okadaic acid  (Merck & Co)

Journal: Nucleic Acids Research

Article Title: AURKB-driven dissolution of CIZ1–RNA assemblies from the inactive X chromosome in mitosis

doi: 10.1093/nar/gkag018

Figure Lengend Snippet: Dissolution of CIZ1–Xi assemblies in mitosis. ( A ) Model of CIZ1–RNA assemblies surrounding and protecting the modification status of underlying chromatin [ , ]. ( B ) Illustrative immunofluorescence images of female murine D3T3 cells stained for CIZ1 via N-terminal epitopes (CIZ1-N, red, detected with pAb 1794), revealing large protein assemblies at the inactive X chromosome (white arrows) that are not detected in mitosis. DNA is shown in blue, bar is 5 µm. ( C ) Diagram illustrating loss in mitosis and the early G1 phase window during which reformation of CIZ1–Xi assemblies takes place, determined previously using cells that were synchronized in G1 phase by release from arrest with nocodazole . ( D ) Map showing conserved putative AURKB phosphorylation sites between murine and human CIZ1 (circles) displayed on full-length murine CIZ1 ( NP_082688.1 .) The location of epitopes of CIZ1 antibodies used throughout is shown above. Conserved prion-like domains (PLD1 and PLD2) are in red , zinc fingers 1–3 in cyan (ZnF_C2H2 SM00355, ZF_C2H2 sd00020, and ZF_C2H2 sd00020), acidic region (Ac) in yellow, matrin-3 homology domain (MH3) in orange (ZnF_U1, smart00451), and h37/m38 amino-acid C-terminal tail in blue. The sequence context and identity of three conserved AURKB phosphorylation sites in the extreme C-terminus are shown. ( E ) Frequency of cells with CIZ1–Xi assemblies (red) or nucleus-wide SAFA (blue) in cells passing through the stages of mitosis indicated, for D3T3 cells and female primary embryonic fibroblasts (PEFs at p3), in the presence and absence of the AURKB kinase inhibitor barasertib at 0.1 and 1 µM. Results show the average of 3–4 independent replicates within one experiment for each line, with SEM. n indicates the number of nuclei inspected (PEF grey, 3T3 black). Statistical analysis of CIZ1–Xi frequency in anaphase cells shows one-way ANOVA with Tukey post hoc test within each cell type, where * <.05, ** <.01, *** <.001. Below, example immunofluorescence images of D3T3 cells through mitosis, with and without 1 µM barasertib. Cells were stained for the N-terminal domains of CIZ1 (CIZ1-N, red) and SAFA (green). DNA is shown in blue, bar is 5 microns. ( F ) Upper histogram shows the proportion of cells with CIZ1-marked Xi in cycling populations of female D3T3 cells after the indicated times exposed to 300 nM Okadaic acid, visualized via CIZ1-N (red). Lower, histograms show the effect of the indicated concentrations of tautomycin for 15 h, stained for CIZ1-N or the ‘tail’ epitope in the C-terminal end of CIZ1 (CIZ1-C, rabbit pAb). Comparison of technical replicates is by t-test, where * <.05, ** <.01, *** <.001. Error bars show SEM. Below, example images showing H3K27me3-marked Xi chromatin in cells stained for CIZ1-N or CIZ1-C at 15 h with or without tautomycin. Bar is 5 microns.

Article Snippet: Okadaic acid , CST , Cat#5934S.

Techniques: Dissolution, Modification, Immunofluorescence, Staining, Phospho-proteomics, Zinc-Fingers, Sequencing, Comparison

Journal: Nucleic Acids Research

Article Title: AURKB-driven dissolution of CIZ1–RNA assemblies from the inactive X chromosome in mitosis

