Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: cAMP increases Na+/K+-ATPase activity, protein abundance at the plasma membrane and the distance traveled by the Na+/K+-ATPase-containing vesicles in A549-GFPα1 cells. (A) A549-GFPα1 cells were incubated in the absence (CT) or presence of 50 μM forskolin (FSK) for 10 minutes and the Na+/K+-ATPase activity was measured as 86Rb+ uptake. Graph represents mean ± s.e.m. of three experiments. (B) A549-GFPα1 cells were incubated as in A, and the Na+/K+-ATPase abundance at the basolateral plasma membrane was determined by western blot of the BLM fraction using a specific antibody against GFP. E-cadherin was used as a loading control. Graph represents mean ± s.e.m. of three experiments. A representative western blot is shown. (C) The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Vesicles were randomly selected from those that showed plus-end-directed displacement. Left panel shows a representative image of A549-GFPα1 cells. Arrowhead indicates the vesicle whose trajectory is shown in the right panel before (CT) and after FSK treatment (FSK). (D) Average contour length traveled by the vesicles as a function of time. The black line represents control vesicles; at 60 seconds, upon addition of FSK (red line), the vesicles move at a faster rate. The average contour length is determined by averaging over many trajectories as described in the Materials and Methods. **P<0.01; ***P<0.001. Scale bars: 10 μm and 2 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Activity Assay, Incubation, Western Blot, Labeling, Single-particle Tracking, Software

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The three isoforms of myosin-V are expressed in A549 cells. (A) RT-PCR using mRNA obtained from A549 and HeLa cells. Primers used for the amplification are described in supplementary material Table S6. (B) Cell lysates from A549 and HeLa cells were obtained and analyzed by western blot with specific antibodies against the three myosin-V isoforms. A representative western blot is shown. (C) The particulate fraction (100,000 g pellet) of A549-GFPα1 cells was loaded onto a flotation sucrose gradient and eight fractions were recovered. The distribution of the proteins of interest was analyzed by western blotting with specific antibodies. A representative western blot is shown. Rab5 and Rab7 are used as markers of early and late endosomes, respectively. (D) Gradients obtained in C were scanned and the marker content was digitally quantified as indicated. Results are expressed as percentage of the total amount of protein.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Marker

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Myosin-Va and myosin-Vc colocalize with Na+/K+-ATPase. (A) A549-GFPα1 cells were incubated in the absence or presence of 50 μM FSK for 10 minutes, basolateral membranes (BLM) and intracellular compartments (IC) were isolated and the Na+/K+-ATPase abundance was determined by western blot using a specific antibody against GFP. E-cadherin and actin were used as loading controls for the BLM and IC fractions, respectively. Graph represents mean ± s.e.m. of three experiments. A representative western blot is shown. (B) The IC fraction of A549-GFPα1 cells was loaded onto a flotation sucrose gradient and eight fractions were recovered. The distribution of the proteins of interest was analyzed by western blotting with specific antibodies. A representative western blot is shown. C+, positive control.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Incubation, Isolation, Western Blot, Positive Control

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The average speed of Na+/K+-ATPase-containing vesicles moving towards the cell periphery is increased in cells expressing a myosin-Va stalk-tail. (A) Live imaging of A549-GFPα1 cells (green) transiently transfected with a dominant-negative myosin-Va that has a m-cherry-tag (red) (m-cherry-DN-Va). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of one vesicle before (CT) and after forskolin treatment (FSK). (B) Average contour length traveled by the vesicles in A as a function of time. The black line represents the control vesicles; FSK was added at time 60 seconds and is represented as a red line. (C) Live imaging of A549-GFPα1 cells (green) transiently transfected with a dominant-negative myosin-Vc that has a m-cherry-tag (red) (m-cherry-DN-Vc). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of one vesicle before (CT) and after forskolin treatment (FSK). (D) Average contour length traveled by the vesicles in C as a function of time. The black line represents control vesicles; FSK was added at 60 seconds and is represented as the red line. Scale bars: 10 μm and 2 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Expressing, Imaging, Transfection, Dominant Negative Mutation, Labeling

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The average speed of Na+/K+-ATPase-containing vesicles moving towards the cell periphery is increased in cells expressing a shRNA against myosin-Va. (A) Live imaging of A549-GFPα1 cells (green) transiently transfected with a shRNA against myosin-Va that has a m-cherry-tag (red) (m-cherry-sh-Va). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of two vesicles (arrowheads) under control (CT) conditions. (B) Live imaging of A549-GFPα1 cells (green) transiently transfected with a shRNA against myosin-Vc that has a m-cherry-tag (red) (m-cherry-shRNA-Vc). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of two vesicles (arrowheads) under control (CT) conditions (C). Graph represents the average contour length traveled by the vesicles as a function of time, calculated as described in methods. The black line represents the m-cherry-sh-Va vesicles and the red line, the m-cherry-sh-Vc vesicles. (D) A549-GFPα1 cells were transfected with a shRNA against myosin-Va or myosin-Vc, cell lysates were isolated and the myosin-Va (left panel) or myosin-Vc (right panel) abundance was determined by western blot using specific antibodies. E-cadherin and tubulin were used as loading controls. Scale bars: 10 μm and 4 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Expressing, shRNA, Imaging, Transfection, Labeling, Isolation, Western Blot

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Dominant-negative myosin-Va mimics cAMP-mediated Na+/K+-ATPase increased activity and recruitment to the plasma membrane in A549-GFPα1 cells. (A) Stable clones expressing myosin-Va tail (DN-Va) and myosin-Vc tail (DN-Vc) were generated as described. Expression of the constructs in the permanent clones was analyzed by western blotting using and antibody against the V5 tag. A representative western blot is shown. (B) A549-GFPα1 cells (CT) and A549-GFPα1 cells permanently transfected with DN-Va and DN-Vc were incubated in the absence or presence of 50 μM FSK for 10 minutes and the Na+/K+-ATPase activity was measured as 86Rb+ uptake. Graph represents mean ± s.e.m. of three different experiments. (C) Control (CT), DN-Va and DN-Vc cells were incubated in the absence or presence of 50 μM FSK for 10 minutes and western blots of the basolateral membrane fraction were performed using a specific antibody against GFP. E-cadherin was used as loading control. A representative western blot is shown. *P<0.05; **P<0.01; n.s., not significant; u.s., unstimulated.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Dominant Negative Mutation, Activity Assay, Clone Assay, Expressing, Generated, Construct, Western Blot, Transfection, Incubation

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Myosin-Va and the Na+/K+-ATPase-containing vesicles colocalize. A549-GFPα1 cells were fixed, permeabilized and blocked. Myosin-Va was visualized by using an anti-myosin-Va antibody and a secondary antibody labeled with Alexa Fluor 568. GFP was directly visualized. Cellular distribution of Na+/K+-ATPase-GFPα1 and myosin-Va was analyzed using a Zeiss LSM 510 laser-scanning confocal microscope and colocalization (blue) was determined using the LSM 510 Meta software.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Labeling, Microscopy, Software

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Microtubules and actin filaments are involved in Na+/K+-ATPase traffic. (A) Live imaging of A549 cells incubated with 10 μM nocodazole for 3 hours. The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Upper panels show a representative immunofluorescence of the microtubule cytoskeleton in control (left) and nocodazole (right) conditions. Lower panel shows the tracking of the movement of one vesicle in control (left) and nocodazole (right) conditions. (B) Live imaging of A549 cells incubated with 5 μM cytochalasin D (Cyto D) for 1 hour. The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Upper panels show a representative immunofluorescence of the actin cytoskeleton under control (left) and cytochalasin D (right) conditions. Lower panel shows the tracking of the movement of one vesicle in control (left) and cytochalasin D (right) conditions. (C) Average contour length traveled by the vesicles as a function of time. The blue line represents the control vesicles; the black line, cells treated with cytochalasin D and the red line, cells treated with nocodazole. Scale bars: 10 μm and 2 μm (inset images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Imaging, Incubation, Labeling, Single-particle Tracking, Software, Immunofluorescence

Journal: Cell systems

Article Title: Quantifying the Central Dogma in the p53 pathway in live single cells

doi: 10.1016/j.cels.2020.05.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: IRDye 800CW Goat anti-Mouse IgG Secondary Antibody , Licor , 926–32210.

