Journal: PLoS ONE

Article Title: An Improved Quantitative Approach for the Assessment of Mitochondrial Fragmentation in Chemoresistant Ovarian Cancer Cells

doi: 10.1371/journal.pone.0074008

Figure Lengend Snippet: ( A ) 3-dimensional top view and side profiles of untreated OV2008 cells, with and without cytological centrifugation prior to fixation. Yellow arrows indicate typical areas of separation that appear between mitochondria after centrifugation. ( B ) Orthogonal sections of the same images seen in (A) taken with identical camera exposure settings. Cells were fixed in 3.7% paraformaldehyde and stained for nuclear (DAPI, blue) and mitochondrial (TOM20, green) signal. ( C ) Comparison of mitochondrial image quality between conventional immunocytochemistry and cytological centrifugation approaches. Images of classical tubular and fragmented mitochondrial morphology becomes visibly sharper after centrifugation due to the minimization of fluorescence originating from background sources beyond the focal plane. The enlargement of the nucleus post-centrifugation is due to flattening of the cells by centripetal force. Scale bars = 5 µm.

Article Snippet: Fluorescence Intensity Profiling (Zeiss LSM software package) is a tool normally used to determine signal-to-background ratios across fluorescent regions of an image .

Techniques: Centrifugation, Staining, Comparison, Immunocytochemistry, Fluorescence

Journal: PLoS ONE

Article Title: An Improved Quantitative Approach for the Assessment of Mitochondrial Fragmentation in Chemoresistant Ovarian Cancer Cells

doi: 10.1371/journal.pone.0074008

Figure Lengend Snippet: ( A ) Fluorescence intensity profiling of a single cell with fragmented mitochondria. Intensity of mitochondrial signal along a linear profile selected by the operator (light blue arrow) is represented quantitatively. (i) Emission peak of a single mitochondrion is indicated graphically (yellow arrow). (ii) Separated mitochondria are evident as independent peaks. (iii) Higher peaks (yellow arrow) suggest the presence of multiple mitochondria, superimposed during centrifugation. 3D image data is layered transparently, with individual mitochondrial number being directly proportional to the intensity of fluorescence. ( B ) Heat mapping of a single cell treated with CDDP (10 µM, 12 h). Differences in z-axis location values of mitochondrial fragments are represented as colors. (i) Magnification (yellow box) revealing distinct mitochondria at different z-axis heights (green vs. orange). (ii) The reverse view of the same cell in (i), indicating further individual fragments (cyan vs blue, yellow arrow). ( C ) Orthogonal section tool showing cross sections of a single cell along the y- axis (inset dotted yellow box, magnified as green box) and x-axis (magnified as red box). Y. (i) Magnification (green box) of y-axis orthogonal section. (ii) Magnification (red box) of x-axis orthogonal section. Yellow arrows indicate spaces separating individual mitochondria.

Article Snippet: Fluorescence Intensity Profiling (Zeiss LSM software package) is a tool normally used to determine signal-to-background ratios across fluorescent regions of an image .

Techniques: Fluorescence, Centrifugation

Journal: PLoS ONE

Article Title: An Improved Quantitative Approach for the Assessment of Mitochondrial Fragmentation in Chemoresistant Ovarian Cancer Cells

doi: 10.1371/journal.pone.0074008

Figure Lengend Snippet: Overview of the novel approach, involving cytological centrifugation, fluorescence analysis and assignment of cut-off scores for mitochondrial fission quantification.

Article Snippet: Fluorescence Intensity Profiling (Zeiss LSM software package) is a tool normally used to determine signal-to-background ratios across fluorescent regions of an image .

Techniques: Centrifugation, Fluorescence

Journal: PLoS ONE

Article Title: COPA and SLC4A4 are Required for Cellular Entry of Arginine-Rich Peptides

doi: 10.1371/journal.pone.0086639

Figure Lengend Snippet: Analysis of FITC-9R uptake in HeLa cells transfected with siRNAs against COPA, SLC4A4, ATP8B3, and CX40.1. (A) FITC-9R uptake of HeLa cells transfected with 4 individual siRNAs that were selected by primary screening. Scale bars = 10 µm. (B) Fluorescence intensity was measured by MetaMorph. Error bars represent SD from three independent experiments.

Article Snippet: The fluorescence intensity of HeLa cells was measured by MetaMorph, which showed that COPA siRNA1, COPA siRNA2, SLC4A4 siRNA1, and SLC4A4 siRNA2 reduced the internalization of FITC-9R compared with scrambled siRNA ( ).

