Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Internalization of fluorescein isothiocyanate-conjugated monoclonal anti-human CCR7 induced by recombinant CCL19 in intact HEK 293a cells that express recombinant CCR7. nt, non-transfected cells.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Recombinant, Transfection

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Endocytosis of the anti-myc monoclonal antibody (clone 4A6, conjugated to AlexaFluor-488, final concentration in the culture medium 3.3 nM) as determined by co-treatment with the CCL19-myc construction in intact HEK 293a cells that optionally and transiently expressed CCR7. A control conditioned medium (CM) or authentic CCL19 were used as control stimuli. The undiluted CM were transferred for a 30-min incubation period before rinsing and observation. Original magnification ×1000.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Concentration Assay, Control, Incubation

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Cytofluorometry of HEK 293a cells that optionally expressed CCR7, sequentially detached and further stimulated with the undiluted CM of other cells producing CCL19-myc, authentic CCL19, or CM of untransfected cells, as indicated (30 min incubation at 37°C). Left: distributions based on the counting of 10,000 cells. A threshold of autofluorescence was defined using control cells with no fluorophore. It was surpassed only under one set of experimental conditions (arrow). Right: mean fluorescence of cells in replicated experiment. ANOVA indicated that the values were heterogeneous ( p < 10 -4 ). * p < 0.01 vs. top-most value (control CM, untransfected recipient cells) by Dunnett’s test.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Incubation, Control, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Epifluorescence microscopy studies in cells co-expressing β-arrestin 1 -Cherry and CCR7 and stimulated as indicated (stimulant and duration). Stimulation always included the fluorescent anti-myc monoclonal antibody 4A6 (green signal). Either CCL19-myc CM or authentic CCL19 rarely condensed β-arrestin 1 at the level of plasma membranes of endosomes (arrowhead indicates possible co-localization with the CCL19-myc-antibody cargo). Cells that expressed well the red-emitting transgene were assumed to co-express co-transfected CCR7 and were evaluated for morphology ( n = 16–49 eligible cells per group). The average number of red condensed structures was 0.4 ± 0.4 in cells treated with control CM and did not vary significantly in all other groups (Kruskal–Wallis test). The average number of green condensed structures in cells treated with control CM was 0 per cell and varied significantly according to treatments ( p < 10 -4 , Kruskal–Wallis test; p values for Dunn’s multiple comparison test for each value vs. that of control CM reported thereafter). There were 8.3 ± 1.7 green specs per cell ( p < 0.05), 25.8 ± 3.2 ( p < 0.001), 33.1 ± 4.4 ( p < 0.001), and 0 (N.S.) in cells treated with CCL19-myc CM for 5, 10, or 30 min, or with recombinant CCL19, respectively.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Epifluorescence Microscopy, Expressing, Clinical Proteomics, Transfection, Control, Comparison, Recombinant

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Epifluorescence microscopy studies in cells co-expressing CCR7 and β-arrestin 2 -GFP and stimulated as indicated (stimulant and duration). Stimulation did not include the fluorescent anti-myc antibody. Either CCL19-myc CM or authentic CCL19 frequently condensed β-arrestin 2 at the level of plasma membrane or endosomes. Right: number of green intracellular condensed structures (“specs”) per cell ± SEM. Numbers close to bars indicate the number of evaluated HEK 293a cells. The Kruskal–Wallis test indicated that the values were heterogenous ( p < 10 -4 ). The effect of each treatment vs. the effect of control CM was evaluated using Dunn’s multiple comparison test. * p < 0.01; ** p < 0.001.

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Epifluorescence Microscopy, Expressing, Clinical Proteomics, Membrane, Control, Comparison

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Epifluorescence microscopy studies in cells co-expressing one of three forms of Rab5-Cherry fusion proteins and CCR7 and stimulated for 30 min with either CCL19-myc or control CM. Co-localization between Rab5-positive corpuscles and the CCL19-myc-antibody cargo was observed (arrowheads). The dominant positive (GTP-locked) mutant of the fusion protein produced typical giant vacuoles that included the green light-emitting cargo. The dominant negative Rab5-GDP-locked-Cherry inhibited the endocytosis of the green cargo (see text for numerical analysis).

