Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: The Journal of comparative neurology

Article Title: Whole Mouse Brain Reconstruction and Registration to a Reference Atlas with Standard Histochemical Processing of Coronal Sections

doi: 10.1002/cne.24602

Figure Lengend Snippet: (a) Coronal sections from the brain are collected in a plate of 10 wells, with successive sections being placed in sequence in well 1 through 10 and then repeated such that each well contains a 1 in 10 series through the brain. Sections are processed for immunohistochemical labeling and then mounted onto slides, with each slide containing a 1 in 10 series, which are imaged. (b) The BrainMaker software (MBF Bioscience) first outlines the individual section on each slide and then renumbers them to put them in sequence for the whole brain. Beginning with section 1 adjacent sections in the whole brain series are then overlain and aligned automatically and adjusted manually. (c) The aligned whole brain image is reconstructed into a stack of images, which may be viewed in coronal (XY), topdown (XZ), sagittal (YZ) or 3D views

Article Snippet: Once the brain sections have been imaged, they are registered and reconstructed into a whole brain image volume using the BrainMaker software (MBF Bioscience, Williston, VT).

Techniques: Sequencing, Immunohistochemical staining, Labeling, Software

Journal: Immunity

Article Title: Secondary influenza challenge triggers resident memory B cell migration and rapid relocation to boost antibody secretion at infected sites

doi: 10.1016/j.immuni.2022.03.003

Figure Lengend Snippet: Alveolar plasma cells are derived from memory B cells (A) Experimental outline for (A–C). (B) TPLSM images of CD19 −/− mice transferred with BAT B cells as described in (A) before and after rechallenge. PCs are highlighted using Imaris-created spots (yellow). (C) PCs L values at r = 200. (D) Alveolar PC density in infected or uninfected sites. Right, data represented as the fold-change difference. (E) Left, experimental outline. Right, PCs L values in untreated resting memory mice or rechallenged mice treated with anti-CD40L. Statistical analysis were made using an unpaired t test (C), a paired t test (D), and a Mann-Whitney U test (E). Error bars represent SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also Figure S4 .

Article Snippet: InVivoMAb anti-mouse CD40L, Clone MR-1 , BioXCell , Cat# BE0017-1; RRID: AB_1107601.

Techniques: Clinical Proteomics, Derivative Assay, Infection, MANN-WHITNEY

Journal: Immunity

Article Title: Secondary influenza challenge triggers resident memory B cell migration and rapid relocation to boost antibody secretion at infected sites

doi: 10.1016/j.immuni.2022.03.003

Figure Lengend Snippet:

Article Snippet: InVivoMAb anti-mouse CD40L, Clone MR-1 , BioXCell , Cat# BE0017-1; RRID: AB_1107601.

Techniques: Control, Purification, Virus, Clone Assay, Generated, Recombinant, Liposomes, Gene Expression, Software, Imaging, Light Microscopy

Journal: Cell reports

Article Title: MCT1 Deletion in Oligodendrocyte Lineage Cells Causes Late-Onset Hypomyelination and Axonal Degeneration

doi: 10.1016/j.celrep.2020.108610

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Adult brain dissociation kit , Miltenyi , Cat#130–107-677.

