = 3/group) are mean + SD. ** p -value <0.05, *** p -value <0.001 calculated by ANOVA. B Left: Matrices demonstrating MDA-MB-231 and T47D cell lines treated with different concentrations of miR-183-5p and NMS-P937, then analyzed to determine cell mortality (% inhibition). Right: Bliss score shows a synergistic effect present on cell mortality between miR-183-5p overexpression and NMS-P937 treatment.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Transfection, Negative Control, Inhibition, Over Expression

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A MDA-MB-231, BT549, T47D, and ZR-75-1 cells treated with NMS-P937 for 48 h, +/- miR-183-5p transfection for 72 h. Total protein lysates were analyzed by Western blot. Blots were stained for PLK1, DNMT1, p-STAT3 (Tyr 705), p-p53 (Ser15), Bcl-XL, cleaved caspases 7 and 3, cleaved PARP and vinculin. B – E Protein band densitometry normalized to vinculin. Data ( N = 3/group) are presented as mean + SD.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Transfection, Western Blot, Staining

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A MDA-MB-231, BT549, T47D, and ZR-75-1 cells treated with NMS-P937 for 48 h, +/- si-PLK1 for 72 h. Total protein lysates were analyzed by Western blot. Blots were stained for PLK1, DNMT1, p-STAT3 (Tyr 705), p-p53 (Ser15), Bcl-XL, cleaved caspases 7 and 3, cleaved PARP and vinculin. B – E Protein band densitometry normalized to vinculin. Data ( N = 3/group) are presented as mean + SD.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Western Blot, Staining

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A Representative flow cytometry images of breast cancer cell lines treated with NMS-P937 for 48 h, +/- si-PLK1 transfection for 72 h. Cells were stained with Annexin-FITC and PI. The cell populations of interest were those undergoing Late (Q2) or early (Q4) apoptosis. B , C Bar graphs representing the percentage of cells undergoing early (B) or late (C) apoptosis. Cells were either treated with negative control miRNA (NC), si-PLK1 only, NMS-P937 only (25 nM or 75 nM), or si-PLK1 + NMS-P937. Data ( N = 4/group) are presented as mean + SD. * p -value <0.05, ** p -value <0.001 compared to negative control by Student’s unpaired t test.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Flow Cytometry, Transfection, Staining, Negative Control

Journal: Molecular oncology

Article Title: PLK1 and FoxM1 expressions positively correlate in papillary thyroid carcinoma and their combined inhibition results in synergistic anti-tumor effects.

doi: 10.1002/1878-0261.13610

Figure Lengend Snippet: Fig. 1. Tissue microarray (TMA) based immunohistochemistry analysis of PLK1 and FoxM1 in papillary thyroid cancer (PTC) patients. PTC TMA spots showing overexpression of PLK1 (A) and FoxM1 (C). In contrast, another set of TMA spots showing reduced expression of PLK1 (B) and FoxM1 (D). 209/0.70 objective on an Olympus BX 51 microscope (Olympus America Inc, Center Valley, PA, USA) with the inset showing a 409 0.85 aperture magnified view of the same TMA spot (scale bar = 500 lm). (E) Recurrence-free survival (RFS). Kaplan–Meier survival plot showing statistically significant poor RFS in PLK1 high expression cases compared to PLK1 low expression (P = 0.0009).

Article Snippet: Plasmid DNA encoding human PLK1 and shRNA targeting human PLK1 were purchased from Origene (Rockville, MD, USA).

Techniques: Microarray, Immunohistochemistry, Over Expression, Expressing, Microscopy

Journal: Molecular oncology

Article Title: PLK1 and FoxM1 expressions positively correlate in papillary thyroid carcinoma and their combined inhibition results in synergistic anti-tumor effects.

doi: 10.1002/1878-0261.13610

Figure Lengend Snippet: Fig. 2. Inhibition of PLK1 impedes cell proliferation and induces cell cycle arrest and apoptosis. (A) Basal expression of PLK1 in Nthy-ori 3–1 and PTC cell lines. Proteins were extracted from Nthy-ori 3–1 cell line and three PTC cell lines and subjected to immunoblotting using indicated antibodies (n = 3). (B, C) Volasertib inhibits cell viability. PTC cells (104) were exposed to increasing doses of volasertib for 24 and 48 h and cell viability was assessed using MTT. Data were presented as mean SD (n = 8). (D, E) Cell cycle analysis of PTC cells treated with volasertib. BCPAP and TPC-1 cells were exposed to 250 and 500 nM volasertib for 48 h. Following incubation, either cells were analyzed for cell cycle fractions by flow cytometry (D) or proteins were extracted from cells and were subjected to immuno-blotting to analyze cell cycle markers (E). Data were presented as mean SD (n = 3). (F, G) Volasertib induces apoptosis in PTC cell lines. PTC cells were exposed to indicated doses of volasertib for 48 h. Following incubation, either cells were stained with annexin-V/PI and analyzed by flow cytometry (F) or proteins were extracted from cells and were subjected immuno-blotting using antibodies as indicated (G). Data were presented as mean SD (n = 3). (H) Effect of volasertib on Bax activation in PTC cells. PTC cells were treated with volasertib for indicated time course. Following treatment, cells were lysed in 1% Chaps lysis buffer and subjected to immuno-precipitation with anti-Bax antibody (sc-493; Santa Cruz) and immunoblotted with anti-Bax antibody (B8554; SIGMA-ALDRICH), (n = 3). Statistical analyses were conducted employing two-tailed Student’s t-tests. *P < 0.05.