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Epifluorescence Microscopy, Expressing, Control, Mutagenesis, Produced, Dominant Negative Mutation

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Epifluorescence microscopy studies in cells co-expressing Rab7-Cherry and CCR7 and stimulated as indicated (stimulant and duration). Co-localization between Rab7-positive corpuscles and the CCL19-myc-antibody cargo was not highly frequent (arrowheads), but increased in frequency as a function of incubation duration (see text for numerical analysis).

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Epifluorescence Microscopy, Expressing, Incubation

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Presence of CCR7 in A375 melanoma cells. (A) RT-PCR for CCR7 in the human melanoma A375 cell line. (B) c-Fos induction in A375 cells stimulated as indicated for 1 h (typical immunoblot and histograms representing the densitometry of replicated experiments).

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Pharmacology

Article Title: C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells

doi: 10.3389/fphar.2013.00122

Figure Lengend Snippet: Detection of endogenous CCR7 in A375 cells using detection of the endocytosed CCL19-myc-4A6 antibody complex using theTyramide Signal Amplification (TSA) system that enzymatically generates AlexaFluor-488 labeling. Epifluorescence and transmission, original magnification: ×1000. Right: proportion of cells with a mean fluorescence intensity above a set threshold (Photoshop level ≥ 50/255) in large photographic records of A375 cells stained as in the left of figure (the number of evaluated cells is indicated next to each histogram). The proportions were compared with that of control cells without antibody (top-most histogram, χ 2 test; * p < 10 -4 ).

Article Snippet: Using these cells, a first CCR7 imaging strategy was based on staining intact and live HEK 293a cells previously transfected with the vector encoding for the receptor with carboxyfluorescein-conjugated monoclonal anti-human CCR7 (CD197; clone 150503, final concentration 5 μg/ml; R&D Systems, Minneapolis, MN, USA).

Techniques: Amplification, Labeling, Transmission Assay, Fluorescence, Staining, Control

Journal: bioRxiv

Article Title: Engineering of Co-Surfactant-Free Bioactive Protein Nanosheets for the Stabilisation of Bioemulsions Enabling Adherent Cell Expansion

doi: 10.1101/2022.10.30.514404

Figure Lengend Snippet: A) Schematic representation of the functionalisation protocol. B) Epifluorescence microscopy images of BSA or BLG emulsions functionalised with SAMSA fluorescein (green, A685) with or without sulfo-SMCC. C) Mean fluorescence intensity of the coupled SAMSA. Scale bars, 100 μm. Error bars are s.e.m.; n = 3.

Article Snippet: The reagent sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate (sulfo-SMCC 22322) and the SAMSA Fluorescein, 5-((2-(and-3)-S-(acetylmercapto) succinoyl) amino) Fluorescein, mixed isomers (A685) are purchased from Thermofischer Scientific.

Techniques: Epifluorescence Microscopy, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Mitochondrial Membrane Potential and Nuclear Changes in Apoptosis Caused by Serum and Nerve Growth Factor Withdrawal: Time Course and Modification by (−)-Deprenyl

doi: 10.1523/JNEUROSCI.18-03-00932.1998

Figure Lengend Snippet: Estimation of cell survival, nuclear chromatin condensation, nuclear DNA fragmentation, and mitochondrial membrane potential. A1, Typical interference contrast micrograph of methylene blue-stained PC12 cells that were partially neuronally differentiated in M/S+N for 6 d and were then washed to remove trophic proteins and immediately replaced in M/S+N to reestablish trophic support at 12 hr before fixation for histology.A2, Similar micrograph for cells that were washed and trophically withdrawn by placement in M/O at 12 hr before fixation.B, Fluorescence photomicrographs of in situ nuclei stained with Hoechst 33258 at 12 hr after washing and placement into M/O. Normal Hoechst 33258-stained nuclei showed a diffuse, granular substructure with division by fine septate-like structures (inset 1). Apoptotic nuclei showed dense staining characterized by the formation of shrunken, intensely fluorescent lobular structures, those being apoptotic bodies (inset 2) or ring-like structures (inset 3). C, Interference contrast micrograph of cells reacted for in situ detection of nuclear DNA fragmentation using the Apop Tag method at 12 hr after washing and placement into M/O. The method revealed nuclei that were shrunken in comparison to those without evidence of DNA fragmentation. Nuclear DNA fragmentation is evident in three of the nuclei, the external plasma membranes of which appear intact using interference contrast microscopy. D, Typical epifluorescence micrograph of CMTMR fluorescence for a partially neuronally differentiated PC12 cell grown in M/S+N and fixed 24 hr after washing and replacement into M/S+N. CMTMR fluorescence from individual mitochondria was discernible using epifluorescence microscopy with 1000×, 1.3 NA objective. However, the thickness of the optical sections resulted in the superimposition of the fluorescence signals from adjacent mitochondria.E, Confocal micrograph of a cell in M/S+N at 24 hr after washing. A pinhole value of 50 produced optical sections that were sufficiently thin to allow for fluorescence measurements from individual mitochondria and the immediately adjacent cytoplasm without superimposition of fluorescence from nearby mitochondria.

