Journal: The Journal of Experimental Medicine

Article Title: RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance

doi: 10.1084/jem.20130790

Figure Lengend Snippet: RGMb can be expressed on the cell surface but is primarily localized intracellularly. (a) RGMb cell surface expression on human breast cancer cell lines (MDA-231, SKBR-3, and MCF-7), the nonsmall cell lung cancer cell line NCI-H226, and the renal cancer cell line SN12C was analyzed by flow cytometry with mAb 1H6 (red) or isotype control (blue). (b) RGMb expression by Western blotting using mAb 1H6. The loading amount was 60–80 µg/lane except for 0.5 µg/lane for 300-hRGMb. (c) SKBR-3 cells were stained for RGMb (1H6-biotin) or isotype control plus streptavidin-Alexa Fluor 488 (green), and then with Phalloidin-TRIC to label F-actin, and analyzed by confocal microscopy. Bars, 25 µm. (d) RGMb expression by Western blotting using mAb 1H6. The loading amount was 60–80 µg/lane, except for 0.5 µg/lane for 300-mRGMb cells. (e) Splenocytes from naive mice and mice treated with FLT-3L as well as 300-mPD-L2 cells (negative control) were stained for surface markers CD3, CD19, and CD11c. Then intracellular flow cytometry staining with PE-conjugated RGMb mAb 9D1 (red) or rat IgG2a (blue) was used to analyze RGMb expression in these cells. (f) RGMb expression in unstimulated cells from the indicated mouse organs by qRT-PCR. Data are mean ± SEM; n = 2; *, P < 0.05; **, P < 0.01. Ordinary one-way ANOVA followed by Dunnett’s multiple comparison test. (g) RGMb expression in lung AM and IM by Western blotting as in d. All data are representative of two or more experiments.

Article Snippet: Images were taken using a Nikon d -Eclipse C1 confocal microscope equipped with a Melles Griot 488 Ion Laser (with a 515/30 emission filter), a Melles Griot 543 Laser (with a 590/50 emission filter), a Melles Griot 640 Laser, and the Confocal Acquisition Software Nikon EZ-C1 version 3.90.

Techniques: Expressing, Flow Cytometry, Control, Western Blot, Staining, Confocal Microscopy, Negative Control, Quantitative RT-PCR, Comparison

Journal: Journal of Biological Research

Article Title: Cortical microtubule orientation in Arabidopsis thaliana root meristematic zone depends on cell division and requires severing by katanin

doi: 10.1186/s40709-018-0082-6

Figure Lengend Snippet: Cortical microtubule orientation in the meristematic zone of untreated ( a ) and aphidicolin-treated ( b – d ) roots. Single CLSM sections through the boundary between protoderm and cortex are presented, so that in the center of each figure cortex cells can be observed. In all the images of this work the root tip is oriented towards the bottom of the page. In control root ( a ) prominent transverse orientation of cortical microtubules can be observed in interphase cells (arrowheads). At 12 h of treatment ( b ) transverse microtubule orientation persists (arrowheads), while dividing cells still exist (arrow points to a mitotic cell). After 24 h of treatment ( c ), cell divisions have ceased and cortical microtubules exhibit random orientation (cells within the brackets are cortex cells viewed at external cortical plane), which is also observed after 48 h of treatment ( d ; cells within brackets). In the latter image, vacuolation of meristematic cells in also prominent (asterisks show vacuoles). Representative decipher-graphs of microtubule alignment in meristematic cells of control ( e ), 12 h ( f ), 24 h ( g ) and 48 h ( h ) aphidicolin treatments. Decipher-graphs show that microtubules are transversely oriented (doubled-headed arrow) in the control and after 12 h of aphidicolin treatment ( e , f ), while the frequency of longitudinal and randomly oriented microtubules increased upon prolonged treatments ( g , h , respectively). Scale bar 10 μm

Article Snippet: The preparations were examined with a Nikon D-Eclipse C1 or a Zeiss LSM780 confocal laser scanning microscope (CLSM), with the appropriate filters for FITC, and micrographs were acquired with each manufacturer’s software.