Techniques: Recombinant, Imaging, Software, Single Cell Tracking

Journal: bioRxiv

Article Title: Apical-driven cell sorting optimised for tissue geometry ensures robust patterning

doi: 10.1101/2023.05.16.540918

Figure Lengend Snippet: A. Schematic representation and immunostaining images of blastocysts and ICMs at stages E3.5 and E4.5. GATA6 and GATA4 are used as markers for PrE fate (green), and NANOG and SOX2 are used as markers for EPI fate (magenta) at stages E3.5 and E4.5, respectively. B. Quantification of total cell numbers in the ICM from blastocysts and isolated ICMs at stage E3.5, blastocysts and isolated ICMs at stage E4.5, and isolated ICMs cultured in vitro for 24 hours from stage E3.5 to E4.5. n=33, 30, 40, 21, 31 embryos for the different groups, respectively. Independent samples t-test between E3.5 blastocysts and E3.5 ICMs, p =0.106. One-way ANOVA between E4.5 Blastocysts, E4.5 ICMs, and E3.5 ICMs+24hr, p =0.145. C. Representative time-lapse imaging of ICMs isolated from E3.5 blastocysts expressing PrE-specific H2B-GFP ( Pdgfrα H2B-GFP , green) and ubiquitous H2B-mCherry ( R26-H2B mCherry , magenta), out of total 8 datasets from 3 independent experiments. Time is indicated in hh:mm, t=00:00 corresponds to start of live-imaging at stage E3.5+3hours, following completion of immunosurgery. D. Schematic representation of single-cell tracking of EPI and PrE cells from isolated ICMs from (C). Line plots indicating radial distances of all cells from one representative ICM until E4.0 stage. Colour of the line indicates cell fate – PrE, green and EPI, magenta. Shaded regions show spatial dispersion as mean ±SD of cell position along ICM radial axis. The geometric centroid of the ICM is considered as d=0.0 and ICM outer surface is considered as d=1.0 to normalise cell position across samples. Time-series plots for cell position were smoothed using a rolling average. E. Quantification of sorting score for isolated ICMs between stage E3.5 and E4.0. Data from n=8 ICMs. F. Line plots for radial cell position versus time from tracking of PrE and EPI cell movements in isolated ICMs. Time-series plots for cell position were smoothed using a rolling average. Cell tracking data pooled from n=160 PrE cells and n=133 EPI cells from 8 ICMs. G. Schematic diagram for analysis of PrE and EPI cell movements. Cell displacement is measured along the radial axis between consecutive timepoints and classified as inward or outward movement depending on the direction of displacement. H. Polar plots indicating preferential direction of cell movements among PrE and EPI. Cell position is plotted along radial axis, time is plotted along angular axis. Measurements are binned according to initial radial cell position and time. The mean displacement of each interval is plotted, colour indicates direction of movement. Scale bars 20μm. ns, non-significant

Article Snippet: Primary antibodies against GATA6 (R&D systems, AF1700), GATA4 (R&D systems, BAF2606), SOX2 (Cell Signaling, 23064), bi-phosphorylated myosin regulatory light chain (ppMRLC) (Cell Signaling, 3674), and Laminin (Novus Biologicals, NB300-14422) were diluted at 1:200.

Techniques: Immunostaining, Isolation, Cell Culture, In Vitro, Imaging, Expressing, Single Cell Tracking, Dispersion, Cell Tracking Assay

Journal: bioRxiv

Article Title: Apical-driven cell sorting optimised for tissue geometry ensures robust patterning

doi: 10.1101/2023.05.16.540918

Figure Lengend Snippet: A. Immunofluorescence image of a 3x blastocyst at stage E3.75 showing laminin distribution around PrE cells. White dotted line, ICM-cavity interface. White arrowhead marks GATA6-expressing nucleus of a PrE cell enriched for laminin expression. B. Immunofluorescence image of a 3x blastocyst at stage E3.75 showing PKCλ+ζ distribution in PrE cells. White arrowhead marks leading edge of a PrE cell with PKCλ+ζ localisation. C. Immunofluorescence image of an E3.75 ICM showing PKCλ+ζ localisation in PrE and EPI cells. White dotted lines mark cell boundaries. Yellow line indicates the line segments from cell inner edge (towards ICM centroid) to cell outer edge (towards ICM-fluid interface) along which fluorescence intensity is measured. D. Line plots for normalised fluorescence intensity of PKCλ+ζ in individual inside cells from E3.75 isolated ICMs. Colour of the line indicates GATA6-expression level of the cell. n=260 cells from 32 ICMs. Each of the thin lines corresponds to measurement from one cell. Bold line and shaded region indicate mean±SD of aPKC intensity for GATA6-high and GATA6-low cells. E. Schematic description of polarisation index. Polarisation index is calculated as the ratio between mean aPKC intensity at 1/4 th distance from outer edge and mean aPKC intensity at 1/4 th distance from inner edge. Boxplots for comparison of the polarisation index in PrE (GATA6 high) versus EPI cells (GATA6 low). GATA6 expression level is categorised as high or low by thresholding the bimodal distribution of GATA6 fluorescence intensity. Colour of the line indicates GATA6-expression level of the cell. n=136 GATA6-high and 124 GATA6-low cells from 32 ICMs. One-way ANOVA, p =6.03e -20 . F. Scatterplot of polarisation index of cells versus radial distance of the cell from the ICM centroid. Colour of the datapoint indicates GATA6-expression level of the cell. Black dotted line, linear regression with Pearson’s R=0.079, p =0.205. n=260 cells from 32 ICMs. G. Immunofluorescence images of control and Gö6983-treated E4.0 isolated ICMs and quantification of sorting score. n=16,24 ICMs for control and Gö6983-treated ICMs respectively. Independent samples t-test, p = 8.01e -04 H. Immunofluorescence images of representative WT, Prkci +/+ ;Prkcz -/- , and Prkci +/- ;Prkcz -/- E4.5 blastocysts and quantification of number of ectopic PrE cells in E4.5 blastocysts from each group. n= 25, 17, 12 blastocysts for WT, Prkci +/+ ;Prkcz -/- , and Prkci +/- ;Prkcz -/- respectively. Mann-Whitney U test, p = 2.43e -04 , 6.36e -04 Scale bars 20μm. ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: Primary antibodies against GATA6 (R&D systems, AF1700), GATA4 (R&D systems, BAF2606), SOX2 (Cell Signaling, 23064), bi-phosphorylated myosin regulatory light chain (ppMRLC) (Cell Signaling, 3674), and Laminin (Novus Biologicals, NB300-14422) were diluted at 1:200.