Techniques: Recombinant, TaqMan Assay, Imaging, Software

Journal: The Journal of Cell Biology

Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

doi: 10.1083/jcb.201709153

Figure Lengend Snippet: Behavior of the anti-RPB1 mAb in U2OS cells. (A) After transduction with Alexa Fluor 488–labeled anti-RPB1 antibodies, cells were imaged after 6 h of incubation and then every hour over a period of 20 h (see Video 1 for all time points). Bar, 15 µm. (B) Increasing amounts of Alexa Fluor 488–labeled anti-RPB1 mAb were transduced in U2OS cells and fixed 24 h after electroporation. A typical nucleus recorded in each case after counterstaining with DAPI is shown. Bar, 5 µm. (C) Binding capacity of anti-RPB1 mAb in U2OS cells. Cells were electroporated with 0 (mock), 0.5, 2, and 4 µg anti-RPB1 mAb and whole-cell extracts prepared 24 h after transduction (INPUT) were mixed with protein G beads. Bound and unbound material was analyzed by Western blotting. The blot shows the fraction of antibody-bound Pol II molecules adsorbed on the beads (beads) or left in the supernatant (SN), and detected with a secondary antibody. (D) After transduction with Alexa Fluor 488–labeled anti-RPB1 mAb (2 µg), cells were treated with or without CSK buffer. The histogram shows the mean fluorescence intensity of the nucleus of nontreated (−CSK) and CSK-treated (+CSK) cells 24 h (Elec 24h) or 48 h (Elec 48h) after electroporation. A classical anti-RPB1 mAb IF experiment was performed as additional control (IF). The +CSK signal is represented as the percentage of the mean intensity of the −CSK signal. Error bars represent the SD obtained with 10 recorded cells for each condition. All images were acquired by confocal microscopy on one single z plane.

Article Snippet: The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 μg/ml gentamicin.

Techniques: Transduction, Labeling, Incubation, Electroporation, Binding Assay, Western Blot, Fluorescence, Control, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

doi: 10.1083/jcb.201709153

Figure Lengend Snippet: Visualization of endogenous transcription factors and phosphorylated H2AX with VANIMA. (A) The labeled mAbs binding specifically to the transcription factors RPB1, TAF10, and TBP were transduced in U2OS cells, and their localization in the cells was monitored by confocal microscopy 24 h after treatment. A single z plane is shown for each condition. The pictures represent a typical nucleus recorded in each case after fixation of the cells and subsequent counterstaining with DAPI. (B) Same as in A, except that the experiments were performed with the corresponding labeled Fab fragments. (C) Increasing amounts of Alexa Fluor 488–labeled anti-TAF10 mAb (green) were transduced in U2OS cells and fixed 24 h after electroporation (anti-TAF10 Electroporation). To verify binding of the antibody to TAF10, a competition assay was performed afterward by adding a constant amount (2 µg) of the same antibody but Alexa Fluor 568–labeled as IF antibody (red, anti-TAF10 IF; see also Fig. S1 C for quantification). DAPI staining is shown in gray. (D) The labeled Fab raised against γH2AX was transduced as in B, and its localization was recorded after treatment of the electroporated cells with either NCS (for 15 min) or HU (for 48 h). Control, nontreated cells. A typical nucleus is represented in each case. (E) After transduction with Alexa Fluor 488–labeled anti-γH2AX Fab (5 µg) and treatment with HU, cells were treated with or without CSK buffer before fixation. The histogram shows the mean fluorescence intensity of the nucleus of nontreated (−CSK) and CSK-treated (+CSK) cells 24 h (Elec 24h) or 48 h (Elec 48h) after electroporation. The +CSK signal is represented as the percentage of the mean intensity of the −CSK signal. Error bars represent the SD obtained with 10 recorded cells for each condition. Bars, 5 µm.

Article Snippet: The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 μg/ml gentamicin.

Techniques: Labeling, Binding Assay, Confocal Microscopy, Electroporation, Competitive Binding Assay, Staining, Control, Transduction, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