Techniques: Control

Journal: Journal of Biological Research

Article Title: Cortical microtubule orientation in Arabidopsis thaliana root meristematic zone depends on cell division and requires severing by katanin

doi: 10.1186/s40709-018-0082-6

Figure Lengend Snippet: Cortical microtubule orientation at the external face of epidermal cells in the transition (i.e. under the shootward part of the lateral root cap; a , c ) and fast elongation ( b , d ) zones of aphidicolin-treated roots. All images are maximum projections of serial CLSM sections. After 24 h of treatment, microtubules are transverse (arrowheads) in the transition ( a ) and fast elongation ( b ) zone, while after 48 h of treatment they appear randomly oriented in both zones ( c , d ). Representative decipher-graphs of microtubule alignment in transition and elongation zone cells of control roots ( e ), and after 24 h ( f ) and 48 h ( g ) of aphidicolin treatment. Decipher-graphs show that microtubules are transversely oriented (doubled-headed arrow) in the control and after 24 h of aphidicolin treatment ( e and f , respectively), while the frequency of longitudinal and/or randomly oriented microtubules increased after 48 h of treatment ( g ). Scale bar 10 μm

Article Snippet: The preparations were examined with a Nikon D-Eclipse C1 or a Zeiss LSM780 confocal laser scanning microscope (CLSM), with the appropriate filters for FITC, and micrographs were acquired with each manufacturer’s software.

Techniques: Control

Journal: Journal of Biological Research

Article Title: Cortical microtubule orientation in Arabidopsis thaliana root meristematic zone depends on cell division and requires severing by katanin

doi: 10.1186/s40709-018-0082-6

Figure Lengend Snippet: Cortical microtubule orientation in cortex cells of wild-type ( a , e ) and p60-katanin mutants ( b – d , f – h ). All high magnification images ( e – h ) are maximum projections of serial CLSM sections through the cortical cytoplasm. Cortex cell files, derived from the areas defined by rectangular frames on low magnification images ( a – d ), were released after application of gentle pressure on the whole mount root specimens. Fairly transverse cortical microtubules can be observed in Col-0 cells ( e ). In ktn1 - 2 ( f ), lue1 ( g ) and fra2 ( h ), though a general transverse pattern exists, several microtubules exhibit random orientation, while foci of microtubule convergence can be discerned as well. Representative decipher-graphs of microtubule alignment in meristematic cortex cells of Col-0 ( i ), ktn1 - 2 ( j ), lue1 ( k ) and fra2 ( l ). Generally, transverse microtubule orientation prevailed in all the cases (doubled-headed arrow). However, in the katanin mutants an increase in the frequency of longitudinal and/or randomly oriented microtubules was noticed. Scale bars 50 μm ( a – d ), 10 μm ( e – h )

Article Snippet: The preparations were examined with a Nikon D-Eclipse C1 or a Zeiss LSM780 confocal laser scanning microscope (CLSM), with the appropriate filters for FITC, and micrographs were acquired with each manufacturer’s software.

Techniques: Derivative Assay, Gentle

Journal: Infection and Immunity

Article Title: A Candida albicans Mannoprotein Deprived of Its Mannan Moiety Is Efficiently Taken Up and Processed by Human Dendritic Cells and Induces T-Cell Activation without Stimulating Proinflammatory Cytokine Production

doi: 10.1128/iai.00669-08

Figure Lengend Snippet: FIG. 1. Uptake of rMP65 fluor by different cell types. (A) MN, MDM, DC, PMN, and PBL treated for 30 min at 37°C with rMP65 fluor (5 g/ml) or rCpPLD fluor (5 g/ml) were analyzed by flow cytometry to assess rMP65 and rCpPLD uptake. The rMP65 fluor uptake by different cell types is expressed as the mean fluorescence intensity (MFI). Data are expressed as means SEM for three independent experiments. *, P 0.05; **, P 0.001 (rMP65 fluor-treated cells versus rCpPLD fluor-treated cells). (B) rMP65 fluor, nMP65 fluor, and rCpPLD fluor uptake by DC, tested using confocal microscopy. DC were treated with 25 g/ml of rMP65 fluor, rCpPLD fluor, or nMP65 fluor for 2 h at 37°C. The cells were then fixed and analyzed with a confocal microscope. (C) MDM or DC were stimulated for 30 min at 37°C with different concentrations of rMP65 fluor (0.5, 1, 2.5, 5, 25, and 50 g/ml). The rMP65 fluor uptake by different cell types is expressed as the MFI. Data are expressed as means SEM for three independent experiments. *, P 0.05; **, P 0.001 (rMP65-treated cells versus untreated cells). (D) MDM were pretreated with various concentrations of RGDS or control peptide for 15 min, and then rMP65 fluor (25 g/ml) was added. Data are expressed as means SEM for three independent experiments. *, P 0.05 (RGDS-treated cells versus untreated cells).

Article Snippet: Images were collected on a Nikon C1 laser scanning confocal unit (Nikon D-Eclipse C1) attached to an inverted fluorescence microscope (Nikon Eclipse TE 2000-U).

Techniques: Cytometry, Confocal Microscopy, Microscopy, Control