Article Snippet: Plasmid DNA encoding human PLK1 and shRNA targeting human PLK1 were purchased from Origene (Rockville, MD, USA).

Techniques: Inhibition, Expressing, Western Blot, Cell Cycle Assay, Incubation, Cytometry, Staining, Activation Assay, Lysis, Immunoprecipitation, Two Tailed Test

Journal: Molecular oncology

Article Title: PLK1 and FoxM1 expressions positively correlate in papillary thyroid carcinoma and their combined inhibition results in synergistic anti-tumor effects.

doi: 10.1002/1878-0261.13610

Figure Lengend Snippet: Fig. 3. PLK1 interacts with FoxM1 in vitro. (A) Basal expression of PLK1 and FoxM1 in PTC cell lines. Proteins were isolated from Nthy-ori 3–1 cell line and three PTC cell lines and immunoblotted with antibodies against PLK1, FoxM1, and GAPDH (n = 3). (B) PLK1 interact with FoxM1. Cell lysates extracted from PTC cells were immunoprecipitated with PLK1 or IgG antibody. Interaction of endogenous PLK1 and FoxM1 was detected by immunoblotting (n = 3). (C) FoxM1 interact with PLK1. Cell lysates extracted from PTC cells were immunoprecipitated with FoxM1 or IgG antibody. Interaction of endogenous FoxM1 and PLK1 was detected by immunoblotting (n = 3). (D) Forced expression of PLK1 triggers the activation of FoxM1 expression. Nthy-ori 3–1 cells were transfected with either an empty vector or PLK1 cDNA for 48 h. Proteins were extracted from overexpression clones and were subjected immunoblotting using antibodies as indicated (n = 3). (E) Knockdown of PLK1 inhibits FoxM1. PTC cells underwent transfection with two distinct PLK1 shRNA sequences, and proteins from the selected clones were later analyzed using immunoblotting (n = 3). (F) Volasertib treatment reduce the expression of PLK1 and FoxM1 in PTC cells. PTC cells were treated with specified doses of volasertib for 48 h. Proteins were extracted after cell lysis and were subjected to immunoblotting, as indicated (n = 3). (G, H) Inhibition of FoxM1 has no effect on PLK1 expression. PTC cells were either transfected with FoxM1 siRNA (20 nM) or treated with thiostrepton (2.5 and 5 lM). Following 48 h, the cells were lysed, and the extracted proteins were subsequently subjected to immunoblotting using antibodies as indicated (n = 3).

Article Snippet: Plasmid DNA encoding human PLK1 and shRNA targeting human PLK1 were purchased from Origene (Rockville, MD, USA).

Techniques: In Vitro, Expressing, Isolation, Immunoprecipitation, Western Blot, Activation Assay, Transfection, Plasmid Preparation, Over Expression, Clone Assay, Knockdown, shRNA, Lysis, Inhibition

Journal: Molecular oncology

Article Title: PLK1 and FoxM1 expressions positively correlate in papillary thyroid carcinoma and their combined inhibition results in synergistic anti-tumor effects.

doi: 10.1002/1878-0261.13610

Figure Lengend Snippet: Fig. 4. Combined inhibition of PLK1 and FoxM1 markedly attenuates PTC cell growth in vitro. (A–B) Volasertib and thiostrepton synergistically inhibits clonogenicity. PTC cells (5 9 102) after volasertib and thiostrepton treatments were seeded into each of three dishes (60 mm diameter), and grown for an additional 10 days, then stained with crystal violet and colonies were counted. Data were presented as mean SD (n = 3). Statistical analyses were performed using two-tailed Student’s t-tests. *P < 0.05. (C) Volasertib and thiostrepton synergistically induce apoptosis. PTC cells were exposed to specified doses either of volasertib and thiostrepton, single or in combination, for 48 h. Subsequently, these cells were stained with annexin-V/PI and analyzed by flow cytometry. Data were presented as mean SD (n = 3). Statistical analyses were performed using two-tailed Student’s t-tests. *P < 0.05. (D) Volasertib and thiostrepton synergistically induce the cleavage of caspase-3 and PARP. PTC cells after 48 h of indicated treatment, lysed and proteins were subjected to immunoblot analysis using antibodies as indicated (n = 3).