Techniques: Transfection, Fluorescence

Journal: PLoS ONE

Article Title: COPA and SLC4A4 are Required for Cellular Entry of Arginine-Rich Peptides

doi: 10.1371/journal.pone.0086639

Figure Lengend Snippet: (A) RNA interference for COPA and SLC4A4. Interference of each gene was executed by two distinct gene-specific siRNAs. HeLa cells were cultured with each siRNA for 72 h. FITC-9R was added 1 h at 37°C before observation, and cells were observed with confocal microscopy. Scale bars = 10 µm. (B) Fluorescence intensity was measured by MetaMorph. Error bars represent SD from three independent experiments.

Article Snippet: The fluorescence intensity of HeLa cells was measured by MetaMorph, which showed that COPA siRNA1, COPA siRNA2, SLC4A4 siRNA1, and SLC4A4 siRNA2 reduced the internalization of FITC-9R compared with scrambled siRNA ( ).

Techniques: Cell Culture, Confocal Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: COPA and SLC4A4 are Required for Cellular Entry of Arginine-Rich Peptides

doi: 10.1371/journal.pone.0086639

Figure Lengend Snippet: (A) HeLa cells were transfected with COPA and/or SLC4A4 siRNAs and cultured for 72 h. Then FITC-9R was added to cells and incubated for 1 h at 37°C. Cells were observed with confocal microscopy. Scale bars = 10 µm. (B) Fluorescence intensity was measured by MetaMorph. Error bars represent SD from three independent experiments.

Article Snippet: The fluorescence intensity of HeLa cells was measured by MetaMorph, which showed that COPA siRNA1, COPA siRNA2, SLC4A4 siRNA1, and SLC4A4 siRNA2 reduced the internalization of FITC-9R compared with scrambled siRNA ( ).

Techniques: Transfection, Cell Culture, Incubation, Confocal Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: COPA and SLC4A4 are Required for Cellular Entry of Arginine-Rich Peptides

doi: 10.1371/journal.pone.0086639

Figure Lengend Snippet: (A) Confocal images of EGFP-COPA and EGFP-SLC4A4 localization in HeLa cells. Scale bars, 10 µm. Right Panel, high magnification merge images. Scale bars = 2 µm. (B) Confocal microscopy images of double fluorescence imaging show the co-localization of FITC-9R with COPA or SLC4A4 in HeLa cells. HeLa cells identified by DIC (differential interference contrast), and signaling with FITC-9R (9R) (green) were also positive for COPA (red), and co-localization was evident when images were merged (yellow). Similarly, the expression of SLC4A4 co-localized with FITC-9R is shown. pDsRed empty vector was used as a negative control and did not show co-localization with FITC-9R. Scale bars, 20 µm. (C) High magnification images from Fig. 5A. Scale bars = 10 µm.

Article Snippet: The fluorescence intensity of HeLa cells was measured by MetaMorph, which showed that COPA siRNA1, COPA siRNA2, SLC4A4 siRNA1, and SLC4A4 siRNA2 reduced the internalization of FITC-9R compared with scrambled siRNA ( ).

Techniques: Confocal Microscopy, Fluorescence, Imaging, Expressing, Plasmid Preparation, Negative Control

Journal: PLoS ONE

Article Title: COPA and SLC4A4 are Required for Cellular Entry of Arginine-Rich Peptides

doi: 10.1371/journal.pone.0086639

Figure Lengend Snippet: HeLa cells were transfected COPA or SLC4A4 siRNAs, and after 72-9R or FITC-TAT were added and incubated for 1 h at 37°C. Cells were observed with confocal microscopy (A, C). Scale bars = 10 µm. Fluorescence intensity of FITC-9R and FITC-TAT were measured by MetaMorph (B, D). Error bars represent SD from three independent experiments.

Article Snippet: The fluorescence intensity of HeLa cells was measured by MetaMorph, which showed that COPA siRNA1, COPA siRNA2, SLC4A4 siRNA1, and SLC4A4 siRNA2 reduced the internalization of FITC-9R compared with scrambled siRNA ( ).

Techniques: Transfection, Incubation, Confocal Microscopy, Fluorescence

Journal: ACS Omega

Article Title: Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays

doi: 10.1021/acsomega.6b00143

Figure Lengend Snippet: Molecular model of mannose/ConA-A488 binding. Mannose with α-5-pentylphosphonic acid was covalently bound to the ACG slide surface. Fluorescence-tagged concanavalin A (ConA-A488) was then bound to mannose specifically. Con A is a lectin tetramer with subunit dimension of 42 × 40 × 39 Å 3 . Each subunit has a mannose binding site. Geometrically, only two binding sites per molecule are available for mannose binding. The most effective mannose/ConA-A488 binding should give the strongest fluorescence intensity, and this model system has been used to optimize the AAO surface for the glycan microarray.

