Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A) Representative images of G2 phase hTERT-RPE1 cells expressing cellular Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs or inhibitors. (B) Box and Whisker plot for FRET measurement-based quantification of cellular Plk1 activity calculated from G2 phase cells represented in (A) derived from N=3 independent experiments comprising n= 115: siControl, 124: siBora, 117: siCep192, 122: siCenexin, 132: siCep192 + siCenexin and 125: Plk1i cells. (C) Representative images of G2 phase hTERT-RPE1 cells expressing centrosomal Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs or inhibitors. (D) Box and Whisker plot for FRET measurement-based quantification of centrosomal Plk1 activity calculated from G2 phase cells represented in (C) derived from N=4 independent experiments comprising n= 183: siControl, 183: siBora, 181: siCep192, 183: siCenexin, 185: siCep192 + siCenexin and 192: Plk1i cells. Data presented as the entire range with values between first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars = 5 µm.

Article Snippet: These cells were later transfected again with either Plk1-FRET sensor c-jun substrate plasmid (Addgene Plasmid # 45203) or c-jun based Plk1 FRET sensor tagged to PACT domain at c-terminus enabling centrosomal localisation (Addgene Plasmid # Plasmid #106907) using X-tremeGENETM 9 (Merck: XTG9-RO) transfection reagent according to manufacturer’s instructions.

Techniques: Expressing, Activity Assay, Whisker Assay, Derivative Assay

Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A) Representative images of G2 phase hTERT-RPE1 cells expressing cellular Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs. (B) Box and Whisker plot for FRET measurement-based quantification of cellular Plk1 activity calculated from G2 phase cells represented in (A) derived from N=3 independent experiments comprising n= 120: siBora + siCep192, 135: siBora + siCenexin, 127: siBora + siCep192 + siCenexin, 124: siAurora and 121: siPlk1 cells. (C) Representative images of G2 phase hTERT-RPE1 cells expressing centrosomal Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs or inhibitors. (D) Box and Whisker plot for FRET measurement-based quantification of centrosomal Plk1 activity calculated from G2 phase cells represented in (C) derived from N=4 independent experiments comprising n= 181: siBora + siCep192, 187: siBora + siCenexin, 181: siBora + siCep192 + siCenexin, 182: siAurora and 182: siPlk1 cells. (E-G) Western Blot confirming the depletion of indicated proteins after indicated siRNA treatments. Data represent the entire range with values between first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars = 5 µm.

Article Snippet: These cells were later transfected again with either Plk1-FRET sensor c-jun substrate plasmid (Addgene Plasmid # 45203) or c-jun based Plk1 FRET sensor tagged to PACT domain at c-terminus enabling centrosomal localisation (Addgene Plasmid # Plasmid #106907) using X-tremeGENETM 9 (Merck: XTG9-RO) transfection reagent according to manufacturer’s instructions.

Techniques: Expressing, Activity Assay, Whisker Assay, Derivative Assay, Western Blot

Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A-H) Representative time stamp images and quantifications from FRAP movies from G2 phase Plk1-EGFP tagged hTERT-RPE1 cells treated with non-targeting siRNA control (A; quantification of recovery in (B)) , Cep192 siRNA (C quantification of recovery in (D)) , Cenexin siRNA (E quantification of recovery in (F)) and siCep192 + siCenexin (G quantification of recovery in (H)) . (I) Box and Whisker plot for measurement of mobile Plk1 pool on centrosomes under indicated conditions (N=3 independent experiments; n= 43, 44, 43 and 45 for siControl, siCep192, siCenexin and siCep192 + siCenexin, respectively.) (J) Box and Whisker plot for measurement of mobile Plk1 pool on centrosomes after Plk1 inhibition derived from N=3 independent experiments comprising n= 43, and 53 individual cells for DMSO and Plk1i, respectively. (K) Representative immunofluorescence images of hTERT-RPE1: Plk1-EGFP cells treated with indicated siRNAs and stained for Pericentrin (Red) and EGFP (Green). (L) Box and Whisker plot for measurement of Plk1 levels on centrosomes during G2 phase under the conditions indicated in (K) generated from N=5 independent experiments comprising n= 287: siControl, 279: siBora, 276: siCep192 and 287: siCenexin cells. Data presented as the entire range with values between first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars Images: 5 µm and zoomed insets: 0.5 µm.