Techniques: Immunofluorescence, Expressing, Fluorescence, Isolation, Comparison, Control, MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: MRC1 and LYVE1 expressing macrophages in vascular beds of GNAQ p.R183Q driven capillary malformations in Sturge Weber syndrome

doi: 10.1186/s40478-024-01757-4

Figure Lengend Snippet: EC-R183Q promote significant THP1 cell adhesion under static and laminar flow-induced condition. a Fluorescence-labeled THP1 cells were incubated with EC-WT and EC-R183Q under static conditions (N = 10). Adherent cells were quantified after 1 h. P -value was calculated by two-tailed t -test. Phase-contrast images of EC-WT (top) or EC-R183Q (bottom) incubated with THP1 cells (green) at 1 h incubation. Scale bar = 50 µm. b Schematic of live-cell imaging set up. A flow rate of 0.5 ml/min was setup using a tabletop syringe pump with a 20 ml syringe Luer-Lock tip. After 5 min of recording, a switch system was used to deliver pre-stained THP1 cells under continuous uninterrupted flow for 30 min. c Time-lapse imaging of THP1 cells (yellow) adhesion to EC-WT (top), and EC-R183Q (bottom) under laminar flow. Images are at time point = 0, 10, 20, and 30 min. Scale bar = 200 µm. N = 6 independent experiments were performed. d Quantification of THP1 cell adhesion under flow over 10, 20, and 30 min. Mann Whitney test was performed to calculate p-value at each time point. e Proteome profiler cytokine array on conditioned media from EC-WT (top) and EC-R183Q (bottom) after incubation in 2% fetal bovine serum EBM2 media for 24 h. Altered protein levels between EC-WT and EC-R183Q are boxed. Protein levels were quantified by measuring dot intensity using FIJI (right). Three independent experiments were performed. f Intercellular adhesion molecule 1 (ICAM1, grey), UEAI (red), and nuclei counterstaining for DAPI (blue) in the SWS brain sections (n = 4). Scale bar = 50 µm. g Time-lapse imaging of THP1 cell (yellow) adhesion to EC-R183Q treated with IgG2A isotype control (top), and EC-R183Q treated with anti-ICAM1 antibody (bottom) under laminar flow. Images are at time point = 0, 10, 20, and 30 min. Scale bar = 200 µm. N = 5 independent experiments were performed. h Quantification of single cell tracking of THP1 cells under flow over 10, 20, and 30 min. Mann Whitney test was performed to calculate p-value at each time point

Article Snippet: The cytokine array was performed on conditioned media using Proteome Profiler human cytokine array (Cat# ARY005B, R&D Systems) in accordance with the manufacturer protocol.

Techniques: Fluorescence, Labeling, Incubation, Two Tailed Test, Live Cell Imaging, Staining, Imaging, MANN-WHITNEY, Control, Single Cell Tracking

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A ) A flowchart of the chemical screen demonstrating the major steps. ( B ) Raw data of cell size and cell cycle stage measured from a single control well in the screen. The scatter plot represents single-cell measurements of cell size and three markers of cell cycle stage; Cdt1 (mKO2-hCdt1), Geminin (mAG-hGem) and DNA (DAPI). Every point in the plot represents measurements on one single cell. Cell size is represented by a color scheme depicted by the colorbar on the right. White arrows are added to guide the reader along the cell cycle trajectory. ( C ) Average size of early G1 cells is negatively correlated with the fraction of cells in early G1. The scatterplot displays the result from one example 384-well plate. Each point on the plot corresponds to one particular screened condition (screened compound or control), and represents the average size of early G1 cells in that condition versus the proportion of cells in G1. Red circles highlight the conditions that significantly affect the size of early G1 cells and/or the proportion of cells in G1. The arrows designate examples of on-axis and off-axis compounds (also see ). ( D ) Distribution of correlation coefficients between average size of early G1 cells and the fraction of cells in G1, calculated for all screened plates (as described in Materials and methods - Analysis of the compound screen), demonstrating that the two variables are significantly negatively correlated (p<10 −16 ). ( E, F ) Ranked p-values from the target enrichment analysis of on-axis and off-axis compounds, respectively (Fisher’s exact test). Components of the mTOR pathway and p38 MAPK pathway, which are highlighted, are among the top-ranked hits of on-axis and off-axis phenotypes, respectively. The Matlab script used to perform the target enrichment analysis is presented in . 10.7554/eLife.26947.009 Figure 1—source data 1. The screen metadata used to identify on-axis and off-axis outliers. 10.7554/eLife.26947.010 Figure 1—source data 2. The analysis script to visualize on-axis and off-axis outliers using . 10.7554/eLife.26947.011 Figure 1—source code 1. The Matlab script used to perform the target enrichment analysis.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Control

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Components from the p38 pathway (highlighted) were highly enriched. Specifically, MK2/MAPKAPK2, a direct downstream substrate of p38 is the top-ranking genes that associate with increased cell size variability.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques:

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: To estimate the cell size variability that results from inhibition of a specific protein, z-scores were averaged from all screen compounds targeting that protein. An average cell size variability was calculated for each of the target proteins and ranked from small to large. Components of the p38 MAPK pathway (highlighted in red) are ranked among the top proteins with increased z-scores in cell size variability.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Inhibition

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A ) Quantifying the coordination of cell size and G1 length. Samples of unsynchronized cells were treated with increasing concentrations of rapamycin (a rapamycin concentration series: 0, 0.03, 0.3, 3 and 30 nM) for a period of 24 hr, and then stained and imaged to quantify cell size and cell cycle stage on a single-cell basis. Each data point (circle) corresponds to a different concentration of rapamycin and shows the average size of early G1 cells and the proportion of cells in G1 resulting from that treatment. Populations treated with higher concentrations of rapamycin had smaller cells and higher fractions of cells in G1, resulting in a robust negative correlation. Rapamycin concentrations are redundantly represented by both the size of the circles and their color, as shown in the colorbar. The small white circles represent control populations that were treated with DMSO, rather than rapamycin. Calculation of the average size and the proportion of G1 cells, in each of the represented samples, was performed by classifying single cells into cell cycle stage as depicted in . Each data point was measured from an unsynchronized population with a minimum of 7000 cells. Additional details on the experiment and analysis is provided in the Materials and methods section. ( B ) The experiment described in panel A is repeated with (red) or without (blue) a chemical inhibitor of p38 (SB203580, 5 μM). The negative correlation between the size of early G1 cells and the proportion of cells in G1 is apparent in populations not treated with SB203580 (blue) but not in the populations that are treated with SB203580. The blue and red trend lines represent linear regressions. ( C ) Western-blots of whole cell lysates from populations that were treated with different combinations of SB203580, rapamycin and Torin-2. The experimental procedure used here are the same as those used to generate the data shown in panel A and B. The increased levels of phopho-p38 in the population that is treated with SB203580 (a p38 inhibitor) should not be interpreted as a lack of efficacy of SB203580. Rather, these higher levels of phopho-p38 are explained by a negative feedback in the p38 pathway , and the fact that while p38 inhibitors prevent p-p38 from phosphorylating its downstream substrates, these inhibitors do not block phosphorylation of p38 itself by upstream regulators . ( D ) Inhibition of the p38 MAPK pathway, but not the MAPK/ERK or SAPK/JNK pathways, disrupts the correlation between the average size of early G1 cells and the proportion of cells in G1. Results were obtained with the same assay used to create panel A and B. Larger circle size indicates higher rapamycin concentration. The rapamycin concentration series includes: 0, 0.03, 0.1, 0.3, 3 and 30 nM. The results shown here are representative of three independent experiments. ( E ) Fitted slopes corresponding to the trends shown in . Error bars represent 90% confidence intervals. For each compound treatment, its fitted slope is compared with the slope of the control (DMSO) from the same experiment. Significance was calculated with one-tailed Student’s t -test (H 0 : slope drug <= slope control ). The meta data and source code used for this analysis and visualization of results is presented in . 10.7554/eLife.26947.017 Figure 2—source data 1. Measurements of cell size and cell cycle stages from the chemical inhibitor experiments as shown in , and .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Concentration Assay, Staining, Control, Western Blot, Blocking Assay, Phospho-proteomics, Inhibition, One-tailed Test