doi: 10.1083/jcb.201709153

Figure Lengend Snippet: The mAbs do not inhibit premRNA transcription, cell cycle progression, cell proliferation and do not induce apoptosis. (A) U2OS cells electroporated but without antibodies (UT elec), electroporated and treated with α-amanitin (α-ama), electroporated with a control antibody binding to bacterial MBP (anti-MBP), or electroporated with the mAbs recognizing specifically RPB1, TAF10, or TBP (anti-RPB1, anti-TAF10, or anti-TBP). 24 h after electroporation, total RNA was isolated, and the expression of Pol I, Pol II, and Pol III genes was analyzed by RT-qPCR. Pol III transcripts were used for normalization. Newly synthesized RNA of the indicated genes was quantified with validated primer pairs (Table S2). The histograms correspond to the mean values obtained with three independent experiments. (B) The mean values of the three independent experiments shown in A are represented as a heatmap reflecting unchanged relative expression in black, up-regulation in green, and down-regulation in red. (C) U2OS cells were electroporated as in A, and cell cycle progression was monitored by propidium iodide staining and FACS analysis 24 or 48 h after electroporation. The cell cycle phases were normalized to cells electroporated without antibody. (D) U2OS cells were electroporated as in A, and their capacity of proliferation was monitored 24 h after transduction by EdU incorporation and FACS. The electroporated cells without the addition of antibody were used as control. The color code is as in A. (E) The cells were treated as in A, except an apoptosis test was performed 24 h after electroporation. Apoptosis induced by the addition of 10 µM H 2 O 2 was taken as reference (100%). In each panel, the error bars represent the biological SD obtained from three independent replicates. UT, untreated cells.

Article Snippet: The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 μg/ml gentamicin.

Techniques: Control, Binding Assay, Electroporation, Isolation, Expressing, Quantitative RT-PCR, Synthesized, Staining, Transduction

Journal: The Journal of Cell Biology

Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

doi: 10.1083/jcb.201709153

Figure Lengend Snippet: Visualization of transcription factors with VANIMA by super-resolution microscopy. (A) The labeled mAbs binding to the transcription factors RPB1, TAF10, and TBP (yellow) were transduced in U2OS cells, and their localization in the cells was monitored 24 h after transduction by 3D-SIM. The pictures show a typical nucleus recorded in each case after fixation and DAPI (gray) treatment (Videos 2–4). The Z maximum intensity projections of five slices show the labeled mAbs with (right half) or without (left half) DAPI counterstaining (gray). The solid white lines depict the nuclear contour. Bottom: Magnification of the white regions of interest, under the corresponding image. (B) The nuclei shown correspond to transduced U2OS cells as in A, except that transductions were performed with the corresponding labeled Fab fragments. Bars, 2 µm.

Article Snippet: The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 μg/ml gentamicin.

Techniques: Super-Resolution Microscopy, Labeling, Binding Assay, Transduction

Journal: The Journal of Cell Biology

Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

doi: 10.1083/jcb.201709153

Figure Lengend Snippet: Quantification of transcription factor distribution in single cells by using VANIMA and super-resolution microscopy. (A) U2OS cells were transduced with Alexa Fluor 488–labeled anti-RPB1 mAb and then treated with Flavo (2 µM) for 1 h or not (Untreated). 24 h after treatment the cells were fixed and analyzed by 3D-SIM. The number of individual spots and their volume in individual nuclei were quantified by using Fiji/ImageJ and Matlab software. The graph shows the percentage of spots with a given volume in untreated (red) and treated cells with Flavo (blue) acquired from 10 individual cells for each condition. (B) Same treatment and analysis as in A, but an Alexa Fluor 488–labeled anti-TAF10 antibody was transduced. (C) Spot volumes were extracted from A and B, and the percentage of spots of RPB1 and TAF10 with a volume >10 −2 μm 3 in the untreated (red) and Flavo (blue) treated cells is shown. The error bars represent the SE from 10 individual cells for each condition. (D) Total number of RPB1 and TAF10 spots in 10 individual nuclei for each condition are represented. (E) Mean cluster size of the RPB1 or TAF10 spots in 10 individual cells for each condition is shown. (F) Total spot volume of RPB1 and TAF10 in 10 individual nuclei for each condition is represented. All black boxes in D–F represent the means and their SEs for each sample. All p-values were calculated by using the two-sample t test.

Article Snippet: The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 μg/ml gentamicin.