Article Snippet: These cells were later transfected again with either Plk1-FRET sensor c-jun substrate plasmid (Addgene Plasmid # 45203) or c-jun based Plk1 FRET sensor tagged to PACT domain at c-terminus enabling centrosomal localisation (Addgene Plasmid # Plasmid #106907) using X-tremeGENETM 9 (Merck: XTG9-RO) transfection reagent according to manufacturer’s instructions.

Techniques: Control, Whisker Assay, Inhibition, Derivative Assay, Immunofluorescence, Staining, Generated

Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A-D) Representative time stamp images and quantifications from FRAP movies from G2 phase Plk1-EGFP tagged hTERT-RPE1 cells treated with DMSO (A : quantification of Plk1 recovery on centrosomes in (B)) and Plk1i ( (C : quantification of Plk1 recovery on centrosomes in (D)) . Data obtained from N=3 independent experiments consisting of n=43 and 53 DMSO and Plk1i treated cells, respectively. (E) Representative immunofluorescence images of hTERT-RPE1: Plk1-EGFP cells treated with indicated siRNAs and stained for Pericentrin (Red) and EGFP (Green). (F) Box and Whisker plot for measurement of Plk1 levels on centrosomes during G2 phase under the conditions indicated in (E) generated from N=5 independent experiments comprising n= 276: siBora + siCep192, 287: siCep192 + siCenexin, 286: Bora + siCenexin and 282: siBora + siCep192 + siCenexin cells. Data presented as the entire range with values between first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars Images: 2 µm and zoomed insets: 0.5 µm.

Article Snippet: These cells were later transfected again with either Plk1-FRET sensor c-jun substrate plasmid (Addgene Plasmid # 45203) or c-jun based Plk1 FRET sensor tagged to PACT domain at c-terminus enabling centrosomal localisation (Addgene Plasmid # Plasmid #106907) using X-tremeGENETM 9 (Merck: XTG9-RO) transfection reagent according to manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Whisker Assay, Generated

Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A-C) Flow cytometry-based quantification of cell cycle profile of hTERT-RPE1 (WT), hTERT-RPE1: Usp28 - / - and hTERT-RPE1: Usp28 - / - 0:0 cells revealing percentage of cells in G1 (A) , S (B) and G2 (C) phases derived from N=4 independent experiments with each treatment comprising around 30,000 cells. (D) Flow cytometry-based quantification of cell cycle profile or RPE1 cells treated with Control vs Cep192 siRNA showing percentage of cells in G1, S and G2 phases obtained from N=5 independent experiments with each treatment comprising of 30,000 cells. (E) Representative images of S phase hTERT-RPE1 cells expressing cellular Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs or inhibitors. (F) Box and Whisker plot for FRET measurement-based quantification of cellular Plk1 activity calculated from S phase cells represented in (E) derived from N=4 independent experiments comprising n= 203: siControl, 204: siBora, 215: siCep192, 219: siCenexin, 215: Plk1i cells. The leftmost G2 Phase – siControl bar shown is replica of siControl data shown in for side-by-side comparison purposes. G) Representative images of S phase hTERT-RPE1 cells expressing centrosomal Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs or inhibitors. (H) Box and Whisker plot for FRET measurement-based quantification of cellular Plk1 activity calculated from S phase cells represented in (G) derived from N=3 independent experiments comprising n= 163: siControl, 187: siBora, 187: siCep192, 184: siCenexin, 178: Plk1i cells. The leftmost G2 Phase – siControl bar shown is replica of siControl data shown in for side-by-side comparison purposes. Data presented as the entire range with values between first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars, Images: 5 µm and zoomed insets: 0.5 µm.

Article Snippet: These cells were later transfected again with either Plk1-FRET sensor c-jun substrate plasmid (Addgene Plasmid # 45203) or c-jun based Plk1 FRET sensor tagged to PACT domain at c-terminus enabling centrosomal localisation (Addgene Plasmid # Plasmid #106907) using X-tremeGENETM 9 (Merck: XTG9-RO) transfection reagent according to manufacturer’s instructions.

