plk4 inhibitor centrinone  (Tocris)

Journal: Nature Communications

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

doi: 10.1038/s41467-025-64804-1

Figure Lengend Snippet: a Schematic of the protocol used to modulate CENP-E activity in cells with variable centriole numbers per spindle pole, generated by continuous Plk4 inhibition. Numbers on the left denote number of centrioles per spindle pole. b Diagram showing progressive centriole depletion after continuous Plk4 inhibition by 300 nM centrinone. c Representative images of live RPE-1 cells expressing CENP-A-GFP and centrin1-GFP (color-coded by depth, color bar) with varying centriole counts (white circles), 5 minutes after end of prometaphase spindle elongation, under indicated treatments. d , e Quantification of polar chromosome number d , and their residence time at spindle poles e , after prometaphase spindle elongation, comparing cells with different centriole numbers. Category “1” includes centriolar poles from both 1:1 and 1:0 spindles. Colored points represent individual cells; black lines show the mean, with light and dark gray areas marking 95% confidence intervals for the mean and standard deviation, respectively. Number of cells per condition: 14, 15, 22, 16, 14, 18, 16, 16, 12, 12, 16, 16 ( d , e ), each pooled from ≥3 independent biological replicates. f Number of polar chromosomes after treatments in cells with varying centriole numbers, fixed prior to measurement. Pooled data from ≥3 independent biological replicates. Number of kinetochores and cells is given in the figure. Dispersion measures as in ( d , e ). g STED microscopy images of RPE-1 cells immunostained for α-tubulin (gray), expressing CENP-A-GFP and centrin1-GFP (color-coded by depth). All images are maximum projections. Statistics: two-tailed ANOVA with post-hoc Tukey’s HSD test. Symbols indicate: n.s., p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; inh., inhibited; depl., depleted; siRNA, small interfering RNA; KC, kinetochore; ctrl., control. Source data are provided as a Source Data file.

Article Snippet: Plk4 inhibitor Centrinone (MedChemExpress, K i value 0.16 nM) at a final concentration of 300 nM, was added 1-5 days before the imaging as noted in the manuscript.

Techniques: Activity Assay, Generated, Inhibition, Expressing, Standard Deviation, Dispersion, Microscopy, Two Tailed Test, Small Interfering RNA, Control

Journal: bioRxiv

Article Title: Multiple clustered centrosomes in antigen-presenting cells foster T cell activation without MTOC polarization

