graph of percent csltp1–ans complex fluorescence  (OriginLab corp)

Journal: Blood

Article Title: A dual role for the class III PI3K, Vps34, in platelet production and thrombus growth

doi: 10.1182/blood-2017-04-781641

Figure Lengend Snippet: Defective platelet production and granule distribution in Vps34-deficient platelets. (A) Whole blood platelet count was measured by using a HORIBA ABX Micros 60 analyzer (mean ± SEM; n = 38 mice for WT and 49 for Pf4-Cre-Pik3c3lox/lox mice [Vps34]); ***P < .001 vs WT according to 2-tailed Student t test) (left). Quantification of the percentage of mice with a mean platelet volume ranging from 4 to 7 µm3 and from 7 to 10 µm3 (mean ± SEM; n = 38 mice for WT and 49 for Pf4-Cre-Pik3c3lox/lox mice [Vps34]) (middle). TEM of resting platelets (right). Images are representative of 5 mice of each genotype. Scale bar represents 2 µm. (B) Mice were intravenously injected with a dylight488–anti-GPIbβ immunoglobulin derivative antibody. The percentage of labeled platelets in blood samples was measured at various time points after injection. (C) Thrombocytopenia in mice was induced by intraperitoneal injection of anti-GPIbα antibody (left). The platelet count was measured in blood samples collected 6 hours after injection (time = 0) and at various time points. The mouse serum TPO level was quantified by immunoassay (middle). Platelets were incubated with 50 ng/mL of TPO at the indicated times and after fixation with a rat antibody against the extracellular domain of Mpl and an anti-rat Alexa Fluor488 antibody (right). The graph is expressed as the percentage of the mean fluorescence intensity (MFI) resting (0) values after flow cytometry analysis (mean ± SEM; n = 4-6 mice of each genotype, *P < .05 vs WT according to 2-way ANOVA). (D) TEM of resting platelets. Images are representative of 5 mice of each genotype. Scale bar represents 1.5 µm. Arrows indicate α-granules. Platelet α- and dense (δ)-granule numbers and mean areas were measured on TEM images by using ImageJ software (mean ± SEM; n = 5 mice of each genotype; *P < .05; ***P < .001 vs WT according to 2-tailed Student t test).

Article Snippet: Overall, these data indicate a role for Vps34 in PI3P production and in the regulation of the endocytic/endosomal pathway in MKs with consequences for fibrinogen trafficking and, in turn, granule biogenesis. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3. caption a7 Defective intracellular trafficking and PI3P production in Vps34-deficient MKs. (A-C) MK uptake of transferrin-Alexa Fluor 546 (A) or fibrinogen-Alexa Fluor 488 (B-C) was observed by confocal (A) or superresolution structured illumination microscopy (B-C) at different incubation time points. (A-B) Graphs represent fluorescence intensity quantified on a MK z -stack by ImageJ or Imaris software. (C) Representative 3-dimensional surface rendering of z -stacks acquired after 960 minutes of fibrinogen uptake are shown.

Techniques: Injection, Labeling, Incubation, Fluorescence, Flow Cytometry, Software

Journal: Blood

Article Title: A dual role for the class III PI3K, Vps34, in platelet production and thrombus growth