Techniques: Flow Cytometry, Derivative Assay, Control, Expressing, Activity Assay, Whisker Assay, Comparison

Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A) Schematic representation of experimental setup for investigating the role of Plk1 activators in checkpoint recovery. (B) Representative field images of hTERT-RPE1 cells treated with indicated siRNAs and stained for α-tubulin, Pericentrin and DAPI. (C) Quantification representing mitotic index after DNA damage recovery in cells under depletion different conditions shown in (B) obtained from N=4 independent experiments consisting of n=1136: siControl, 1398: siBora, 976: siCep192, 1359: siCenexin, 1157: siCep192 + siCenexin and 972: siPlk1 cells. (D) Representative field images of hTERT-RPE1 cells treated with indicated siRNAs and stained for α-tubulin, Pericentrin and DAPI. (E) Quantification representing mitotic index in cells without doxorubicin pulse under different depletion conditions shown in (D) obtained from N=3 independent experiments consisting of n=1413: siControl, 838: siBora, 827: siCep192, 1412: siCenexin, 636: siCep192 + siCenexin and 1803: siPlk1 cells. Data presented as the entire range with values between first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars: 5 µm.

Article Snippet: These cells were later transfected again with either Plk1-FRET sensor c-jun substrate plasmid (Addgene Plasmid # 45203) or c-jun based Plk1 FRET sensor tagged to PACT domain at c-terminus enabling centrosomal localisation (Addgene Plasmid # Plasmid #106907) using X-tremeGENETM 9 (Merck: XTG9-RO) transfection reagent according to manufacturer’s instructions.

Techniques: Staining

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) Engineered U2OS cell expressing mTurquoise2-NDC80 and β-tubulin-TC-FlAsH. NDC80 (gray), mTurquoise2 (blue) and TC-FlAsH (green). ( B ) Two-photon microscopy images of the engineered U2OS cells not exposed to FlAsH (top) and exposed to FlAsH (bottom). 3 μm scale bar. mTurquoise2 (blue) and FlAsH (green). ( C ) Example fluorescence decay curves of mTurquoise2-NDC80 in the engineered U2OS cells not exposed to FlAsH (left, green circle) and exposed to FlAsH (right, orange triangle), plotted with the best-fit single-exponential decay models (black and blue dotted lines). Corresponding weighted residuals (the deviation of data from model, divided by the square root of the number of photons) are plotted below after being smoothened to display systematic deviations. ( D ) The fluorescence decay curve of mTurquoise2-NDC80 in the presence of FlAsH labeling (orange triangle, same as ( C )), plotted with the best-fit two-exponential model (blue solid line). The single-exponential model fit to the fluorescence decay curve in the absence of FlAsH labeling (black dotted line) plotted together for comparison. Corresponding smoothened weighted residual (described above) for the two-exponential model is plotted below. Long- and short-lifetime exponentials correspond to the mTurquoise2-NDC80 populations in non-FRET state and FRET state, respectively, and their relative amplitudes give the fraction of each population. To facilitate the comparison, the fluorescence decay curves in the absence and presence of FlAsH labeling were normalized such that they asymptotically overlap. Data points and source FLIM data are available in Figure 1-Data .

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Expressing, Microscopy, Fluorescence, Labeling, Comparison

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) to ( D ) Schematic descriptions, example cell images, and example mTurquoise2 fluorescence decay curves from three different FRET-negative control experiments and a nocodazole treatment experiment. mTurquoise2 fluorescence decay curves (blue circles) are plotted with best-fit single- (black dotted line) or two-exponential decay model (black solid line), and the associated weighted residuals are plotted below (blue curve). 3 μm scale bar. ( A ) Negative control 1. Nuf2 N-terminally labeled with mTurquoise2, and no FlAsH labeling. ( B ) Negative control 2. Nuf2 C-terminally labeled with mTurquoise2 (far from kMT), and no FlAsH labeling. ( C ) Negative control 3. Nuf2 C-terminally labeled with mTurquoise2 (far from kMT), and β-tubulin C-terminally labeled with FlAsH. ( D ) Nocodazole treatment experiment. Nuf2 N-terminally labeled with mTurquoise2, and β-tubulin C-terminally labeled with FlAsH. Cell was incubated with 5 µM nocodazole for >10 min to depolymerize microtubules. ( E ) Boxplot of fluorescence lifetimes estimated from single-exponential models fit to the negative control fluorescence decays. n = 32, 11, and 6 cells for Neg Ctrl 1, 2, and 3, respectively.