Article Snippet: PC12 cells (American Type Culture Collection, Rockville, MD) were propagated in minimum essential medium (MEM) (Life Technologies, Gaithersburg, MD) with 10% horse serum (Life Technologies), 5% fetal bovine serum (Life Technologies), 2 m m l -glutamine (Life Technologies), 50 U/ml penicillin, and 50 μg/ml streptomycin (PC12 media).

Techniques: Membrane, Staining, Fluorescence, In Situ, Comparison, Clinical Proteomics, Microscopy, Epifluorescence Microscopy, Produced

Journal: The Journal of Neuroscience

Article Title: Mitochondrial Membrane Potential and Nuclear Changes in Apoptosis Caused by Serum and Nerve Growth Factor Withdrawal: Time Course and Modification by (−)-Deprenyl

doi: 10.1523/JNEUROSCI.18-03-00932.1998

Figure Lengend Snippet: Nuclear DNA fragmentation demonstrated with BODIPY-dUTP simultaneously with CMTMR staining to estimate ΔΨM. A1, A2, Low-power epifluorescence micrographs of the same image fields of cells after 12 hr in M/O that were immunoreacted for tubulin (A1) and also reacted using the BODIPY-dUTP method for detecting nuclear DNA fragmentation (A2). Confocal laser images of identical fields for tissue dually reacted with an anti-histone antibody are shown inB1 and C1, and BODIPY-dUTP is shown inB2 and C2. D1–D3, E1–E3, Identical confocal image fields for serial image planes (separated by ∼1.0 μm) through a group of partially neuronally differentiated PC12 cells in M/O at 6 hr after washing. Cultures are stained for BODIPY-dUTP nuclear staining and CMTMR mitochondrial fluorescence, respectively.

Article Snippet: PC12 cells (American Type Culture Collection, Rockville, MD) were propagated in minimum essential medium (MEM) (Life Technologies, Gaithersburg, MD) with 10% horse serum (Life Technologies), 5% fetal bovine serum (Life Technologies), 2 m m l -glutamine (Life Technologies), 50 U/ml penicillin, and 50 μg/ml streptomycin (PC12 media).

Techniques: Staining, Fluorescence

Journal: PLoS Pathogens

Article Title: Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission

doi: 10.1371/journal.ppat.1006061

Figure Lengend Snippet: (A) Vero cells were mock-transfected (Uninf.) or transfected with WT SINV or Y400K RNA, incubated at 37°C for 8 h, and fixed. Cells were permeabilized and stained with antibodies to detect the viral E2 envelope protein (red) and α-tubulin (blue), and with phalloidin to detect F-actin (green). Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of three independent experiments, which are quantitated in (B). Arrows indicate two examples of intercellular extensions; note that each is positive for all three markers and is in contact with a neighboring cell. Bar = 20 μm. (B) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for E2, actin and tubulin and their contact with a neighboring cell (see ). Graph in B shows the mean and standard deviation of three independent experiments, with 10 cells quantitated in each sample including the uninfected cells. ** P<0.01, ****P<0.0001.

Article Snippet: Vero cells (obtained from ATCC) were cultured at 37°C in Dulbecco’s modified Eagle’s medium containing 10% FCS, 100 U penicillin/ml, and 100 μg streptomycin/ml.

Techniques: Transfection, Incubation, Staining, Confocal Microscopy, Infection, Standard Deviation

Journal: PLoS Pathogens

Article Title: Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission

doi: 10.1371/journal.ppat.1006061

Figure Lengend Snippet: (A) Vero cells stably expressing GFP-actin were infected with WT-mCherry SINV and incubated for 7 h at 37°C. The cells were then imaged using the TIRF microscope in the wide-field mode and both the 561-nm and 488-nm lasers. Images were acquired every 10 s for 60 min (see ). Arrows indicate stable contacts established between an infected cell and a neighboring cell, and marker lines are used to follow cell position during migration. Bar = 20 μm. (B-E) Vero cells were infected with WT SINV, incubated at 37°C for 9 h, fixed, processed for SEM, and imaged using a Zeiss Supra 40 field emission SEM. (B) A representative image of infected cells. Bar = 10 μm. (C) SEM image of the region indicated by the dashed white box in panel B. Bar = 10 μm. (D and E) SEM images of the regions indicated by the dashed white boxes in panel C. Arrowheads indicate virus-sized structures. Bar = 1 μm.