doi: 10.1101/2024.07.18.604057

Figure Lengend Snippet: ( A ) DNA staining of mature CETN2-GFP expressing BMDCs to determine nuclear ploidy. Left: gating strategy for identification of 2N and 4N DCs and histogram of DNA content distribution of CD11c + cells. Right: quantification of 2N and 4N cells according to DNA content. Graph displays mean values ± s.d. of 18 independent experiments. ( B ) Inhibition of PLK4 activity by Centrinone. CETN2-GFP expressing BMDCs were treated with either 250 or 500 nM Centrinone during differentiation (from day 0 on) and centriole numbers were determined according to CETN2-GFP + foci in mature BMDCs. Left: confocal images of Centrinone-treated and control cells. Merged channels of CETN2-GFP (green) and DAPI (blue) are shown. White arrowheads indicate cells without centrioles. Scale bars, 5 μ m. Right: quantification of centriole numbers after Centrinone treatment. Graph displays mean values ± s.d. of three independent experiments with at least N = 200 cells analyzed per condition. Picture created with BioRender. ( C ) Immunostaining of mature CETN2-GFP BMDCs against γ-tubulin after 250 nM Centrinone treatment. Merged and individual channels of CETN2-GFP (green), γ-tubulin (red) and DAPI (blue) are shown. Scale bars, 5 μ m. White arrowheads indicate cells without centrioles but with prominent γ-tubulin foci. Right: quantification of γ-tubulin signal intensity in mature CETN2-GFP expressing BMDCs after Centrinone treatment. Graph shows normalized values relative to cells with two centrioles ± s.d. Each data point represents one cell derived from one representative experiment out of three independent experiments. Dotted line drawn at 1.0. ****, P < 0.0001 (one-way Anova with Dunnett’s multiple comparisons). ( D ) Expansion microscopy of mature control or Centrinone-treated (500nM) CETN2-GFP BMDCs. Top panels: NHS-Ester staining. Bottom panels: merged channels of acetylated (ac)-tubulin (green) and γ-tubulin (magenta). Images are shown as top view and right view. Scale bars, 1 µm. ( E ) Immunostaining of MTs in Centrinone-treated and control cells. Upper panel: mature CETN2-GFP (green) BMDCs were confined under agarose and immunostained against γ-tubulin (white) and α-tubulin (red). Nuclei were counterstained with DAPI (blue). Scale bars, 10 µm. Magnifications of the indicated regions show individual channels of CETN2-GFP (green) and γ-tubulin (white). Scale bars, 2 µm. Lower panel: quantification of MTOCs (left) and MT filaments emanating from defined regions around centrosomes (right) in mature CETN2-GFP expressing BMDCs after Centrinone treatment. Left graph shows median and distribution of data points of at least three independent experiments. ns, non-significant (Kruskal-Wallis test with Dunn’s multiple comparisons). Right graph shows median, interquartile range and minimum to maximum values of at least three independent experiments. ns, non-significant (one-way Anova with Dunnett’s multiple comparisons). In both graphs each data point represents one cell. ( F ) Quantification of proliferating T cells after Centrinone treatment according to CFSE labeling. DCs were treated with the indicated concentrations of Centrinone and loaded with or w/o antigen. Graph displays mean values ± s.d. Each data point represents one independent experiment with at least N = 10.000 cells analyzed per condition. Cells were derived from three different mice. *, P < 0.0332; **, P < 0.0021 (two-way Anova with Dunnett’s multiple comparisons).

Article Snippet: To prevent new pro-centriole formation cells were cultured in the presence of the PLK4 inhibitor Centrinone (Tocris; 250 nM or 500 nM) or control (solvent DMSO) during differentiation and maturation.

Techniques: Staining, Expressing, Inhibition, Activity Assay, Control, Immunostaining, Derivative Assay, Microscopy, Labeling

Journal: Molecular Oncology

Article Title: NUAK1 governs centrosome replication in pancreatic cancer via MYPT1 / PP1β and GSK3β ‐dependent regulation of PLK4

doi: 10.1002/1878-0261.13425

Figure Lengend Snippet: NUAK1 regulates PLK4 expression. (A) Quantification of centrosome number in 50 mitotic Mia PaCa‐2 cells per treatment group per experiment, measured by γ‐tubulin IF, fixed 10.5 h post‐release from thymidine block, and treated at 6 h post‐release with 10 μ m HTH‐01‐015 or vehicle control. Mean ± SEM of three independent experiments shown. T ‐test. (B) Immunoblot of PLK4 total protein in asynchronous Mia PaCa‐2 cells transfected with NUAK1, or non‐targeting (si Ctrl), siRNAs for 24 h. Representative of three independent experiments. (C) Immunoblot of PLK4 total protein in asynchronous Mia PaCa‐2 cells treated with the indicated concentrations of HTH‐01‐015 for 1 h. Representative of three independent experiments. (D) Immunoblot of PLK4 total protein in asynchronous Mia PacCa‐2 cells pre‐treated for 1 h with centrinone or vehicle, followed by 1 h treatment with HTH‐01‐015, vehicle, or HTH‐01‐015 + centrinone combined. Representative of three independent experiments. (E) Quantification of centrosome number by γ‐tubulin IF in mitotic Mia PaCa‐2 cells treated with Centrinone, HTH‐01‐015, both, or vehicle ctrl, from time of release from thymidine block. Cells were fixed for analysis at 10.5 h post‐release. Fifty cells were scored per treatment group per experiment. Mean ± SEM of three independent experiments shown. One‐way ANOVA with post hoc Tukey's test. (F) Quantification of incidence of nuclear aberrations in Mia PaCa‐2 cells treated with centrinone, HTH‐01‐015, both, or vehicle ctrl, from time of release from thymidine block. Cells were fixed for analysis at 13 h post‐release. One hundred cells were scored per treatment condition per experiment. Mean ± SEM of three independent experiments shown. One‐way ANOVA with post hoc t ‐test. For all panels, P value; ** P < 0.01; **** P < 0.0001.