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Fluorescence, Negative Control, Labeling, Incubation

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) Fluorescence decay curves of cells expressing mTurquoise2-TC in the absence (green circle) and the presence (orange triangle) of FlAsH. A single-exponential model (black solid line) was fit to the fluorescence decay curve in the absence of FlAsH. For easier comparison, the fluorescence decay curves were normalized such that they asymptotically overlap. ( B ) The conformational ensemble of the flexible tether between mTurquiose2 and TC were modeled by Monte Carlo protein simulations, and the distance, r , between mTurquiose2 (blue cartoon) and TC-FlAsH (green ball) was estimated. This distribution is denoted by p ( r ). ( C ) The measured fluorescence decay of mTurquoise2-TC-FlAsH (orange triangles, same as ( A ) but not normalized) plotted with the best-fit decay model (black dotted line, model described in the box and derived in Materials and methods). Associated weighted residual (deviation of model from data, divided by the square root of the number of photons) plotted below. Fitting the decay model to the data estimated the Förster radius to be 5.90 ± 0.10 nm (SE).

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Fluorescence, Expressing, Comparison, Derivative Assay

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) The conformational ensemble of the flexible tether between mTurquoise2 and Nuf2 (red) and the disordered C-terminal tails of beta-tubulins around the NDC80 (green) were modeled by large-scale Monte Carlo protein simulations, which were then used to calculate the distances, r i , between the mTurquoise2 and the TC motifs. FlAsH labeling was assigned to the TC motifs with 26.1% probability (which is the measured labeling fraction of beta-tubulin). Fluorescence lifetimes of the mTurquoise2 were calculated for randomly sampled sets of distances, r → = ( r i ) , based on which fluorescence decay curves were simulated. ( B ) (top) Fluorescence decay curves for various distances between NDC80 and MT were simulated and then were fit using single- and two-exponential decay models. Difference in Bayesian information criteria (BIC) between single- and double-exponential models is plotted against the NDC80-MT distance. Data points are mean and SD. (bottom) Example simulated fluorescence decay curves (green dots) for 0, 4, and 9 nm NDC80-MT distances are plotted with the best-fit single- (blue line) and two-exponential (red line) models. Corresponding smoothened weighted residuals plotted below. ( C ) Fluorescence decay curves for various NDC80 binding fractions ( f b ) were simulated and fit by using two-exponential decay model to estimate FRET fraction ( f FRET ). (left) Three example simulated fluorescence decay curves (green dots) for 0, 30, and 60% binding fractions with the best-fit two-exponential models (red line), and the corresponding smoothened weighted residuals plotted below. (right) NDC80 FRET fractions ( f FRET ) plotted against NDC80 binding fractions ( f b ) (blue dots), and the linear fit (black line). Gray-shaded area represents the uncertainty in the slope, which was determined from the uncertainties in the measured beta-tubulin labeling fraction and Förster radius (see Supplemental experiments in Materials and methods).

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Labeling, Fluorescence, Binding Assay

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) (left) kMTs predominantly depolymerize at leading kinetochores and polymerize at trailing kinetochores. (right) K-K distance is a proxy for centromere tension. Measuring NDC80-kMT binding along with the kinetochore movement and K-K distance therefore reveals how NDC80-kMT binding is related to the kMT dynamics and centromere tension. ( B ) Image of a metaphase cell with mTurquoise2-NDC80 (blue) and β-tubulin-TC-FlAsH (green), and kinetochore tracking (yellow circles) and pairing (red lines) results. 3 μm scale bar. ( C ) NDC80 FRET fraction vs. kinetochore speed for leading (green circle) and trailing (orange triangle) kinetochores (n = 17 cells, 681 kinetochores/data point). Data points are the mean, y-error bars the SEM, and the x-error bars the interquartile ranges within groups of kinetochores with similar velocities. ( D ) NDC80 FRET fraction vs. K-K distance for untreated cells (green circle, n = 17 cells, 984 kinetochores/data point), cells treated with 10 μM taxol (orange triangle, n = 7 cells, 525 kinetochores/data point), and cells treated with 5 μM STLC (purple square, n = 16 cells, 493 kinetochores/data point). For STLC data, only poleward-facing kinetochores are plotted (see for comparison between poleward and anti-poleward kinetochores). Data points are the mean, y-error bars the SEM, and the x-error bars the interquartile ranges within groups of kinetochores with similar K-K distances. Gray area is the 95% confidence interval for the linear fit to the combined data. ( E ) Histograms of K-K distances for the untreated (top, green), taxol-treated (middle, orange), and STLC-treated (bottom, purple) cells. 3 μm scale bar in the cell images of mTurquoise2-NDC80 (blue) and beta-tublin-TC-FlAsH (green). ***p-value (Welch’s t-test) less than 10 −30 . Data points and source FLIM data are available in Figure 3-Data .