doi: 10.1093/nar/gkag018

Figure Lengend Snippet: AURKB site modification in mitosis. ( A ) Western blot showing denatured proteins in whole cell lysates collected from untransfected D3T3 cells, or populations expressing full-length mouse GFP-m845 WT or GFP-m845 DDD , after immunostaining for CIZ1-N or CIZ1-C (tail epitope), or β-actin, and GFP as indicated. ( B ) Western blot showing denatured endogenous proteins in chemically treated D3T3 cells to achieve cell cycle enrichment in mitosis (M, nocodazole), S phase (S, thymidine), or a phosphatase-suppressed state (okadaic acid). Immunoblotting for C-terminal tail and N-terminal CIZ1 indicates a reduction in tail epitope, compared to untreated cells, during arrest in metaphase, or after phosphatase inhibition. Histone H3 is shown as a loading control. Cy, cycling. ( C ) Illustration showing data interpretation in which the CIZ1 tail AURKB site cluster is phosphorylated in mitosis, driving dispersal of CIZ1 from Xi assemblies. ( D ) Female D3T3 cells in stages of mitosis as indicated, immunostained for CIZ1-N or CIZ1-C and co-stained for SAFA. Right, histograms show frequency of retention in interphase (I), prophase (P), metaphase (M), or anaphase (A), where N indicates replicate analyses and n nuclei scored. Lower, by metaphase CIZ1-N and CIZ1-C are significantly different ( P < .00017), student’s t-test. ( E ) Experimental overview of in vitro kinase reactions using purified recombinant human CIZ1 C-terminal fragment C179 and purified AURKB. Middle, products analysed by western blot with C-terminal CIZ1 epitope-defined antibodies, showing changes in reactivity in response to exposure to increasing concentrations of AURKB kinase. Graph shows band intensities relative to untreated C179 control. Products were also analysed by mass spectrometry .

Article Snippet: Okadaic acid , CST , Cat#5934S.

Techniques: Modification, Western Blot, Expressing, Immunostaining, Inhibition, Control, Staining, In Vitro, Purification, Recombinant, Mass Spectrometry

Journal: The EMBO Journal

Article Title: Molecular requirements for PLK1 activation by T-loop phosphorylation

doi: 10.1038/s44318-025-00681-0

Figure Lengend Snippet: ( A ) Pull-down of mNeonGreen-PLK1 WT or mNeonGreen-PLK1 K208R from mitotic HeLa cell lysates with or without RNAi-mediated Bora depletion, as schematized. The bar plot represents the arithmetic mean of three replicates, and black dots are the normalized values (over PLK1 signal) of signal intensity for each replicate. OE overexpressed; siBora siRNA-mediated depletion of Bora. The bar plot represents the arithmetic mean of three replicates, and black dots are the normalized values (over PLK1 signal) of signal intensity for each replicate. The error bar is the standard deviation. ( B ) Pull-down performed as in ( A ), except for the addition of Okadaic acid (100 nM OA, 1 h before cell harvesting), with or without Bora depletion, as schematized. The bar plot represents the arithmetic mean of three replicates, and black dots are the normalized values (over PLK1 signal) of signal intensity for each replicate. OE overexpressed, siBora siRNA-mediated depletion of endogenous Bora, PPPi phosphoprotein phosphatase inhibitor. The bar plot represents the arithmetic mean of three replicates, and black dots are the normalized values (over PLK1 signal) of signal intensity for each replicate. The error bar is the standard deviation. ( C ) HeLa cell lines expressing mNeonGreen-PLK1 WT or mNeonGreen-PLK1 K208R were synchronized with a double-thymidine arrest, released in the cell cycle, and followed as they entered mitosis. Dot plots show the distribution of mitotic entry times of individual cells after double-thymidine release (DTR). Each dot represents an individual cell, the horizontal line represents the mean, and the vertical line the 95% CI of the mean. Statistical comparison was performed as indicated in the legend for Fig. . OE overexpressed, siBora/siPLK1 siRNA-mediated depletion of endogenous Bora and endogenous PLK1, PLK1i Polo-like kinase 1 inhibitor (BI2536). The experiments were interrupted after 24 h of filming due to the significant effects on cell viability caused by phototoxicity after this time point. The exact P values were: 1–2 < 0.0001; 1–3 < 0.0001; 1–4 < 0.0001; 2–3 > 0.9999; 2–5 > 0.9999; 3–6 = 0.6884; 4–5 < 0.0001; 4–6 < 0.0001; 5–6 > 0.9999. ( D ) In vitro assay with Aurora A:Bora and PLK1 WT , PLK1 R95A , PLK1 E121A, , PLK1 R95A-K208R , and PLK1 E121A-K208R. . After 60 min at 30 °C, activation loop phosphorylation of PLK1 was monitored. ( E ) Pull-down from mitotic cell lysates to assess the extent of T-loop phosphorylation of mNeonGreen-PLK1 WT , mNeonGreen-PLK1 E121A , and mNeonGreen-PLK1 E121A-K208R . The experimental regime is as in Fig. . The bar plot represents the arithmetic mean of three replicates, and black dots are the normalized values of signal intensity for each replicate. The error bar is the standard deviation. OE overexpressed. .

Article Snippet: Okadaic acid , Merck , Cat#04906845001.

Techniques: Standard Deviation, Cell Harvesting, Expressing, Comparison, In Vitro, Activation Assay, Phospho-proteomics