Article Snippet: Plasmid DNA encoding human PLK1 and shRNA targeting human PLK1 were purchased from Origene (Rockville, MD, USA).

Techniques: Inhibition, In Vitro, Staining, Two Tailed Test, Cytometry, Western Blot

Journal: Molecular oncology

Article Title: PLK1 and FoxM1 expressions positively correlate in papillary thyroid carcinoma and their combined inhibition results in synergistic anti-tumor effects.

doi: 10.1002/1878-0261.13610

Figure Lengend Snippet: Fig. 5. Inhibition of PLK1 decreases the self-renewal ability of spheroids generated from PTC cells. (A, B) Knockdown of PLK1 impairs the self-renewal capacity of spheroids. PTC cells were transfected with PLK1 shRNA and subsequently subjected to a sphere-forming assay (scale bar = 1 mm). The total number of spheroids in the entire well was quantified. Data were presented as mean SD (n = 3). (C) Knockdown of PLK1 reduces the stem cell characteristics of spheroids. PTC cells were transfected with PLK1 shRNA and cultured in a sphere-forming medium. Subsequently, proteins were extracted from the spheroids and analyzed via immunoblotting using antibodies as indicated (n = 3). (D) Forced PLK1 expression enhances the growth of spheroids. Nthy-ori 3–1 cells were transfected with either an empty vector or PLK1 cDNA, and subsequently, these cells were subjected to a sphere-forming assay. The total number of spheroids in the entire dish was quantified. Data were presented as mean SD (n = 3). (E) Forced expression of PLK1 enhances the stem cell properties of spheroids, as validated through immunoblotting employing stem cell markers. Nthy-ori 3–1 cells were transfected with either an empty vector or PLK1 cDNA and subsequently cultured in a sphere-forming medium. Proteins were isolated from spheroids and immunoblotted with antibodies against PLK1, FoxM1, CD44, CD133, NANOG, and GAPDH (n = 3). (F) Volasertib and thiostrepton synergistically reduces spheroid growth. PTC cells were exposed to specified doses of volasertib and thiostrepton, either single or in combination, for 48 h. Following this treatment, the cells were subjected to a sphere-forming assay. Spheroids in the entire dish were counted. Data were presented as mean SD (n = 3). (G) PTC cells were exposed to specified doses either of volasertib and thiostrepton, single or in combination, for 48 h. Subsequently, these treated cells were cultured in a sphere-forming medium and proteins isolated from spheroids were subjected to immunoblotting using antibodies as indicated (n = 3). Statistical analyses were performed using two-tailed Student’s t-tests. *P < 0.05.

Article Snippet: Plasmid DNA encoding human PLK1 and shRNA targeting human PLK1 were purchased from Origene (Rockville, MD, USA).

Techniques: Inhibition, Generated, Knockdown, Transfection, shRNA, Cell Culture, Western Blot, Expressing, Plasmid Preparation, Isolation, Two Tailed Test

Journal: Molecular oncology

Article Title: PLK1 and FoxM1 expressions positively correlate in papillary thyroid carcinoma and their combined inhibition results in synergistic anti-tumor effects.

doi: 10.1002/1878-0261.13610

Figure Lengend Snippet: Fig. 6. Volasertib and thiostrepton synergistically inhibit PTC tumor growth in vivo. TPC-1 cells were subcutaneously injected into the flanks of 6-week-old NU/J mice (4 9 106 cells per mouse). Once the tumors reached approximately 100 mm3 in size, the mice were subjected to intraperitoneal treatment with the specified doses of volasertib (20 mgkg1) and thiostrepton (20 mgkg1), either administered individually or in combination. This treatment regimen was repeated twice a week and continued for a duration of 30 days. (A) Representative images of tumors from each group of mice. (B) The tumor volume measurements were taken every week. The average (n = 5) tumor volume (SD) in each group of mice was calculated, *P < 0.05. (C) Following 4 weeks of treatment, the mice were euthanized, and the mean tumor weight (SD) was determined for each group, *P < 0.05. (D) Tissue lysates from tumors were immuno-blotted with antibodies against PLK1, FoxM1, PARP, Caspase-3, Cleaved-caspase-3, Bcl-2, Bcl-xL, and GAPDH. (E) Western blots were quantified and data are the mean SD (n = 3). Statistical analyses were performed using two-tailed Student’s t-tests. *Statistically significant difference compared to vehicle control with P < 0.05. #Statistically significant difference compared to volasertib treatment with P < 0.05. @Statistically significant difference compared to thiostrepton treatment with P < 0.05.