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Binding Assay, Comparison

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) (top) Cell images showing mTurquoise2-NDC80 (blue) and beta-tubulin-TC-FlAsH (green). (bottom) Time course of NDC80 FRET fraction in response to Aurora B inhibition by 3 μM ZM447439 (n = 15 cells). ( B ) Images of cells with mTurquoise2-NDC80 (blue) and beta-tubulin-TC-FlAsH (green) after depleting endogenous Hec1 by siRNA and expressing siRNA-insensitive WT or three different phosphomimetic mutants of Hec1: 9A-, 2D-, and 9D-Hec1 (see Materials and methods). ( C ) NDC80 FRET fraction of cells whose endogenous Hec1 are replaced with WT or phosphomimetic Hec1 (see Materials and methods). Black dots are from individual cells and red error bars are mean ± SEM. n = 19, 22, 12, and 17 cells for WT-, 9A-, 2D-, and 9D-Hec1. *p<0.1; **p<0.01; ***p<0.001; ****p<0.0001. ( D ) Time course of NDC80 FRET fraction of 2D-Hec1-expressing cells in response to Aurora B inhibition by 3 μM ZM447439 (n = 12 cells). ( E ) (top) Cell images color-coded with Aurora B sensor non-FRET fraction. (bottom) Time course of the non-FRET fraction of the cytoplasmic Aurora B FRET sensor in response to 3 μM ZM447439 (n = 10 cells). In (A), (D) and (E), black squares and error bars are the weighted mean and SEM of the data points (green circles) in equally spaced time intervals of 1 min. Red solid and dashed lines are the best-fit exponential decay models and their 95% confidence intervals, respectively. 5 μm scale bar for all images. (F)NDC80 FRET fraction (from (A)) and NDC80 binding fraction (converted from the FRET fraction) plotted against the fraction of phosphorylated Aurora B phosphorylation sites in NDC80 (converted from Aurora B FRET sensor non-FRET fraction in (E)). Red solid and dashed lines are the best-fit NDC80-kMT binding model (derived in Mathematical modeling in Materials and methods) and its 95% confidence interval. Data points and source FLIM data are available in Figure 4-Data .

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Inhibition, Expressing, Binding Assay, Phospho-proteomics, Derivative Assay

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) NDC80 FRET fraction vs. K-K distance for 9A-Hec1-expressing cells with no drug treatment (green circle, n = 12 cells, 803 kinetochores/data point), with 10 μM taxol treatment (orange triangle, n = 9 cells, 1113 kinetochores/data point), or with 5 μM STLC treatment (purple square, n = 10 cells, 855 kinetochores/data point). For STLC data, only poleward-facing kinetochores are included (see for comparison between poleward-facing and anti-poleward-facing kinetochores). Data points are the mean, y-error bars the SEM, and the x-error bars the interquartile ranges within groups of kinetochores with similar K-K distances. Gray area is the 95% confidence interval for the linear fit to the combined data. ( B ) Histograms of K-K distances for the untreated 9A-Hec1 cells (top, green), untreated cells with endogenous Hec1 (top, black line), 9A-Hec1 cells treated with taxol (middle, orange), and 9A-Hec1 cells treated with STLC (bottom, purple). 3 μm scale bar in the cell images of mTurquoise2-NDC80 (blue) and beta-tubulin-TC-FlAsH (green). ***p<10 −6 (Welch’s t-test). ( C ) Haspin kinase phosphorylates histone H3 at Thr3 (H3T3), which recruits the chromosome passenger complex (CPC, red) to centromeres. 5-Iodotubercidin (5-ITu) inhibits haspin kinase, thereby displacing Aurora B from centromeres. ( D ) Spinning-disk confocal microscopy images of cells expressing mNeonGreen-Nuf2 (green) and INCENP-mCherry (red) before (top) and after (bottom) haspin inhibition by 10 μM 5-ITu treatment. 3 μm scale bar. ( E ) NDC80 FRET fraction vs. K-K distance for cells treated with 10 μM 5-ITu (green circle, n = 15 cells, 1170 kinetochores/data point), for cells treated with both 10 μM 5-ITu and 10 μM taxol (orange triangle, n = 3 cells, 359 kinetochores/data point), and for cells treated with 10 μM 5-ITu and 5 μM STLC (purple square, n = 12 cells, 564 kinetochores/data point). For 5-ITu + STLC data, only poleward-facing kinetochores are included (see for comparison between poleward-facing and anti-poleward-facing kinetochores). Data points are the mean, y-error bars the SEM, and the x-error bars the interquartile ranges within groups of kinetochores with similar K-K distances. Gray area is the 95% confidence interval for the linear fit to the combined data. ( F ) Histograms of K-K distances for the 5-ITu-treated (top, green), untreated (top, black line), 5-ITu + taxol treated (middle, orange), and 5-ITu + STLC treated cells (bottom, purple). 3 μm scale bar in the cell images of mTurquoise2-NDC80 (blue) and beta-tubulin-TC-FlAsH (green). ***p<10 −6 (Welch’s t-test). ( G ) NDC80 FRET fraction and ( H ) the non-FRET fraction of Nuf2-targeted Aurora B FRET sensor (proxy for Aurora B activity at NDC80) for different drug treatments. Each data point (gray circle) corresponds to an individual cell, and the error bar (red) shows the mean and SEM. P-values from two-sided Welch’s t-test. Data points and source FLIM data are available in Figure 5-Data .