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A–C ) Scatterplots displaying relationship between average growth rate in G1 stage with G1 duration for individual cells in DMSO control, p38 inhibition and mTOR inhibition. ( D–F ) Scatterplots displaying relationship between average growth rate over cell cycle with cell cycle duration for individual cells in the three conditions. r indicates Spearman’s correlation coefficient. ( G–I ) Distribution of average growth rate in G1 (before Geminin rise), S/G2 (after Geminin rise) and over the entire cell cycle for the three tested conditions. While mTORC1 inhibition by Rapamycin decreases growth rate, p38 inhibition by SB203580 does not significantly affect growth rate in G1. The meta data and source code used in this analysis is presented in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Control, Inhibition

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A ) Live cells subject to p38 inhibition (SB203580) or to mTORC1 inhibition (rapamycin) were followed with time-lapse microscopy to monitor proliferation over a period of 50 hr. mTOR inhibition significantly slowed rates of proliferation, while p38 inhibition increased rates of proliferation. ( B ) As an alternative method to assay cell cycle lengths, populations of cells were treated with p38 inhibitors and samples were fixed every 20 hr over a period of 3 days (see Materials and methods -Estimation of cell proliferation durations and growth rate from bulk measurements). Proportion of cells in the different cell cycle stages, in each of the collected samples, were calculated based on the cell cycle indicators depicted in . Consistently, p38 inhibitors accelerate proliferation by shortening the duration of G1 but not the durations of S or G2 (also see ). The meta data and source code used for this cell cycle analysis is presented in . ( C–F ) Live cells were imaged by time-lapse microscopy for a period of 50 hr to obtain growth trajectories of single cells over the course of their entire cell cycle. Computer generated image processing and cell tracking were performed, as described in Materials and methods - Automated lineage tracking and analysis, to obtain single cell growth curves. Nuclear size was used as a proxy of cell size, as has been validated in . Cells that were successfully tracked throughout their entire cell cycle were collected to calculate the cell cycle durations and cell size dynamics. ( G–I ) Scatterplots displaying relationship between nuclear size at birth and G1 duration for individual cells that are subject to chemical inhibition of p38 ( H ), chemical inhibition of mTOR ( I ), and a control population treated with DMSO ( G ). Every single point corresponds to the birth size and G1 length of a single live cell that was followed by time-lapse microscopy. Also shown are means and errorbars (SEM) of average G1 length calculated for different cell size bins. The dashed line shows the result of linear regression with the binned data. ( J ) Slopes obtained by the linear regression shown in ( G–I ) Error bars indicate 95% confidence bounds. The results shown here are representative of two independent experiments. The single-cell tracking data from the live-cell imaging experiments and the source code for analysis and visualization of the results is presented in . 10.7554/eLife.26947.022 Figure 3—source data 1. Estimation of cell cycle duration and growth rate from bulk measurements of fixed cell populations. 10.7554/eLife.26947.023 Figure 3—source data 2. Measurements of single-cell dynamics of cell size captured by live-cell imaging.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Inhibition, Time-lapse Microscopy, Cell Cycle Assay, Generated, Cell Tracking Assay, Control, Single Cell Tracking, Live Cell Imaging

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Cells were treated with indicated inhibitors for 24 hr before collecting lysates. Anisomycin was added to select wells 1 hr prior to making lysates, to activate MAPK pathways. All inhibitors were used at the ‘high dose’ indicated in and . ( A ) Cells treated with p38 inhibitors display a lower level of p-HSP27 (downstream of p38). The p38 inhibitors induce a higher level of p-p38. This is due to negative feedback in the p-p38 pathway, and the fact that p38 inhibitors prevent p-p38 from phosphorylating downstream substrates, but do not block phosphorylation of p38 itself by upstream regulators. ( B, C ) Cells treated with JNK or MEK I/II inhibitor inactivate the corresponding pathway under Anisomycin induction. The influence of the inhibitor is not obvious under control condition probably due to low basal activation of the pathways.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Blocking Assay, Phospho-proteomics, Control, Activation Assay

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Measurements collected in the same experiment as . ( A ) Scatterplot comparing cells of negative control (DMSO) with cells under p38 inhibition (treated with indicated inhibitor and concentration). Each data point was measured from a cell population with a minimum of 7000 cells. The rapamycin concentration range is as follows: 0, 0.03, 0.1, 0.3, 3 and 30 nM. The results are representative of three independent experiments. ( B ) The slope between size and proportion of cells in G1 is either disturbed or weakened. p-Values were calculated with one-tailed Student t -test (H 0 : slope of control >= slope of compound treatment). The meta data and source code used for the analysis and visualization of the results is presented in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Negative Control, Inhibition, Concentration Assay, One-tailed Test, Control

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: The p38 inhibitors and three higher concentrations shown here are also included in and . ( A ) Cells treated with only rapamycin concentration series (blue) display negative correlation between cell size and proportion of cells in G1. However, the negative correlation between cell size and proportion of cells in G1 disappears or weakens when cells are co-treated with p38 inhibitors and the rapamycin concentration series (red). The blue and red lines show the result of linear regression. The rapamycin concentration range is as follows: 0, 0.03, 0.1, 0.3, 3 and 30 nM. ( B ) The fitted slope of measurements shown in ( A ) For each compound treatment, its fitted slope is compared with the slope of the control (DMSO) from the same experiment. Significance was calculated with one-tailed Student t -test (H 0 : slope drug <= slope control ). The meta data and source code used for the analysis and visualization of the results is presented in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Concentration Assay, Control, One-tailed Test

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A–C ) Scatterplots displaying relationship between nuclear size at birth with cell cycle duration for individual cells in DMSO control, p38 inhibition and mTOR inhibition. The points with error bar shows mean and SEM by binning cells with similar size. The dashed line shows the result of linear regression with the binned data. Measurements were obtained from time-lapse experiments as indicated in . (D) Bar plot comparing the slopes of the linear regression between size and G1 duration as shown in panel A–C. Error bar indicates 95% confidence bounds. The meta data and source code used to in this analysis is presented in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Control, Inhibition