Article Snippet: Plasmid DNA encoding human PLK1 and shRNA targeting human PLK1 were purchased from Origene (Rockville, MD, USA).

Techniques: In Vivo, Injection, Western Blot, Two Tailed Test, Control

Journal: Oncogene

Article Title: The CHFR mitotic checkpoint protein delays cell cycle progression by excluding Cyclin B1 from the nucleus.

doi: 10.1038/sj.onc.1208428

Figure Lengend Snippet: Figure 5 Analysis of Plk1 and Aurora-A in HCT116 neo- and CHFR-expressing cells exposed to nocodazole. The cells were synchronized in G1/S, released into the cell cycle (0 h), exposed to nocodazole (4 h) and examined 8–13 h after release from the cell cycle block, as indicated. (a,c) Plk1 and Aurora-A (Aur A) protein levels. G2/early prophase (adherent, Ad) cells were separated from prometaphase (nonadherent, NA) cells by mitotic shake-off before the extracts were prepared. (b,d,e) Immunofluorescence analysis for Plk1, Aurora-A (AurA) and Aurora-A phosphorylated on Thr288 12 h after release from the G1/S block. The centrosomes (arrow- heads) were marked by staining for g-tubulin. In (e),the cell marked with an asterisk is in prometaphase

Article Snippet: Antibodies to cell cycle markers were obtained from commercial vendors, as follows: Plk1 and H3 phosphorylated on Ser 10 from Upstate Biothechnology; Cdc2, Aurora-A, Aurora-B, Cyclin A and Cyclin B1 from Transduction Labs; Lamins A/C and B from Santa Cruz; Aurora-A phosphorylated on Thr288 and phospho-Thr-Pro from Cell Signaling Technologies.

Techniques: Expressing, Blocking Assay, Staining

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: Oncogene

Article Title: Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

doi: 10.1038/onc.2012.309

Figure Lengend Snippet: The mitotic kinase Plk1 is overexpressed in LNCaP-AI cells. ( a ) LNCaP and LNCaP-AI PCa cells were cultured in full media (FBS) or androgen-depleted media (csFBS). Cell growth over 7 days was determined by an MTS (OD 490 nm) growth assay. Results from triplicate samples (mean ± SD) in a representative experiment from n = 3 independent experiments are shown. ( b ) Lysates (20 μg) from LNCaP and LNCaP-AI cells grown in full media were immunoblotted for the androgen receptor (AR) and mitotic proteins as shown. α-tubulin was used as a loading control. ( c ) Relative Plk1 protein levels were determined (Plk1/tubulin and normalized against the value in LNCaP cells) (mean ± SD, n = 3 experiments).

Article Snippet: The following antibodies were used: androgen receptor (Upstate); Plk1, Cdc25C and caspase-3 (Santa Cruz Biotechnology); Plk1 (Origene), pT210-Plk1 and p62/sequestosome1 (BD Biosciences), Sgo2 and LC3 (Bethyl Laboratories), BubR1 (Novus Biologicals), PICH (Abnova), cyclin B1 (abcam), phospho-Histone H3 (Millipore), cleaved PARP-1 (Cell Signaling), α-tubulin (GeneTex), β-tubulin2.1 (Sigma-Aldrich), CREST serum (Dr. Bill Brinkley, Baylor College of Medicine), and secondary antibodies conjugated to Texas Red and Alexa Fluor 488 (Molecular Probes).

Techniques: Cell Culture, Growth Assay, Control

Journal: Oncogene

Article Title: Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

doi: 10.1038/onc.2012.309

Figure Lengend Snippet: Plk1 is elevated and active in LNCaP-AI cells. Cells were cultured in either full media (FBS) or androgen-depleted media (csFBS). ( a ) FACS analysis of randomly growing cells and cells synchronized by nocodazole treatment to enrich for G2/M phase population. ( b ) FACS analysis of cells costained with Plk1 and a mitotic marker, phospho-histone H3 (pH3), in randomly cycling cells and in cells synchronized by a nocodazole treatment. pH3(-) represent non-mitotic cells; pH3(+) represent mitotic cells. ( c ) Lysates (20 μg) from cells enriched in G2/M by nocodazole treatment were analyzed by immunoblotting with antibodies shown. After scanning the blots (NIH ImageJ), protein levels were measured (protein/tubulin and normalized against the value in LNCaP cells). Similar data were obtained in n = 3 experiments.