Article Snippet: Cells in log phase growth were treated with the indicated concentrations of 5 or 10 μ m of NUAK1 inhibitor (HTH‐01‐015, Tocris, Bristol, UK), GSK3 inhibitor 3 μ m – (CHIR99021, Tocris), PLK4 inhibitor 100 n m (Centrinone, MedChem Express, Monmouth Junction, NJ, USA) throughout the study.

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

Journal: Molecular Oncology

Article Title: NUAK1 governs centrosome replication in pancreatic cancer via MYPT1 / PP1β and GSK3β ‐dependent regulation of PLK4

doi: 10.1002/1878-0261.13425

Figure Lengend Snippet: NUAK1 regulation of PLK4 is mediated by GSK3β. (A) Quantification of centrosome number by γ‐tubulin IF staining in synchronised Mia PaCa‐2 cells transfected with MYPT1, or non‐targeting (siC), siRNAs. Fifty cells scored per condition per experiment. Mean ± SEM of three independent experiments shown. One‐way ANOVA with post hoc t ‐test. (B) Immunoblot of total PLK4 in Mia PaCa‐2 cells transfected with MYPT1, versus non‐targeting (siCtrl), siRNA. Representative of three independent experiments. (C) Immunoblot of Ser9 phospho‐GSK3β in Mia PaCa‐2 cells transfected with NUAK1, versus non‐targeting, siRNA. Representative of three independent experiments. (D) Immunoblot of Ser9 phospho‐GSK3β in Mia PaCa‐2 cells treated with 10 μ m HTH‐01‐015 or DMSO vehicle for 4 h. Representative of three independent experiments. (E) Immunoblot of total PLK4 in Mia PaCa‐2 cells transiently transfected with constitutively active (S9A) or kinase‐dead (K85R) mutant GSK3β expression vector, compared with empty vector (EV). Representative of three independent experiments. (F) Immunoblots for endogenously expressed PLK4, NUAK1, and GSK3β in lysates and anti‐PLK4 immunoprecipitates, or IgG control IPs, of untreated asynchronous Mia PaCa‐2 cells. Representative of two independent experiments. (G) Immunoblot of total PLK4 in Mia PaCA‐2 cells pre‐treated for 1 h with 3 μ m CHIR‐99021 (GSK3i) or DMSO vehicle, followed by 10 μ m HTH‐01‐015 ± 3 μ m CHIR‐99021 for 1 h immediately prior to harvest. Representative of three independent experiments. (H) Quantification of centrosome number by γ‐tubulin IF in mitotic Mia PaCa‐2 cells treated with CHIR‐99021 (GSK3i), HTH‐01‐015 (HTH), both, or vehicle ctrl, from time of release from thymidine block. Cells were fixed for analysis at 10.5 h post‐release. Fifty cells were scored per treatment condition per experiment. Mean ± SEM of three independent experiments shown. One‐way ANOVA with post hoc Tukey's test. (I) Quantification of incidence of nuclear aberrations in Mia PaCa‐2 cells treated with CHIR‐99021 (GSK3i), HTH‐01‐015 (HTH), both, or vehicle ctrl, from time of release from thymidine block. HTH‐01‐015 and ctrl values are the same as Fig. as experiment was performed simultaneously. Cells were fixed for analysis at 13 h post‐release. One hundred cells were scored per treatment group per experiment. Mean ± SEM of three independent experiments shown. One‐way ANOVA with post hoc Tukey's test. For all panels, P value; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells in log phase growth were treated with the indicated concentrations of 5 or 10 μ m of NUAK1 inhibitor (HTH‐01‐015, Tocris, Bristol, UK), GSK3 inhibitor 3 μ m – (CHIR99021, Tocris), PLK4 inhibitor 100 n m (Centrinone, MedChem Express, Monmouth Junction, NJ, USA) throughout the study.

Techniques: Staining, Transfection, Western Blot, Mutagenesis, Expressing, Plasmid Preparation, Control, Blocking Assay