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Cells were transfected with siRNA as indicated and subsequently assayed with a rapamycin concentration series (0, 0.03, 0.1, 0.3, 3 and 30 nM) as described in to assay the correlation of size and G1 length. Larger circle size indicates higher concentrations of rapamycin. ( A ) Knocking down p38α/βpartially weakens the negative correlation between cell size and proportion of cells in G1, while knockdown of p38γ/δ drastically disturbs the correlation. ( C ) The negative correlation between cell size and proportion of cells in G1 is disturbed when cells are transfected with siRNA against MKK3/4/6 but not MKK7. Each data point in is measured on an unsynchronized population with a minimum of 3000 cells. The results shown in are representative of two and three independent experiments with duplicates or triplicates. ( B, D ) Fitted slopes of the trends shown in . Error bars indicate 90% confidence intervals. Analysis is performed with the same method as indicated in . The meta data and source code to analyze and visualize the genetic knock down results is presented in . 10.7554/eLife.26947.027 Figure 4—source data 1. Binding activity (Kd’s in nM) of the p38 inhibitors used in the study against each of the p38 isoforms. Kd values in the table were extracted from ). As marked in that paper, blank fields indicate combinations that were tested, but for which binding was weak (Kd >10 μM), or not detected in a 10 μM primary screen. 10.7554/eLife.26947.028 Figure 4—source data 2. Measurements of cell size and cell cycle stage from the knockdown experiments as shown in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Transfection, Concentration Assay, Knockdown, Binding Assay, Activity Assay

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Western-blot of cell lysates from conditions shown in confirms efficiency of knockdown of MKKs ( A ) or p38 isoforms ( B ).

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Western Blot, Knockdown

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: The bar plot and error bar display mean and SEM across three replicate Western-blot experiments. Treatment of rapamycin or Torin-2 increases both p-p38 and p-CREB, confirming that activity in the p38 pathway is upregulated under mTORC1 inhibition. SB203580, a p38 inhibitor, significantly reduces the phosphorylation of CREB (downstream of p38), confirming that p38 activity is inhibited. p27 is a negative regulator of G1 progression. mTOR inhibition by rapamycin or Torin-2 upregulates p27 activity, which promotes a longer G1. Strikingly, cells co-treated with p38 inhibitor and mTOR inhibitor have lower p27 compared to treatment of p38 inhibitor alone. This may be the mechanism by which p38 inhibition disturbs the cells’ ability to compensate their small size with longer G1.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Western Blot, Activity Assay, Inhibition, Phospho-proteomics

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A ) Cells were treated with either 50 nM of Torin-2 or DMSO (control) for 20 hr, followed by drug wash-out and media replacement. Cells undergoing mTOR inhibition, on average, decrease in size and slow their proliferation rate. Following release from mTOR inhibition, cells grow but maintain a low proliferation rate until their normal size is reached. Cells resume a wild type rate of proliferation only when their size reaches the size of the untreated population. ( B ) Western blots of whole cell lysates collected at time points ranging from 0 to 20 hr post release from mTOR inhibition. Levels of mTOR pathway activity recover within 1 hr after Torin-2 wash-out. By contrast, activity of p38 remains upregulated in the Torin-treated cells compared with controls, and gradually fades away only as cells recover their wild-type size. ( C ) Cells simultaneously expressing reporters of both p38 MAPK and JNK were treated with a series of rapamycin concentrations, as in . Each data point (circle) corresponds to the average G1 cell size and the average level of MAPK activity (JNK and p38) that corresponds to a given concentration of rapamycin. As positive controls, we include populations that were co-treated with the p38 inhibitor, SB203580 (orange circles). Higher concentrations of rapamycin (bigger circle size) result in smaller cells with higher activity of p38 (top left panel). Unlike p38, activity of JNK was not upregulated in proportion to cell size (left bottom panel). Also shown are the correlations of MAPK activity (JNK and p38) and cell size at 6 hr post release from mTOR inhibition (right panels) (also see ). Each data point represents average values of cell size and MAPK activity of the G1 cells subpopulation from an unsynchronized population with a minimum of 3000 cells. Results shown here are representative of three independent experiments. The meta data and source code used to analyze and visualize the correlation between cell size and KTR readout is presented in . ( D ) Western-blots of whole cell lysates from samples collected at 0 or 6 hr post release from a 22 hr treatment with either 50 nM Torin-2, 1 μM cycloheximide or DMSO (control). ( E ) Western-blots of whole cell lysates from samples collected at 0 or 6 hr post release from a 30-min treatment with either 25 ng/mL anisomycin, hyperosmotic shocks (NaCl and Sucrose) or DMSO (control). 10.7554/eLife.26947.035 Figure 5—source data 1. Measurements of cell size and p38 KTR as shown in and .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Control, Inhibition, Western Blot, Activity Assay, Expressing, Concentration Assay

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: The p38 KTR functions by translocating to the cytoplasm once p38 is activated. Cells were imaged after a 1 day treatment with DMSO (control), 3 nM rapamycin or 5 μM SB203580 (p38 inhibitor), or a 30 min treatment with 25 ng/mL Anisomycin (a stimulator of the p38 pathway). Anisomycin treatment results in reduced fluorescence in the nucleus, and a less defined nuclear boundary as compared to control. By contrast, treatment with SB203580 results in elevated fluorescence in the nucleus, implying a lower p38 activity. Cells subject to rapamycin treatment are smaller in size as compared to control. While these cells display a spectrum of cytoplasmic-to-nuclear localization, rapamycin treatment increases the fraction of cells that display p38 activity, as indicated by the blurry nuclear boundary and increased cytoplasmic localization of the KTR (highlighted by arrows).

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Control, Fluorescence, Activity Assay

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Cells treated with either DMSO (control), 30 nM rapamycin or 1 μM cycloheximide for 1 day, or with 25 ng/mL Anisomycin (a stimulator of the p38 pathway) for 30 min were fixed and imaged. While Anisomycin treatment leads to hyperactivation of the p38 pathway, rapamycin or cycloheximide treatment result in a weak but visible elevation in p-p38 level in the nucleus (also see ).