Article Snippet: The following antibodies were used: androgen receptor (Upstate); Plk1, Cdc25C and caspase-3 (Santa Cruz Biotechnology); Plk1 (Origene), pT210-Plk1 and p62/sequestosome1 (BD Biosciences), Sgo2 and LC3 (Bethyl Laboratories), BubR1 (Novus Biologicals), PICH (Abnova), cyclin B1 (abcam), phospho-Histone H3 (Millipore), cleaved PARP-1 (Cell Signaling), α-tubulin (GeneTex), β-tubulin2.1 (Sigma-Aldrich), CREST serum (Dr. Bill Brinkley, Baylor College of Medicine), and secondary antibodies conjugated to Texas Red and Alexa Fluor 488 (Molecular Probes).

Techniques: Cell Culture, Marker, Western Blot

Journal: Oncogene

Article Title: Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

doi: 10.1038/onc.2012.309

Figure Lengend Snippet: Differential response of LNCaP-AI cells to the Plk1 inhibitor BI2536. ( a ) Cells were cultured in androgen-depleted media and treated with increasing concentrations of BI2536 for 5 days. % Maximal growth relative to non-treated control cells (set at 100%) were analyzed in triplicates (mean ± SD) by MTS assays. Dotted line, IC50 of 0.2 nM BI2536 for LNCaP-AI cells. ( b ) FACS analysis of LNCaP-AI cells treated with or without 0.8 nM BI2536 for 5 days. ( c ) % Trypan-blue positive dead LNCaP-AI cells in (a) were counted in triplicates (mean ± SD). Similar data were obtained in n = 3 experiments.

Article Snippet: The following antibodies were used: androgen receptor (Upstate); Plk1, Cdc25C and caspase-3 (Santa Cruz Biotechnology); Plk1 (Origene), pT210-Plk1 and p62/sequestosome1 (BD Biosciences), Sgo2 and LC3 (Bethyl Laboratories), BubR1 (Novus Biologicals), PICH (Abnova), cyclin B1 (abcam), phospho-Histone H3 (Millipore), cleaved PARP-1 (Cell Signaling), α-tubulin (GeneTex), β-tubulin2.1 (Sigma-Aldrich), CREST serum (Dr. Bill Brinkley, Baylor College of Medicine), and secondary antibodies conjugated to Texas Red and Alexa Fluor 488 (Molecular Probes).

Techniques: Cell Culture, Control

Journal: Oncogene

Article Title: Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

doi: 10.1038/onc.2012.309

Figure Lengend Snippet: Growth inhibition of additional androgen-insensitive prostate cancer cells to the Plk1 inhibitor BI2536. ( a ) Lysates (20 μg) were analyzed for Plk1 levels by western blot analysis. α-tubulin was used as a loading control. *, nonspecific band. ( b - d ) abl, C4-2B and CWR22Rv1 cells were culture in full (FBS) or androgen-depleted (csFBS) media. Cell growth over 5 days was determined by an MTS assay. ( e - g ) Cells were cultured in androgen-depleted media and treated with increasing concentrations of BI2536 for 5 days. % Maximal growth relative to non-treated control cells (set at 100%) were analyzed in triplicates (mean ± SD) by MTS assays. Data in (b – g) are representative of n = 3 experiments.

Article Snippet: The following antibodies were used: androgen receptor (Upstate); Plk1, Cdc25C and caspase-3 (Santa Cruz Biotechnology); Plk1 (Origene), pT210-Plk1 and p62/sequestosome1 (BD Biosciences), Sgo2 and LC3 (Bethyl Laboratories), BubR1 (Novus Biologicals), PICH (Abnova), cyclin B1 (abcam), phospho-Histone H3 (Millipore), cleaved PARP-1 (Cell Signaling), α-tubulin (GeneTex), β-tubulin2.1 (Sigma-Aldrich), CREST serum (Dr. Bill Brinkley, Baylor College of Medicine), and secondary antibodies conjugated to Texas Red and Alexa Fluor 488 (Molecular Probes).

Techniques: Inhibition, Western Blot, Control, MTS Assay, Cell Culture

Journal: Oncogene

Article Title: Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

doi: 10.1038/onc.2012.309

Figure Lengend Snippet: Plk1 inhibition by BI2536 resulted in distinct changes in nuclear morphology and an increase in aneuploidy in LNCaP-AI cells. ( a ) Cells were treated with 0.8 nM BI2536 for 5 days in androgen-depleted media, immunostained for α-tubulin (green) and counterstained with DAPI (blue) for DNA. Arrow, cells in telophase. Bars, 10 μm. ( b ) Nuclear morphologies for cells in A were quantified as mononuclear (normal) or multinuclear (with nuclear vesicles) (mean ± SD, n = 3 experiments). N, cell number. None, no mononuclear cells were detected.