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Expressing, Comparison, Confocal Microscopy, Inhibition, Activity Assay

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet:

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Transfection, Construct, Labeling, Retroviral, Plasmid Preparation, Selection, Marker, Modification, Recombinant, Sequencing, Synthesized, Electroporation, Software, Concentration Assay

Journal: Virology

Article Title: A robust high-throughput fluorescence polarization assay for rapid screening of SARS-CoV-2 papain-like protease inhibitors

doi: 10.1016/j.virol.2022.07.006

Figure Lengend Snippet: The inhibitory activity and mechanism of a natural product anacardic acid (AA) against SARS-CoV-2 PLpro in vitro . (A) The 4-step procedure of the FP screening assay for rapid screening of PLpro inhibitors. Step 1: 29 μL PLpro solution (1 μM) was incubated with 1 μL sample of a natural product (2 mg/mL) for 30 min at RT in a black 96-well microplate. Step 2: 20 μL FP probe solution (30 nM) was added and incubated for 30 min at RT. Step 3: The reaction was quenched by adding 10 μL avidin solution (240 nM) followed by 5 min incubation. Step 4: The mP value was measured by a microplate reader (BioTek). (B) The natural product layout in a black 96-well microplate for a large-scale screening. The positive (GRL0617), negative, and background wells were highlighted. (C) The illustration for the primary screening cycle of a natural product library using the FP screening assay. The red dotted line indicates a baseline (equivalent to 50% inhibition) in the primary screening cycle, and 5 candidate compounds were identified. (D) The chemical structure of anacardic acid. (E) Inhibitory effect of anacardic acid on the PLpro activity in the FP screening assay. (F) Inhibitory effect of anacardic acid on the PLpro activity in the FRET screening assay. GRL0617 was used as a positive control. (G) Inhibitory effect of anacardic acid on the Mpro activity in the FRET screening assay. PF-07321332 served as a positive control. (H) Inhibitory effect of anacardic acid on the PLpro isopeptidase activity. The fluorogenic peptide Z -RLRGG-AMC was used as the substrate for inhibition of PLpro isopeptidase activity by anacardic acid in vitro . GRL0617 served as a positive control. (I) The Lineweaver-Burk double-reciprocal plots for inhibition of PLpro activity by anacardic acid in vitro for the FRET substrate. (J) The secondary plots for a K i value.

Article Snippet: The FRET substrate (Dabcyl-FTLKGGAPTKVTE-Edans; λ ex /λ em = 340/500 nm), FP probe (FITC-FTLKGGAPTKVTK-Biotin; λ ex /λ em = 485/535 nm), FITC-FTLKGG peptide, and the fluorogenic peptide Z -RLRGG-AMC (λ ex /λ em = 340/460 nm) were purchased from GL Biochem (Shanghai, China), respectively.

Techniques: Activity Assay, In Vitro, Screening Assay, Incubation, Avidin-Biotin Assay, Inhibition, Positive Control