Article Snippet: Vero cells (obtained from ATCC) were cultured at 37°C in Dulbecco’s modified Eagle’s medium containing 10% FCS, 100 U penicillin/ml, and 100 μg streptomycin/ml.

Techniques: Stable Transfection, Expressing, Infection, Incubation, Microscopy, Marker, Migration, Virus

Journal: PLoS Pathogens

Article Title: Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission

doi: 10.1371/journal.ppat.1006061

Figure Lengend Snippet: (A) Vero cells were transfected with SINV WT-mCherry RNA (t = 0) and incubated for 6.5 h at 37°C. Cells were labeled for 30 min with CellTracker Green and then uninfected Vero cells were added and the co-cultures were incubated for 2 h at 37°C. Cells were then fixed and imaged by epifluorescence microscopy. Images are representative of three independent experiments, evaluating 10 cell pairs/experiment. Bar = 20 μm. (B) Vero cells expressing the PM-GFP marker were transfected with SINV WT-mCherry RNA and incubated at 37°C for 7 h. Uninfected Vero cells were then added and the co-cultures were incubated for 2 h at 37°C. Cells were fixed, stained with antibodies to detect α-tubulin, and imaged by confocal microscopy. Images from one optical section are shown, and are representative of three independent experiments, 10 cell pairs/experiment. Bar = 20 μm.

Article Snippet: Vero cells (obtained from ATCC) were cultured at 37°C in Dulbecco’s modified Eagle’s medium containing 10% FCS, 100 U penicillin/ml, and 100 μg streptomycin/ml.

Techniques: Transfection, Incubation, Labeling, Epifluorescence Microscopy, Expressing, Marker, Staining, Confocal Microscopy

Journal: PLoS Pathogens

Article Title: Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission

doi: 10.1371/journal.ppat.1006061

Figure Lengend Snippet: (A) Vero cells were mock-infected (Uninf.), transfected with SINV Y400K RNA, or infected with WT SINV, SFV or CHIKV (MOI 20, 10, 10, respectively). Cells were then incubated at 37°C for 11 h, fixed, permeabilized, and stained with antibodies to detect viral envelope proteins (virus GP) and α-tubulin, and with phalloidin to detect F-actin. Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of three independent experiments. Bar = 20 μm. (B) Quantitation of the number of extensions in Vero cells from experiments as described in (A). Intercellular extensions emanating from infected cells were identified based on positive staining for GP, actin and tubulin and contact with a neighboring cell (see ). (C) HUVECs were infected and stained as in (A) and imaged and quantitated as in (B). Graphs in B and C show the mean and standard deviation of three independent experiments, with 10 cells quantitated in each sample including the uninfected cells. * P<0.05, ** P<0.01,*** P<0.001.

Article Snippet: Vero cells (obtained from ATCC) were cultured at 37°C in Dulbecco’s modified Eagle’s medium containing 10% FCS, 100 U penicillin/ml, and 100 μg streptomycin/ml.

Techniques: Infection, Transfection, Incubation, Staining, Virus, Confocal Microscopy, Quantitation Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission

doi: 10.1371/journal.ppat.1006061

Figure Lengend Snippet: (A) Effect of NRAMP downregulation on free virus infection. Vero cells were cultured for 3 days in control media or media containing 200 μg/ml ammonium iron citrate to down-regulate the SINV receptor NRAMP2. Cells were infected with SINV or SFV (MOI = 5) for 2 h at 37°C. 20 mM NH 4 Cl was then added to the medium to prevent secondary infection. Cells were fixed at 24 h post-infection and the ratio of infected to total cells quantitated by staining with antibody to the SINV or SFV E2 protein. The graph represents the mean and standard deviation of three independent experiments, with infection normalized to that of control cells (which was set to 1). * P<0.05. (B) Effect of NRAMP downregulation on formation of intercellular extensions. Vero cells were infected with SINV (MOI = 5) for 5 h at 37°C. Target Vero cells stably expressing the PM-GFP marker were pretreated as in (A) to downregulate NRAMP2, and then plated onto the infected cells at an approximate ratio of 1:1. The co-cultures were incubated for 3 h at 37°C in the continued presence of iron as indicated, fixed, permeabilized, and stained with antibody to SINV E2. Fluorescence microscopy images were acquired with the same exposure time, and images are representative of the results of 3 independent experiments. Bar = 20 μM. (C) Quantitation of the number of extensions in Vero cells from experiments as described in (B). Intercellular extensions emanating from infected cells were identified based on positive staining for the SINV E2 protein and contact with a PM-GFP expressing target cell, as described in the methods. The graph in (C) represents the mean and standard deviation of three independent experiments.