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Control

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: Measurements were obtained from the same experiment as indicated in . Cells express dual reporters of both p38 MAPK and JNK were treated with a concentration series of rapamycin, decreasing cell size to varying extents. For each condition/replicate, cells were partitioned into G1, S and G2 stage according to the cellular DNA readout (see Materials and methods – Cell cycle stages). The activity of p38 MAPK negatively correlates with cell size after mTOR inhibition for cells that are in G1. Cells that are in S and G2 also display a negative correlation, but with a lower correlation coefficient. Interestingly, after cells are released from mTOR inhibition, p38 activity negatively correlates with cell size only among cells that are in G1, but not S or G2. This result supports the hypothesis that cell-size-dependent regulation of p38 activity is exclusive to the G1 phase of cell cycle, which is consistent with its assumed role in regulating G1 duration. The meta data and source code used to analyze and visualize the correlation between cell size and KTR readout among different cell cycle stages is presented in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Concentration Assay, Activity Assay, Inhibition

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A ) Workflow of the experiment. Cells were treated with 50 nM Torin-2 with or without the indicated MAPK inhibitors for 22 hr, and then released from Torin-2 while still being subject to the indicated MAPK inhibitors (red) or DMSO (blue). MAPK inhibitors were administrated at a concentration consistent with the highest corresponding concentration used in and . At 0, 6, 24, 30 and 48 hr post release from Torin-2 treatment, samples were measured for both average cell size (B) and cell count (C). ( B ) Cells treated with DMSO (control) recovered in size within 24 hr and remained at a constant average size thereafter. Cells treated with p38 inhibitors, but not ERK or JNK inhibitors, failed to recover their size, even 48 hr post Torin-2 wash-out. This suggests that p38 inhibitors suppressed the recovery in cell size rather than slowing the kinetics associated with this process. ( C ) Cells treated with inhibitors of p38, but not inhibitors of ERK or JNK, show increased rates of proliferation as compared to control conditions, after being released from mTOR inhibition. Results shown in this figure are representative of two replicate experiments. The measurements and source code for visualization of the results is presented in . 10.7554/eLife.26947.038 Figure 6—source data 1. Cell size dynamics after released from mTOR inhibition.

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Concentration Assay, Cell Counting, Control, Inhibition

Journal: eLife

Article Title: Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length

doi: 10.7554/eLife.26947

Figure Lengend Snippet: ( A ) Workflow of the experiment. Similarly as indicated in , Cells were co-treated with both Torin-2 (50 nM) with or without the indicated MAPK inhibitor for 22 hr. The cells were then released from both inhibitors and grown in regular medium. At 0, 6, 24, 30, and 48 hr post release from the inhibitors, the cells were measured for both cell size ( B ) and cell number ( C ) by Coulter counter. ( B ) Cells in ‘DMSO’ condition recovered in size within 24 hr and stays at this size. Cells with a history of p38 inhibition, but not Erk or JNK inhibition, displayed a delayed size-recovery dynamics. ( C ) Cell proliferation were followed after release from the inhibitors. The results shown in this figure are representative of two replicate experiments. The measurements and source code used to visualize the results shown in this figure is presented in .

Article Snippet: Lentiviral expression vectors encoding the JNK and p38 MAPK Kinase Translocation Reporters (KTR) were a kind gift from Markus Covert (Addgene plasmids No. 59151 and 59155).

Techniques: Inhibition

Journal: medRxiv

Article Title: Insights into KIF11 pathogenesis in Microcephaly-Lymphedema-Chorioretinopathy syndrome: a lymphatic perspective

doi: 10.1101/2023.11.02.23297090

Figure Lengend Snippet: (a) 5 x 10 4 serum starved cells were plated per fibronectin-coated transwell chamber and given 10 hours to migrate in the presence of increasing concentrations of Ispinesib (0 to 200 nM) before fixation and staining of the nuclei with DAPI. Resulting membranes (two examples given on the left) were scored for the movement of nuclei through the transwell membrane, and results represented in a plot (on the right). Error bars represent SD, n = 3 biological replicates (with 2 technical replicates per biological replicate) (b) Single cell tracking migration assay follows wound closure ability over 15 hours in DMSO (Ctrl) and 50nM Ispinesib treated cells. (c) Number of cells in experimental wound-window area for Ctrl and 50nM Ispinesib treated cells over time (frames). (d) Distance and direction of a subset of cells illustrated as star plots of cell tracks for Ctrl and 50nM Ispinesib treated cells. ( b-d ) One representative experiment is shown from n=2. (e) 3D spheroid sprouting assay. Sprouting was stimulated in LECs by VEGFC 150 ng/ml either in the presence of DMSO vehicle or several doses of Ispinesib (25,50,100nM). Average sprout length in µm (28 spheroids analysed) and total number of sprouts per spheroid (15 spheroids analysed) were quantified in each condition. One representative image is shown per condition from n=2 biological repeat. ns=not significant, *p<0.05, **p<0.01, ***p<0.001. Two-tailed unpaired Student’s t-test. Scale bar 100 µm.

Article Snippet: Ispinesib (Tocris), a specific EG5 antagonist, was used at various concentrations and exposure times depending on the nature of the experiment.

Techniques: Staining, Membrane, Single Cell Tracking, Migration, Two Tailed Test

Journal: medRxiv

Article Title: Insights into KIF11 pathogenesis in Microcephaly-Lymphedema-Chorioretinopathy syndrome: a lymphatic perspective

doi: 10.1101/2023.11.02.23297090

Figure Lengend Snippet: (a) Human Phospho-Kinase Array showing changes in phosphorylation levels of 43 kinases. Dots have been magnified for selected kinases with decreased (in blue) or increased (in red) phosphorylation. (b) Network pathway analysis using STRING and Cytoscape reveals known interactions and the directionallity of the phosphorylation change (blue, decreased and red, increased phosphorylation in siRNA KIF11 treated cells). (c) Effect of Ispinesib treatment (50nM) on AKT and MAPK phosphorylation in LECs over time after stimulation with VEGFC (100ng/mL). DMSO (vehicle) treated cells as control. Phosphorylation was analysed with anti-pAKT S473 and anti-p44/42 MAPK Thr202/Tyr204 (upper panels) and levels of AKT and MAPK protein expression with anti-AKT and MAPK antibodies (lower panels). Molecular mass markers (in kDa) is indicated on the left. Blots are quantified (on the right). (d) VEGFR3 , PROX1 , Podoplanin (PDPN), FOXC2 , LYVE1, ZO1, CLDN5 and CDH5 gene expression related to GAPDH in LECs treated with siRNA KIF11 for 48 and 72 hours analysed by qPCR. (e) Western blot analysis and quantification of PROX1 and VEGFR3 expression in LECs treated with siRNA KIF11 for 48 and 72 hours. GAPDH was used as control. ( c-e ) Bars represent mean relative expression ± SEM. ( c and e ) One representative image is shown from n=3-4 experiments. ns=not significant, *P<0.05, **P<0.01, ***P<0.001. Two-tailed unpaired Student’s t-test.

Article Snippet: Ispinesib (Tocris), a specific EG5 antagonist, was used at various concentrations and exposure times depending on the nature of the experiment.

Techniques: Control, Expressing, Western Blot, Two Tailed Test

Journal: Molecular Biology of the Cell

Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway

doi: 10.1091/mbc.E14-08-1332

Figure Lengend Snippet: Kif26b is expressed in ECs and interacts with the Dvl3/Daam1 complex. (A) Quantitative real-time PCR of Kif26b transcripts in HUVECs, HMVECs, HeLa, immortalized HMVECs, and HEK293T (HEK) cells. (B) Endogenous Kif26b appears to colocalize with MTs in ECs. HUVECs immunostained with the antibodies against Kif26b (green) and α-tubulin (red). Bottom, high-magnification images of MTs. Scale bar, 15 μm (top), 5 μm (bottom). (C) Confocal analysis of HUVECs transfected with plasmids coding for Dvl3 with a Myc tag and immunostained with anti-Kif26b (green), anti-Daam1 (red), or anti-Myc (blue) antibodies. Bottom, high-magnification images. Scale bar, 10 μm (top), 3 μm (bottom). (D) Extracts from HeLa cells transfected with each indicated vector were immunoprecipitated with antibody against V5 (for V5-tagged N-MD mutant), Kif26b, or nonspecific immunoglobulin. Immunoprecipitates (IPs) and lysates were then immunoblotted with anti-Myc (for Myc-tagged Kif26b), anti-Kif26b, anti-V5, anti-Daam1, and anti-Dvl3 antibodies. (E) HeLa cells cotransfected with Kif26b-Myc and Dvl3 were treated with either si Control or si Daam1. Lysates were then immunoprecipitated with anti-Kif26b. IPs and lysates were immunoblotted with anti-Myc, anti-Daam1, and anti Dvl3 antibodies.