doi: 10.1182/blood-2017-04-781641

Figure Lengend Snippet: Defective intracellular trafficking and PI3P production in Vps34-deficient MKs. (A-C) MK uptake of transferrin-Alexa Fluor546 (A) or fibrinogen-Alexa Fluor488 (B-C) was observed by confocal (A) or superresolution structured illumination microscopy (B-C) at different incubation time points. (A-B) Graphs represent fluorescence intensity quantified on a MK z-stack by ImageJ or Imaris software. (C) Representative 3-dimensional surface rendering of z-stacks acquired after 960 minutes of fibrinogen uptake are shown. Scale bar represents 5 µm. Graphs represent fibrinogen-positive structure number and volume quantified over time on 3-dimensional images using Imaris software (mean ± SEM; n = 10-60 MKs from 3 mice of each genotype; *P < .05; ***P < .001 vs WT according to 2-way ANOVA). (D-E) Fixed MKs stained with anti-clathrin, anti-EEA1, anti-Rab11, or anti-LAMP1 antibodies followed by corresponding secondary Alexa Fluor488 antibodies were observed by confocal microscopy. Live MKs were stained with LysoTracker Deep Red, fixed, and observed by confocal microscopy. Representative images of a z-stack are shown. Scale bar represents 5 µm. Graphs represent the structure number and area analyzed on a z-stack with ImageJ software (mean ± SEM; n = 30-50 MKs from 3 mice of each genotype; *P < .05; ***P < .001 vs WT according to 2-tailed Student t test). (F) PI3P mass assay performed on MKs as described in “Methods” (mean ± SEM; n = 5; **P < .01 vs WT according to 1-sample Student t test). (G) MKs stained with anti-PI3P and secondary Alexa Fluor488 antibodies were observed by confocal microscopy, and fluorescence intensity was quantified by using ImageJ software (mean ± SEM; n = 40 MKs from 3 mice of each genotype; ***P < .001 vs WT according to 2-tailed Student t test).

Article Snippet: Overall, these data indicate a role for Vps34 in PI3P production and in the regulation of the endocytic/endosomal pathway in MKs with consequences for fibrinogen trafficking and, in turn, granule biogenesis. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3. caption a7 Defective intracellular trafficking and PI3P production in Vps34-deficient MKs. (A-C) MK uptake of transferrin-Alexa Fluor 546 (A) or fibrinogen-Alexa Fluor 488 (B-C) was observed by confocal (A) or superresolution structured illumination microscopy (B-C) at different incubation time points. (A-B) Graphs represent fluorescence intensity quantified on a MK z -stack by ImageJ or Imaris software. (C) Representative 3-dimensional surface rendering of z -stacks acquired after 960 minutes of fibrinogen uptake are shown.

Techniques: Microscopy, Incubation, Fluorescence, Software, Staining, Confocal Microscopy, Mass Assay

Journal: Nature Communications

Article Title: Cryo-electron microscopy structures of pyrene-labeled ADP-P i - and ADP-actin filaments

doi: 10.1038/s41467-020-19762-1

Figure Lengend Snippet: a Phosphate slows the labeling of ADP-actin filaments with N-(1-pyrene)iodoacetamide. Solutions of Mg-ADP-actin filaments were polymerized from 5 µM monomers overnight at 4 °C in 100 mM KCl; 1 mM MgCl 2 ; 10 mM imidazole, pH 7.0; 0.3 mM ADP; 3 mM NaN 3 and preincubated at room temperature with the same volume of either water, 20 mM potassium phosphate or 20 mM potassium sulfate before adding 50 µM N-(1-pyrene)iodoacetamide. The basal fluorescence is from the free N-(1-pyrene)iodoacetamide, and the fluorescence increase is due to the conjugation of N-(1-pyrene)iodoacetamide to the sidechain of C374 in actin filaments. b Effect of phosphate in the buffer on the fluorescence of Mg-ADP-pyrenyl-actin filaments. The fluorescence change in each data point was calculated by subtracting the fluorescence of 120 µL of Mg-ADP-pyrenyl-actin filaments (polymerized from 5 µM monomers) preincubated with 30 µL of water from the fluorescence of 120 µL of Mg-ADP-pyrenyl-actin filaments (polymerized from 5 µM monomers) preincubated with the same volume of phosphate or sulfate. Each sample was incubated for ~1 hour before the measurements. Error bars indicate the standard deviations of five readings on the same sample. The Y-axes are in arbitrary units (A.U.). Data are presented as mean values +/− SD. Source data are provided as a Source Data file.

Article Snippet: The fluorescence graphs (Fig. ) were plotted with OriginLab.