Article Snippet: Vero cells (obtained from ATCC) were cultured at 37°C in Dulbecco’s modified Eagle’s medium containing 10% FCS, 100 U penicillin/ml, and 100 μg streptomycin/ml.

Techniques: Virus, Infection, Cell Culture, Control, Staining, Standard Deviation, Stable Transfection, Expressing, Marker, Incubation, Fluorescence, Microscopy, Quantitation Assay

Journal: PLoS Pathogens

Article Title: Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission

doi: 10.1371/journal.ppat.1006061

Figure Lengend Snippet: (A) Down-regulation of target cell NRAMP. Vero cells were transfected with WT SINV or SFV RNA and incubated at 37°C for 5 h (producer cells). Target Vero cells stably expressing the PM-GFP marker were cultured for 3 days in control media or media containing 200 μg/ml ammonium iron citrate to down-regulate the SINV receptor NRAMP2, and then plated onto the infected cells at an approximate ratio of 1:1 and the co-cultures incubated for 19 h at 37°C in the continued presence of iron as indicated. The % infected target cells was quantitated by staining with antibody to the SINV or SFV E2 protein. The graph represents the mean and standard deviation of three independent experiments, with infection normalized to that of control target cells (which was set to 1). (B) Down-regulation of producer cell NRAMP. Vero cells were pretreated as in Fig 6A to downregulate NRAMP2, transfected with WT SINV or SFV RNA, and incubated at 37°C for 5 h (producer cells). Uninfected Vero cells stably expressing PM-GFP (target cells) were then plated onto the infected cells, and the co-cultures were incubated for 19 h at 37°C in the continued presence of iron as indicated. Infection of target cells was quantitated as in Fig 6A. The graphs in A and B represent the mean and standard deviation of three independent experiments.

Article Snippet: Vero cells (obtained from ATCC) were cultured at 37°C in Dulbecco’s modified Eagle’s medium containing 10% FCS, 100 U penicillin/ml, and 100 μg streptomycin/ml.

Techniques: Transfection, Incubation, Stable Transfection, Expressing, Marker, Cell Culture, Control, Infection, Staining, Standard Deviation

Journal: PLoS Pathogens

Article Title: Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission

doi: 10.1371/journal.ppat.1006061

Figure Lengend Snippet: Vero cells were mock-transfected (Uninf.) or transfected with SFV WT or G91D mutant RNA, incubated at the permissive temperature (28°C) overnight (A) or at the non-permissive temperature (37°C) for 8 h (B), and fixed. Note that different times of incubation at these two temperatures were used to allow virus replication and extension formation. Cells were permeabilized and immunostained to detect the virus glycoproteins (GP) or α-tubulin, and stained with phalloidin-Alexa488 to detect F-actin. Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of the images from three independent experiments. Bar = 20 μm. (C,D) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for GP, actin and tubulin and their contact with a neighboring cell. (E,F) Vero cells were transfected with WT or G91D SFV RNA and incubated at 37°C for 2h (producer cells). At 4 h post-transfection Vero target cells stably expressing the PM-GFP marker were plated onto the infected cells at an approximate ratio of 1:1 and the co-cultures incubated overnight at 37 or 28°C. Cells were then fixed, permeabilized, and immunostained to detect the viral glycoproteins. Epifluorescence microscopy was used to acquire 5 images using the 20X objective. The number of infected PM-GFP-positive target cells was quantitated and expressed as a fraction of the total number of target cells. The graphs in C-F represent the mean and standard deviation of three independent experiments. * P<0.05, ** P<0.01.

Article Snippet: Vero cells (obtained from ATCC) were cultured at 37°C in Dulbecco’s modified Eagle’s medium containing 10% FCS, 100 U penicillin/ml, and 100 μg streptomycin/ml.