Article Snippet: The following antibodies were used: androgen receptor (Upstate); Plk1, Cdc25C and caspase-3 (Santa Cruz Biotechnology); Plk1 (Origene), pT210-Plk1 and p62/sequestosome1 (BD Biosciences), Sgo2 and LC3 (Bethyl Laboratories), BubR1 (Novus Biologicals), PICH (Abnova), cyclin B1 (abcam), phospho-Histone H3 (Millipore), cleaved PARP-1 (Cell Signaling), α-tubulin (GeneTex), β-tubulin2.1 (Sigma-Aldrich), CREST serum (Dr. Bill Brinkley, Baylor College of Medicine), and secondary antibodies conjugated to Texas Red and Alexa Fluor 488 (Molecular Probes).

Techniques: Inhibition

Journal: Oncogene

Article Title: Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

doi: 10.1038/onc.2012.309

Figure Lengend Snippet: Necrostatin-1 attenuates cell death by necroptosis resulting from either Plk1 depletion or Plk1 inhibition in LNCaP-AI cells. ( a ) LNCaP-AI cells were transfected with either siLuciferase or Plk1 RNAi oligos for 5 days. Cell lysates (20 μg) were analyzed for Plk1 levels. α-tubulin was used as a loading control. ( b ) Cells prepared as in (a) or treated with 0.8 nM BI2536 were analyzed after 5 days by an MTS assay. Triplicate samples (mean ± SD) from one of n = 2 experiments are shown. ( c ) Cells were treated as in (b) and counterstained with DAPI to visualize DNA, shown here in black and white for contrast. Arrows, cells in mitosis in control cultures. Single giant cells containing clusters of nuclear vesicles are shown for siPlk1 and BI2536-treated LNCaP-AI cells. Bars, 10 μm. Note differences in scale. ( d ) Cells were transfected with siPlk1 for 5 days. 7.7 μM Necrostatin-1 (Nec-1), the necroptosis inhibitor, was added 3 h prior to siPlk1 transfection and remained throughout the experiment. Triplicate samples (mean ± SD) from MTS assays from one of n = 2 experiments are shown. ( e ) Cells were treated with 0.4 or 0.8 nM BI2536 for 5 days. 5 μM Nec-1 was added 3 h prior to BI2536 addition and remained throughout the experiment. Triplicate samples (mean ± SD) from MTS assays from one of n = 3 experiments are shown. ( f ) Working model of necroptosis induction by Plk1 inhibition in androgen-insensitive PCa cells. See text.

Article Snippet: The following antibodies were used: androgen receptor (Upstate); Plk1, Cdc25C and caspase-3 (Santa Cruz Biotechnology); Plk1 (Origene), pT210-Plk1 and p62/sequestosome1 (BD Biosciences), Sgo2 and LC3 (Bethyl Laboratories), BubR1 (Novus Biologicals), PICH (Abnova), cyclin B1 (abcam), phospho-Histone H3 (Millipore), cleaved PARP-1 (Cell Signaling), α-tubulin (GeneTex), β-tubulin2.1 (Sigma-Aldrich), CREST serum (Dr. Bill Brinkley, Baylor College of Medicine), and secondary antibodies conjugated to Texas Red and Alexa Fluor 488 (Molecular Probes).

Techniques: Inhibition, Transfection, Control, MTS Assay

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Pericentromeric cohesin is deprotected long before sister chromatids separate. During phosphorylation- and Wapl-dependent release of cohesin from chromosome arms, pericentromeric cohesin is protected by associated Sgo1-PP2A. Following the early mitotic degradation of Nek2a and cyclin A2, Sgo1-PP2A relocates to kinetochores. Yet, pericentromeric cohesion persists until securin and cyclin B1 are degraded and separase is activated. A requirement for Nek2a and cyclin A2 for prophase pathway signaling would explain why, in metaphase, Wapl can no longer release cohesin. ( B ) Nek2a and Cdk1/2-cyclin A2 are necessary for Wapl-dependent in vitro displacement of cohesin from chromatin. Chromatin was freshly purified by repeated sedimentation from G2-arrested HeLaK cells, incubated with recombinant Wapl, kinases, and inhibitors (BI2536, ZM-447439, and RO-3306), as indicated, and then re-pelleted. Supernatants and chromatin pellets were analyzed by immunoblotting. ( C ) Wapl and the three kinases Nek2a, Cdk1/2-cyclin A2, and Aurora B-INCENP are sufficient to support the displacement of cohesin from isolated chromatin. Chromatin was freshly purified by repeated sedimentation from G2-arrested HeLaK cells, incubated with recombinant Wapl, Nek2a, Cdk1/2-cyclin A2, Cdk1-cyclin B1, aurora B, and/or Plk1, as indicated, and then re-pelleted. Supernatants and chromatin pellets were analyzed by immunoblotting. .