Techniques: Labeling, Fluorescence, Conjugation Assay, Incubation

Journal: Molecular cell

Article Title: A tethering mechanism controls the processivity and kinetochore-microtubule plus-end enrichment of the kinesin-8 Kif18A

doi: 10.1016/j.molcel.2011.07.022

Figure Lengend Snippet: A) Photobleaching of Kif18A-GFP in a metaphase HeLa cell. Irradiation was targeted to the indicated region (dashed yellow line). Enlarged images show K-MT plus-end fluorescence before and after photobleaching. Time is indicated in seconds and is relative to irradiation. Scale bars, 10 µm and 2 µm (enlarged images). B) Kinetics of Kif18A-GFP fluorescence recovery at a K-MT plus-end. A representative plot of normalized Kif18A-GFP fluorescence at a single kinetochore against time is shown. The recovery of Kif18A-GFP at K-MT plus-ends fit a single exponential (black line). C) Taxol causes equatorial enrichment of Kif18A-GFP. Still images from a video of a HeLa cell stably expressing Kif18A-GFP treated with 10 µM taxol. Time is indicated in min and is relative to taxol addition. Scale bar, 10 µm. D) Kif18A is enriched at kinetochores in taxol-treated HeLa cells. The localizations of endogenous Kif18A (red) and kinetochores (green) in a control HeLa cell or in a cell treated with 10 µM taxol for 15 min are shown. Insets are higher magnification views of the boxed regions. Scale bars, 10 µm and 1 µm (enlarged images). E) Photobleaching of Kif18A-GFP in a taxol-treated metaphase HeLa cell. Time is indicated in seconds and is relative to irradiation. Scale bars, 10 µm and 2 µm (enlarged images). F) Kinetics of Kif18A-GFP fluorescence recovery at a K-MT plus-end in a cell treated with 10 µM taxol. A representative plot of normalized Kif18A-GFP fluorescence at a single kinetochore against time is shown.

Article Snippet: Graphs of fluorescence intensities versus time were generated in SigmaPlot (Systat Software), and the resulting data fit to a single exponential, F t =F 0 +F inf *(1-e −kt ), essentially as described ( Howell et al., 2000 ).

Techniques: Irradiation, Fluorescence, Stable Transfection, Expressing, Control

Journal: Molecular cell

Article Title: A tethering mechanism controls the processivity and kinetochore-microtubule plus-end enrichment of the kinesin-8 Kif18A

doi: 10.1016/j.molcel.2011.07.022

Figure Lengend Snippet: A) Metaphase localization of Kif18A truncation mutants. The localizations of full-length GFP-Kif18A, GFP-Kif18A-N406, Kif18A-N480-GFP, and GFP-Kif18A-C307 in cells co-stained for tubulin (red) and Hec1 (blue) are shown. Scale bar, 5 µm. B) The C-terminal tail domain of Kif18A is required for the motor to accumulate at K-MT plus-ends. Representative linescans showing the distribution of Kif18A (green) along metaphase K-MTs (red) near the kinetochore (Hec1 peak, blue). C) The tail domain of Kif18A increases the dwell time of the motor on spindle MTs. Still images from photoconversion runs of tdEOS-Kif18A-FL and Kif18A-N480-tdEOS are shown. An image of fluorescence from the GFP channel is shown at moment of photoconversion (t=0). Regions that were photoconverted and subjected to analysis are outlined. Time is indicated in sec and is relative to the time of photoconversion. Scale bar, 10 µm. D) Decay kinetics of tdEOS-Kif18A-FL (blue) and Kif18A-N480-tdEOS (pink) fluorescence from the mitotic spindle. Normalized mean fluorescence of photoconverted tdEOS-Kif18A-FL (n=8) and Kif18A-N480-tdEOS (n=11) versus time in sec are shown. Asterisks denote time points corresponding to the final images shown in Figure 2C. Black lines represent fits of the data to single exponentials. Error bars represent SEM.

Article Snippet: Graphs of fluorescence intensities versus time were generated in SigmaPlot (Systat Software), and the resulting data fit to a single exponential, F t =F 0 +F inf *(1-e −kt ), essentially as described ( Howell et al., 2000 ).

Techniques: Staining, Fluorescence