Techniques: Transfection, Mutagenesis, Incubation, Virus, Staining, Confocal Microscopy, Infection, Stable Transfection, Expressing, Marker, Epifluorescence Microscopy, Standard Deviation

Journal: PLoS Pathogens

Article Title: Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission

doi: 10.1371/journal.ppat.1006061

Figure Lengend Snippet: (A) Vero cells were transfected with SFV WT or D188K mutant RNA, incubated at 37°C and fixed at 8 hours post transfection. Cells were permeabilized and immunostained to detect the virus E2 glycoprotein or α-tubulin, and imaged by confocal microscopy. Images from one optical section are shown and are representative of the images from three independent experiments. Bar = 20 μm. (B) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for E2 and tubulin and their contact with a neighboring cell. (C) Vero cells were transfected with WT or D188K SFV RNA and incubated at 37°C for 2 h (producer cells). At 4 hours post transfection Vero target cells stably expressing the PM-GFP marker were plated onto the producer cells at an approximate ratio of 1:1 and the co-cultures incubated at 37°C overnight. Cells were then fixed, permeabilized, and immunostained to detect the viral glycoproteins. Epifluorescence microscopy was used to acquire 5 images using the 20X objective. The number of infected PM-GFP-positive target cells was quantitated and expressed as a fraction of the total number of target cells. The graphs in B and C represent the mean and standard deviation of three independent experiments. *** P<0.001.

Article Snippet: Vero cells (obtained from ATCC) were cultured at 37°C in Dulbecco’s modified Eagle’s medium containing 10% FCS, 100 U penicillin/ml, and 100 μg streptomycin/ml.

Techniques: Transfection, Mutagenesis, Incubation, Virus, Confocal Microscopy, Infection, Staining, Stable Transfection, Expressing, Marker, Epifluorescence Microscopy, Standard Deviation

Journal: PLoS Pathogens

Article Title: Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission

doi: 10.1371/journal.ppat.1006061

Figure Lengend Snippet: (A) Vero cells were transfected with plasmids encoding the SFV structural proteins with or without capsid and with or without the E2 Y399R mutation. At 24 h post-transfection the cells were fixed, permeabilized, immunostained for the SFV E2 protein and stained with phalloidin-Alexa568 to detect F-actin. Images were acquired by confocal microscopy and are representative of three independent experiments. Images from one optical section are shown. Bar = 20 μm. (B) Using the conditions described in (A), the number of intercellular extensions per E2-expressing cell was quantitated based on positive staining for actin and contact with a neighboring cell. The graph represents the mean and standard deviation of three independent experiments. *P<0.05. (C) Vero cells were transfected with a plasmid encoding the SFV structural proteins including capsid, incubated at 37°C for 24 h, fixed, processed for SEM and imaged using a Zeiss Supra 40 field emission SEM. Shown are representative SEM images. Left panel: Bar = 5 μm. Middle panel: Bar = 1 μm. Right panel: 2X zoom of middle panel, bar = 1μm. (D) Vero cells were transfected with a plasmid encoding the SFV structural proteins including capsid, incubated at 37°C for 24 h, fixed, permeabilized, and immunostained for the SFV E2 protein and α-tubulin. The intercellular extensions per expressing cell were quantitated based on their positive staining for tubulin and their contact with another cell, and scored based on expression of SFV E2 in the target cell. The graph represents the mean and standard deviation of three independent experiments. ** P<0.01.

Article Snippet: Vero cells (obtained from ATCC) were cultured at 37°C in Dulbecco’s modified Eagle’s medium containing 10% FCS, 100 U penicillin/ml, and 100 μg streptomycin/ml.

Techniques: Transfection, Mutagenesis, Staining, Confocal Microscopy, Expressing, Standard Deviation, Plasmid Preparation, Incubation

Journal:

Article Title: Identification of Novel Principles of Keratin Filament Network Turnover in Living Cells V⃞

doi: 10.1091/mbc.E03-09-0707

Figure Lengend Snippet: Details of KF network formation in living SW13 cells synthesizing HK8-CFP and HK18-YFP fluorescent hybrids. (A) Epifluorescence microscopy (detection of HK18-YFP) of a characteristic peripheral segment (position of plasma membrane indicated at 3.5 min) highlighting intermediate steps of KF network production. Two KF precursors are marked by color and followed until integration into the filament system. Note the formation of small granules below the cell surface that elongate, fuse, and become part of the KF network. The corresponding movie 5 shows further details. (B) Size changes of a single particle that was tracked from its first detection until integration into the network. For clarity's sake only the pixels associated with that particular particle are shown. Sampling frequency, 30 s. (C) The graph is derived from B, showing a surface view of the altering particle dimensions demonstrating a continuous, nonincremental particle elongation (l) before fusion with the KF network over time (t). (D) 3D reconstruction (voltex presentation) of the HK18-YFP fluorescence as recorded by confocal laser scanning microscopy of a peripheral region revealing spatial characteristics of various intermediates of filament assembly in relationship to intracellular topology (position of cell border demarcated). The volume of the top segment has the dimensions 20 × 20 × 3.5 μm. The bottom cube is a cross section to specifically highlight the increase in diameter of KF precursors only after integration into the peripheral KF network. (E) Plot summarizing the quantitative analysis of 10 time-space diagrams (see, e.g., C) depicting the increase in length (top graph) and diameter (bottom graph). Note the continuous elongation of particles during the entire 20 min in contrast to the increase in diameter that is limited to the first 5 min. Bars, SD. Bars, 1 μm in A and D.

Article Snippet: Cell Lines and cDNA Transfection The following cell lines were used and cultured according to the specifications given by ATCC or the respective references: rat kangaroo kidney PtK2 cells (ATCC CCL-56), Madin-Darby canine kidney (MDCK) cells (clone 20; ATCC CCL-34), bovine kidney-derived MDBK cells (ATCC CCL-22), human mammary adenocarcinoma-derived MCF7 cells (ATCC HTB-22), human hepatocellular carcinoma-derived PLC cells (ATCC CRL8024), human colon carcinoma CaCo-2 cells (ATCC HTB-37), human immortalized HaCaT keratinocytes ( Boukamp et al., 1988 ), and human small-cell carcinoma SW13 cells derived from the adrenal cortex (ATCC CCL-105).

Techniques: Epifluorescence Microscopy, Clinical Proteomics, Membrane, Single Particle, Sampling, Derivative Assay, Fluorescence, Confocal Laser Scanning Microscopy

Journal: Blood

Article Title: The sumoylation pathway is dysregulated in multiple myeloma and is associated with adverse patient outcome

doi: 10.1182/blood-2009-03-211045

Figure Lengend Snippet: Effect of UBE2I-dominant-negative mutant on growth, proliferation, and adhesion to BMSCs. (A) Myc-tagged fusion proteins were expressed in RPMI 8226 cells. Mo indicates mock transfected; Wt, UBE2I wild-type; and DN, UBE2I-DN (dominant negative). Lysates were probed with a Myc-tag specific mAb (top), Ube2I-specific mAb (middle), or Sumo-1 polyclonal Ab (bottom). (B) UBE2I-DN increased γ-radiation–induced apoptosis. Early apoptosis was detected by annexin V staining. Plots indicate percentage of RPMI 8226 cells in early apoptosis 24 hours after treatment with 10 Gy γ-radiation. Data are representative of 3 independent experiments. (C) UBE2I-wt and UBE2I-DN resulted in opposite effects on MM cell growth. Values represent the mean of triplicate measurements using the MTT assay. (D) Ube2I is necessary for MM adhesion to bone marrow stroma. Adhesion of GFP-labeled UBE2I transfectants to normal BMSCs is shown by fluorescence microscopy after 96 hours of incubation under standard conditions. Data are representative of 3 independent experiments. Images were viewed with a Zeiss Axiovert 200 inverted epifluorescence microscope using a 20× objective (37°C; cells were in PBS; FITC fluorescent filter). Images were acquired with a Zeiss AxioCam HRc 14-bit color CCD camera and were processed with Axio Vision software (Version 3.1). (E) UBE2I-DN decreased BMSCs-induced uptake of 3H-thymidine. Values represent the mean of triplicate measurements of 3H-thymidine after 96 hours of coculture.

Article Snippet: Rabbit polyclonal antibody to human NSE2 (BC100-2506) was obtained from Novus Biologicals and was used at a final concentration of 1:500 and rabbit polyclonal antibody to PIAS1 was from Abcam.

Techniques: Dominant Negative Mutation, Transfection, Staining, MTT Assay, Labeling, Fluorescence, Microscopy, Incubation, Inverted Epifluorescence, Software