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: In Vitro, Purification, Sedimentation, Incubation, Recombinant, Western Blot, Isolation

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Recombinant kinases are active as judged by the phosphorylation of model substrates. Plk1, aurora B-INCENP, Nek2a, Cdk1/2-cyclin A2, or Cdk1-cyclin B1 supplemented with their specific inhibitor or carrier solvent DMSO (−) were incubated with the corresponding model substrate in the presence of [γ 33 P]-ATP. Reactions were subjected to SDS-PAGE followed by autoradiography. BI BI2536, ZM ZM-447439, ST staurosporine, RO RO-3306, MBP myelin basic protein. ( B ) Recombinant Nek2a, Cdk1/2-cyclin A2, and Wapl are free of cyclin B1 and Plk1. The preparations of Nek2a, Cdk1/2-cyclin A2, Wapl, Cdk1-cyclin B1, and Plk1 used for the release assays were characterized by immunoblotting using the indicated antibodies. In vitro expressed (IVTT) cyclin B1 served as an additional control.

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: Recombinant, Solvent, Incubation, SDS Page, Autoradiography, Western Blot, In Vitro, Control

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Experimental outline. DNA-mediated chromatin pull-down from G2-arrested HeLaK cells is followed by cohesin eviction experiments. ( B ) Cohesin is eluted from immobilized chromatin upon DNA-cleavage but not high salt treatment. Chromatin beads were treated with benzonase and/or high salt, and retained proteins were detected by immunoblotting. High-salt-washed chromatin (lane 6) was used for subsequent cohesin eviction experiments. ( C ) Wapl-dependent cohesin release in vitro requires Nek2a, Cdk1/2-cyclin A2, and Aurora B but not Cdk1-cyclin B1 and Plk1. Immobilized chromatin was combined with the indicated proteins. Following centrifugation, DNA-beads and supernatant were analyzed by immunoblotting. .

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: Western Blot, In Vitro, Centrifugation

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: ( A ) Nek2a and Cdk1/2-cyclin A2 target juxtaposed but distinct residues within the disordered, C-terminal domain of Pds5b. In vitro expressed Pds5b variants were incubated with the indicated kinases and inhibitors in the presence of [γ- 33 P]-ATP, subjected to SDS-PAGE, and analyzed by autoradiography and immunoblotting. Note that Pds5b is also phosphorylated by aurora B and, weakly, by Plk1. KD kinase-dead Nek2a-Lys37Met; IVTT coupled in vitro transcription-translation. ( B ) Graphical summary of Nek2a- and Cdk1/2-cyclin A2-targeted Pds5b residues and corresponding phosphorylation-resistant variants. ( C ) Ser1209 of Pds5b is phosphorylated by Nek2a but not Cdk1/2-cyclin A2. Following incubation of in vitro expressed Pds5b-WT or −3A with Nek2a or Cdk1/2-cyclin A2, samples were analyzed by immunoblotting using the indicated antibodies. Nek2a-Lys37Met (KD, kinase-dead) served as a negative control. ( D ) The spatiotemporal dynamics of Ser1209-phosphorylation correlate with the recruitment of Wapl to chromatin followed by cohesin release. HeLaK cells synchronously cycling through mitosis were subjected to time-resolved fractionation and immunoblotting. At the time of release from a RO-3306-mediated arrest, cultures were supplemented with a Nek2a inhibitor (NCL00017509) or carrier solvent (mock). .

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: In Vitro, Incubation, SDS Page, Autoradiography, Western Blot, Negative Control, Fractionation, Solvent

Journal: The EMBO Journal

Article Title: Requirement of Nek2a and cyclin A2 for Wapl-dependent removal of cohesin from prophase chromatin

doi: 10.1038/s44318-024-00228-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Recombinant human active PLK1 Protein , R&D Systems , 3804-KS-010.