Article Snippet: The oligonucleotides used were designed by Origene for h-Kif26b (SR310475A, 5′-CCGCUACAAUGCA­GACAAGCCUU-3′; SR307944B, 5′-UGUUUGUACAUAAGAA­CU­GCUAG-3′) and h-Daam1 (SR307929A, 5′-GGAAUUCAACCUGU­AAUAGAUAA-3′, and SR307929B, 5′-AGCUGCGAAA­GUAAACA­UGACUG-3′).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Immunoprecipitation, Mutagenesis

Journal: Molecular Biology of the Cell

Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway

doi: 10.1091/mbc.E14-08-1332

Figure Lengend Snippet: Kif26b controls EC sprouting initiation and elongation in a 3D sprouting assay. (A, B) Kif26b depletion in HUVECs impairs nucleus cell polarization when cells initiate sprouting. (A) Analysis of cell polarization by measuring the angle between the long axis of the nucleus and the tangent to the bead surface. The angle of an idealized polarized cell is close to 90°, whereas the angle of an unpolarized cell is not. (B) Depletion of Kif26b (si Kif26b) significantly decreased this angle compared with control (si Control) conditions. Bars denote minimum to maximum; 20 cells from three experiments. *** p < 0.001 by Student’s t test. (C–F) Depletion of Daam1 (si Daam1) and Kif26b (si Kif26b) impaired HUVEC sprouting, extension, and orientation parameters, which were recovered under Kif26b lentiviral transduction. (C) Control or Kif26b lentivirus–transduced HUVECs were transfected with the indicated siRNA and then replated on beads. After 96 h, cells were fixed and labeled with anti-CD31 antibody (green) and Hoechst nuclear marker (blue). Each representative image is a Z -stack projection of sprouts. Scale bar, 50 μm. (D) Quantification of sprouting surface area and number of sprouts per bead. Bars denote mean ± SEM; ≥20 beads from four experiments. Statistical comparison between groups was performed by a one-way ANOVA followed by Tukey’s test ( p < 0.05) to detect differences between all groups. * p < 0.05, ** p < 0.01, and *** p < 0.001 by one-way ANOVA. (E) Schematic representation of sprout orientation analysis by measuring the angle between the longitudinal axis of a sprout and the tangent to the bead surface. (F) Distribution of sprouting angles in rose plots. Bars and values denote mean ± circular SD; ≥80 sprouts from four experiments.

Article Snippet: The oligonucleotides used were designed by Origene for h-Kif26b (SR310475A, 5′-CCGCUACAAUGCA­GACAAGCCUU-3′; SR307944B, 5′-UGUUUGUACAUAAGAA­CU­GCUAG-3′) and h-Daam1 (SR307929A, 5′-GGAAUUCAACCUGU­AAUAGAUAA-3′, and SR307929B, 5′-AGCUGCGAAA­GUAAACA­UGACUG-3′).

Techniques: Transduction, Transfection, Labeling, Marker

Journal: Molecular Biology of the Cell

Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway

doi: 10.1091/mbc.E14-08-1332

Figure Lengend Snippet: Kif26b impaired EC directional migration toward Wnt3a and increased velocity of cell migration. (A) Wnt3a-conditioned medium induced persistent directional migration of HUVECs. Subjected to a stable Wnt3a gradient (right) or no gradient (left), cell paths were recorded by time-lapse videomicroscopy every 20 min over 18 h, and individual cell tracks were transposed into a trajectory plot (60 cells/experiment). Quantification of mean cell velocity, index of directness, and forward migration index was performed for 60 cells/experiment (bottom). Bars denote mean ± SEM; three experiments. * p < 0.05; ns, not significant; Student’s t test. (B) Kif26b depletion impaired cell directional migration and increased cell velocity, whereas Daam1 depletion impaired all cell migration parameters. For si Control, si Kif26b, and si Daam1 conditions, single-cell tracking was used to map the migratory pathway as before to quantify cell velocity, directness index, and forward migration index for 60 cells/experiment (bottom). Bars denote mean ± SEM; three experiments. Statistical comparison between groups was performed using one-way ANOVA followed by Tukey’s test ( p < 0.05) to detect differences between all groups. * p < 0.05 and ** p < 0.01 by one-way ANOVA.

Article Snippet: The oligonucleotides used were designed by Origene for h-Kif26b (SR310475A, 5′-CCGCUACAAUGCA­GACAAGCCUU-3′; SR307944B, 5′-UGUUUGUACAUAAGAA­CU­GCUAG-3′) and h-Daam1 (SR307929A, 5′-GGAAUUCAACCUGU­AAUAGAUAA-3′, and SR307929B, 5′-AGCUGCGAAA­GUAAACA­UGACUG-3′).

Techniques: Migration, Single Cell Tracking

Journal: Molecular Biology of the Cell

Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway

doi: 10.1091/mbc.E14-08-1332

Figure Lengend Snippet: Kif26b depletion impaired MTOC/Golgi and myosin IIB cell asymmetrical distribution in migrating ECs. (A) si Control, si Kif26b, or si Daam1 HUVECs were fixed and stained 6 h after wounding of a confluent monolayer in the presence of Wnt3a to induce cell migration. Nucleus, MTOC, and Golgi positions were examined by 4′,6-diamidino-2-phenylindole (DAPI; blue), pericentrin (green), and Golgi-97 (red) immunofluorescence. Scale bars, 50 μm. (B) Angles between the nucleus center/MTOC/Golgi axis and wound axis were measured to evaluate cell polarization. (C) Distribution of cell orientation angles in both lenti-GFP control and lenti-kif26b conditions in a rose plot. Bars and values denote mean ± circular SD; 200 cells from three experiments. (D–F) Knockdown of Kif26b (si Kif26b) and Daam1 (si Daam1) impaired myosin IIB cell rear enrichment in migrating cells, whereas Kif26b lentiviral transduction restored myosin IIB accumulation. (D) Cells were stained with anti–myosin IIB (red) and DAPI (blue) and imaged. Scale bars, 50 μm. (E, F) Asymmetrical rear enrichment of myosin IIB was assessed by analyzing the intensity values along the line drawn across the nucleus long axis and cell extension processes perpendicular to the wound line. Intensity profile curve per cell was then plotted, area under curve in the rear ( A R ) and front ( A F ) of the nucleus was calculated, and a rear enrichment index was determined ( i = A R / A F ). Bars denote mean ± SEM; ≥30 cells from three experiments. Statistical comparison between groups was performed using one-way ANOVA followed by Tukey’s test ( p < 0.05) to detect differences between all groups. *** p < 0.001 by one-way ANOVA. ns, not significant.