Article Snippet: The B max in column 7 of Table was estimated using the fluorescence intensity obtained from 1 μM to 100 mM with GraphPad Prism 5.0.

Techniques: Binding Assay, Fluorescence, Glycoproteomics, Microarray

Journal: ACS Omega

Article Title: Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays

doi: 10.1021/acsomega.6b00143

Figure Lengend Snippet: (A) Changes in electrical current for the fabrication of ACG slide no. 5. (B) Image of fluorescence intensities that resulted from the mannose/ConA-A488 binding of ACG slide no. 5. This image shows a 10 × 12 matrix with 10 repeated arrays, with each column of the mannose solution concentration varied (in consecutively 10 times dilution) from 100 mM to 1 pM. (C) Response surface of the modified quadratic model for AAO thickness transformed into a function of voltage and reaction time, Y AAO thickness = a × ( V ) 1/2 + b × (RT) 1/2 ; intercept ≠ 0 ( P < 0.0001). (D) Response surface fluorescence intensity with respect to 100 μM sugar concentration arrayed on the ACG slide surfaces. (E) Response surface of B max (1 μM to 100 mM) derived from model fitting, Y B max = a × V + b × V 2 (with significant P value) given in Table S5 . Both (D) and (E) show an optimal curvature of high fluorescence intensity within the range of this study.

Article Snippet: The B max in column 7 of Table was estimated using the fluorescence intensity obtained from 1 μM to 100 mM with GraphPad Prism 5.0.

Techniques: Fluorescence, Binding Assay, Concentration Assay, Modification, Transformation Assay, Derivative Assay

Journal: ACS Omega

Article Title: Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays

doi: 10.1021/acsomega.6b00143

Figure Lengend Snippet: (A) On-time measurement of the AAO layer formation under various voltages and reaction times. The starting pure Al-coated glass slide was as smooth as glass. The AAO growth (thickness increasing) depends on the voltage at the beginning, and electropolishing (surface smoothing) and extended AAO growth occur later. (B) Fluorescence intensity differences in ACG slide no.5 of RSM (response surface measurement) study vs NHS glass slide at various sugar solution concentrations arrayed on the surface.

Article Snippet: The B max in column 7 of Table was estimated using the fluorescence intensity obtained from 1 μM to 100 mM with GraphPad Prism 5.0.

Techniques: Fluorescence

Journal: ACS Omega

Article Title: Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays

doi: 10.1021/acsomega.6b00143

Figure Lengend Snippet: (A) GenePix scanning images (at PMT 380) of ConA-A488 bound to mannose (1 mM) on ACG slide (A) vs that on NHS glass slide (B). (C) ConA-A488/mannose (1 mM) binding on ACG slide vs NHS-activated glass slide. The fluorescence intensities of the averaged 20 spots for ACG slide vs NHS glass slide. (D) Spot analysis of ACG slides vs NHS-activated glass slides. (E) Confocal microscope (Leica SP8) images of ConA-A488/mannose binding on ACG slide (left) vs NHS-activated glass slide (right).

Article Snippet: The B max in column 7 of Table was estimated using the fluorescence intensity obtained from 1 μM to 100 mM with GraphPad Prism 5.0.

Techniques: Binding Assay, Fluorescence, Microscopy

Journal: ACS Omega

Article Title: Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays

doi: 10.1021/acsomega.6b00143

Figure Lengend Snippet: Optimization Experiment—Factors, Voltage (volt), Reaction Time (s) and Responses of AAO Layer Thickness (nm), Electrical Current (mA), Fluorescence Intensity of 100 μM Mannose Solution Arrayed on Each Slide Surface, and B max Derived from Michaelis–Menten Equation Using GraphPad Prism7.0

Article Snippet: The B max in column 7 of Table was estimated using the fluorescence intensity obtained from 1 μM to 100 mM with GraphPad Prism 5.0.

Techniques: Fluorescence, Derivative Assay

Journal: ACS Omega

Article Title: Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays

doi: 10.1021/acsomega.6b00143

Figure Lengend Snippet: Optimized Reaction Condition for Making the ACG Slide from an Al-Coated Glass Slide Based on the Fluorescence Intensity Resulting from Mannose/ConA-A488 Binding and B max Analysis

Article Snippet: The B max in column 7 of Table was estimated using the fluorescence intensity obtained from 1 μM to 100 mM with GraphPad Prism 5.0.

Techniques: Fluorescence, Binding Assay

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