Techniques: Recombinant, Transduction, Sequencing, Protease Inhibitor, Membrane, Software

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A Left: Renilla luciferase activity analyses in HEK 293 T cells co-transfected with psiCHECK2-PLK1-CDS, psiCHECK2-PLK1-3′UTR, or psiCHECK-PLK1-3′UTR-MUT + miR-183-5p or negative control miRNA (NC) (Ambion). Results are 48 h post transfection. Right: Renilla luciferase activity analyses in HEK 293 T cells co-transfected with psiCHECK2-PLK1-CDS or psiCHECK2-PLK1-3′UTR + miR-18a-3p, miR-100-5p, or negative control miRNA (NC). Data ( n = 3/group) are presented as mean + % SD. B Left: Immunoblots showing PLK1 expression following no treatment (NT), transfection with negative control (NC) or miR-183-5p. Right: RT-qPCR demonstrating expression levels of miR-183-5p following transfection (results are x10 3 ). C Left: Immunoblots showing PLK1 expression following no treatment (NT) or miR-183-5p, following transfection with PLK1 CDS with 3′UTR (CDS + 3′UTR) or PLK1 CDS only (CDS only). Right: RT-qPCR demonstrating expression levels of miR-183-5p following transfection (results are x10 3 for MDA-MB-231, BT549 and ZR-75-1, x10 for T47D). D Western blots show results of the rescue experiments (see material and methods) performed on the MDA-MB-231 (left) and T47D (right) cell lines. Bar graphs are densitometry results of PLK1-fold expression. Vinculin was used as internal control. Data are the mean of at least 3 independent experiments ( N = 3) + SD. ** p -value <0.01 compared to negative control by Student’s unpaired t test. E TCGA, IBCD analysis. Left: Violin plots represent miR-183-5p expression in Cancerous versus Normal breast tissue ( p -value = 3.35 × 10 −21 ) and in Luminal A versus TNBC ( p -value = 0.25). Right: Violin plots depicting PLK1 expression in Cancerous versus Normal breast tissue ( p -value = 4.22 × 10 −20 ) and in Luminal A versus TNBC. ( p -value = 1.01 × 10 −6 ). N = 46 paired luminal A, 15 paired TNBC, 61 cancer vs normal.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Luciferase, Activity Assay, Transfection, Negative Control, Western Blot, Expressing, Quantitative RT-PCR

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A WST8 assay demonstrating the effects of NMS-P937 treatment: 0, 10, 25, 50, 75, 100, and 150 nM after transfection with negative control (NC) or miR-183-5p or si-PLK1. Left hand graphs. TNBC cell lines MDA-MB-231 (top left, p -value = 3.26e-14) and BT549 (top right, p -value = 0.00112). Luminal A cell lines T47D (lower left, p -value < 2e-16) and ZR-75-1 (lower right p -value = 1.93e-6). Right hand graphs. Assay was repeated using si-PLK1. Data ( N = 3/group) are mean + SD. ** p -value <0.05, *** p -value <0.001 calculated by ANOVA. B Left: Matrices demonstrating MDA-MB-231 and T47D cell lines treated with different concentrations of miR-183-5p and NMS-P937, then analyzed to determine cell mortality (% inhibition). Right: Bliss score shows a synergistic effect present on cell mortality between miR-183-5p overexpression and NMS-P937 treatment.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Transfection, Negative Control, Inhibition, Over Expression

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A MDA-MB-231, BT549, T47D, and ZR-75-1 cells treated with NMS-P937 for 48 h, +/- miR-183-5p transfection for 72 h. Total protein lysates were analyzed by Western blot. Blots were stained for PLK1, DNMT1, p-STAT3 (Tyr 705), p-p53 (Ser15), Bcl-XL, cleaved caspases 7 and 3, cleaved PARP and vinculin. B – E Protein band densitometry normalized to vinculin. Data ( N = 3/group) are presented as mean + SD.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Transfection, Western Blot, Staining

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A MDA-MB-231, BT549, T47D, and ZR-75-1 cells treated with NMS-P937 for 48 h, +/- si-PLK1 for 72 h. Total protein lysates were analyzed by Western blot. Blots were stained for PLK1, DNMT1, p-STAT3 (Tyr 705), p-p53 (Ser15), Bcl-XL, cleaved caspases 7 and 3, cleaved PARP and vinculin. B – E Protein band densitometry normalized to vinculin. Data ( N = 3/group) are presented as mean + SD.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Western Blot, Staining

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A Representative flow cytometry images of breast cancer cell lines treated with NMS-P937 for 48 h, +/- si-PLK1 transfection for 72 h. Cells were stained with Annexin-FITC and PI. The cell populations of interest were those undergoing Late (Q2) or early (Q4) apoptosis. B , C Bar graphs representing the percentage of cells undergoing early (B) or late (C) apoptosis. Cells were either treated with negative control miRNA (NC), si-PLK1 only, NMS-P937 only (25 nM or 75 nM), or si-PLK1 + NMS-P937. Data ( N = 4/group) are presented as mean + SD. * p -value <0.05, ** p -value <0.001 compared to negative control by Student’s unpaired t test.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Flow Cytometry, Transfection, Staining, Negative Control