Article Snippet: The oligonucleotides used were designed by Origene for h-Kif26b (SR310475A, 5′-CCGCUACAAUGCA­GACAAGCCUU-3′; SR307944B, 5′-UGUUUGUACAUAAGAA­CU­GCUAG-3′) and h-Daam1 (SR307929A, 5′-GGAAUUCAACCUGU­AAUAGAUAA-3′, and SR307929B, 5′-AGCUGCGAAA­GUAAACA­UGACUG-3′).

Techniques: Staining, Migration, Immunofluorescence, Transduction

Journal: Molecular Biology of the Cell

Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway

doi: 10.1091/mbc.E14-08-1332

Figure Lengend Snippet: Kif26b induced a cell polarity axis through MT stabilization. (A–C) Kif26b and Daam1 knockdown decreased the ratio of cell extension length vs. control conditions. Restored Kif26b rescued the formation of polarized cell extension. (A) Si Control, si Kif26b, and si Daam1 HUVECs were subjected to wound-induced migration under Wnt3a stimulation. Cells were stained with anti–acetylated tubulin (gray) and DAPI (blue). Scale bars, 50 μm. White lines indicate wound edges. (B, C) Ratio of total cell length ( L TOTAL ) to extension length ( L EXT ), to analyze cell polarization toward the wound. Bars denote mean ± SEM; ≥30 cells from three experiments. Statistical comparison between groups was performed using one-way ANOVA followed by Tukey’s test ( p < 0.05) to detect differences between all groups. *** p < 0.001 by one-way ANOVA. (D, E) Kif26b overexpression induced MT stabilization. (D) Control (GFP) or Kif26b-GFP lentivirus–transduced HUVECs were treated with either dimethyl sulfoxide or nocodazole (500 nM) for 5, 15, or 30 min; lysates were subjected to SDS–PAGE and Western blotting with anti–acetylated tubulin and anti–α tubulin antibodies (left). Quantification of fold change was calculated in relation to acetylated tubulin level in GFP-transduced HUVEC basal condition (right). Error bars denote mean ± SEM; three experiments. * p < 0.05, ** p < 0.01 by one-way ANOVA followed by Tukey’s test. ns, not significant. (E) Nocodazole-treated cells were stained with GFP (green) or acetylated tubulin (red) and DAPI (blue) and imaged. Scale bars, 10 μm.

Article Snippet: The oligonucleotides used were designed by Origene for h-Kif26b (SR310475A, 5′-CCGCUACAAUGCA­GACAAGCCUU-3′; SR307944B, 5′-UGUUUGUACAUAAGAA­CU­GCUAG-3′) and h-Daam1 (SR307929A, 5′-GGAAUUCAACCUGU­AAUAGAUAA-3′, and SR307929B, 5′-AGCUGCGAAA­GUAAACA­UGACUG-3′).

Techniques: Migration, Staining, Over Expression, SDS Page, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Kif26b controls endothelial cell polarity through the Dishevelled/Daam1-dependent planar cell polarity–signaling pathway

doi: 10.1091/mbc.E14-08-1332

Figure Lengend Snippet: Kif26b in synergy with Daam1 regulated the balance between the noncanonical PCP and canonical signaling pathway activation in ECs. (A) Kif26b and Daam1 functioned synergistically to activate an AP1 luciferase reporter system. HeLa cells were transfected with control, Kif26b, Kif17, N-MD, or C-term-Kif26b mutants together with control or Daam1 plasmids. Quantification of fold change was determined in relation to control condition activation. Error bars denote SD; three experiments. *** p < 0.001 by one-way ANOVA followed by Tukey’s test. (B) Kif26b and Daam1 block in synergy TOP-flash reporter activation induced by Dvl3. HeLa cells were transfected with control, Kif26b, Daam1, or Kif26b and Daam1 together in combination with Dvl3 plasmid. The graph represents quantification of fold change in the function of Dvl3-induced TOP activation. Error bars denote mean ± SEM; three experiments. Statistical comparison between groups was performed using one-way ANOVA followed by Tukey’s test ( p < 0.05) to detect differences between all groups. *** p < 0.001 by one-way ANOVA. Kif26b and Daam1 depletion impaired Wnt5a-induced cdc42 activation. Activated cdc42 precipitation was performed on depleted cells after 30 min of Wnt5a activation. (C) Lysates of si Control, si Kif26b, or si Daam1 HUVECs activated or with Wnt5a were blotted with cdc42. (D) si Control or si Daam1–treated HeLa cells were transfected with a control plasmid or a plasmid coding for Kif26b-Myc. Lysates were blotted with Daam1, Myc, and cdc42. Activated cdc42 precipitation was then performed using Pak-CRIB peptide and blotted with anti-cdc42 antibody. Fold change in activated cdc42 levels either in si Control basal or Kif26b ectopic expression in si Daam1 conditions was calculated. Error bars denote mean ± SEM; three experiments. ns, not significant, and *** p < 0.05 and * p < 0.05 vs. control, by one-way ANOVA followed by Tukey’s test. Kif26b knockdown in HUVECs increased the canonical signaling pathway through an increase in active β-catenin level. (E) si Control and si Kif26b- or si Daam1–treated HUVECs were subjected to Wnt3a-conditioned medium activation for 6 h. Immunoblotting was performed with antibodies against active β-catenin and total β-catenin. Fold change is reported as a function of active β-catenin/β-catenin ratio in si Control conditions. Error bars denote mean ± SEM. Three experiments. * p < 0.05 by one-way ANOVA followed by Tukey’s test.

Article Snippet: The oligonucleotides used were designed by Origene for h-Kif26b (SR310475A, 5′-CCGCUACAAUGCA­GACAAGCCUU-3′; SR307944B, 5′-UGUUUGUACAUAAGAA­CU­GCUAG-3′) and h-Daam1 (SR307929A, 5′-GGAAUUCAACCUGU­AAUAGAUAA-3′, and SR307929B, 5′-AGCUGCGAAA­GUAAACA­UGACUG-3′).

Techniques: Activation Assay, Luciferase, Transfection, Blocking Assay, Plasmid Preparation, Expressing, Western Blot

Journal:

Article Title: Identification of novel short peptides derived from the ?4, ?5 and ?6 fibrils of type IV collagen with anti-angiogenic properties

doi: 10.1016/j.bbrc.2006.12.231

Figure Lengend Snippet: Effect of the peptides on the migration of HUVECs in a modified Boyden chamber migration assay. Endothelial cells were allowed to migrate for 20 h in the presence of 20 ng/ml VEGF and 30 μg/ml peptide solution, then stained with calcein and counted. The fluorescent signal was scaled so that the figure represents the percentage of migration inhibition. Here 100% represents the negative control (endothelial cells in serum- and growth factor-free medium, data not shown) or the total inhibition of the cell migration and 0% the positive control (migration in the presence of 20 ng/ml VEGF, data not shown) or zero inhibition of cell migration. Vertical bars indicate the standard error. All values are significantly different from 0% at p<0.001. In all cases the standard error for the controls was less than 4% (n=8).

Article Snippet: In-vitro Cell Migration Assay A modified Boyden chamber migration assay (BD Biosciences, San Jose, CA) was used to examine endothelial cell migration in the presence of an activator.

Techniques: Migration, Modification, Staining, Inhibition, Negative Control, Positive Control