Techniques: Super-Resolution Microscopy, Transduction, Labeling, Software

Journal: The Journal of Cell Biology

Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

doi: 10.1083/jcb.201709153

Figure Lengend Snippet: Imaging of phosphorylated H2AX with VANIMA by super-resolution microscopy. (A) The labeled anti-γH2AX Fab (yellow) was transduced in U2OS cells, and its localization in the nucleus was recorded by 3D-SIM after treatment with HU for 48 h (+HU) and staining with DAPI (gray). Untreated cells (−HU) were used as the control. The Z maximum intensity projections of 20 slices show the labeled anti-γH2AX Fab with (right half) or without (left half) DAPI counterstaining (gray). The solid white lines depict the nuclear contour. Bottom panels: magnification of the white regions of interest, under the corresponding image (Videos 5 and 6). Bars, 2 µm. (B) The number of spots presented in the nuclei as shown in A after quantification with Fiji/ImageJ software. Error bars represent the SD obtained with five recorded cells for each condition.

Article Snippet: The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 μg/ml gentamicin.

Techniques: Imaging, Super-Resolution Microscopy, Labeling, Staining, Control, Software

Journal: The Journal of Cell Biology

Article Title: Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

doi: 10.1083/jcb.201709153

Figure Lengend Snippet: Live imaging of transcription factors by using VANIMA. (A) 24 h after electroporation, U2OS cells transduced with Alexa Fluor 488–labeled anti-RPB1 mAb were subjected to live-cell analysis by confocal microscopy focusing on one z section of individual nuclei. They were imaged over a period of 2.5 h and pictures taken every 10 min (Video 7). Arrows point to two larger Pol II cluster examples that move over time. Bar, 5 µm. (B) Imaging by 3D-SIM microscopy of an individual Pol II cluster observed in U2OS after transduction as in A. The images were taken over a period of 37 s every 4.1 s and show a maximum intensity projection of the 3D video (Video 8). Bar, 1 µm. (C) U2OS cells transduced as in A with the labeled anti-γH2AX Fab were subjected to live-cell analysis by spinning-disk confocal microscopy after the addition of NCS to the culture medium. Pictures were taken every 10 min over a period of 4 h (Video 9) and by focusing on a single z plane. The first time point (0 min) corresponds to the time of the drug addition. Arrows point to γH2AX clusters that appear and disappear over time. Bar, 5 µm. (D) Imaging of an individual γH2AX cluster by 3D-SIM microscopy observed in U2OS cells after transduction as in C. Images were recorded over a period of 45 s every 15 s (Video 10). The first time point (0 s) shown was taken 10 min after NCS treatment. Bar, 0.8 µm.

Article Snippet: The human U2OS osteosarcoma cells (HTB-96; American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FCS and 40 μg/ml gentamicin.

Techniques: Imaging, Electroporation, Transduction, Labeling, Cell Analysis, Confocal Microscopy, Microscopy

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Noninvasive Imaging of Therapeutic Gene Expression Using a Bi-directional Transcriptional Amplification Strategy

doi: 10.1038/mt.2008.180

Figure Lengend Snippet: Comparing activity of the Survivin promoter with the viral CMV promoter in different cancer cell lines pCMV-FL and pSurv-FL are PGL3 plasmids where the FL reporter gene is driven by the CMV or Survivin promoter respectively. Firefly activity obtained in different cell lines normalized to microgram protein is given as mean values ± SD. Sensitivity Index = (RLU pSurv-FL /RLU pCMV-FL )×100

Article Snippet: Cell lines and media Colorectal cancer cell lines HCT116 and HT29 were obtained from ATCC while WiDr cells are a kind gift from Dr. Anna Wu (School of Medicine, UCLA).

Techniques: Activity Assay

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Noninvasive Imaging of Therapeutic Gene Expression Using a Bi-directional Transcriptional Amplification Strategy

doi: 10.1038/mt.2008.180

Figure Lengend Snippet: HCT116 cells were transiently transfected with (i) TE buffer (mock), (ii) one-step system (pSurv-FL or pSurv-TR), (iii) G5-based unidirectional TSTA system (pSurv-G5-FL or pSurv-G5-TR) and (iv) G8-based unidirectional TSTA system (pSurv-G8-FL or pSurv-G8-TR). The cells were harvested 24 h after transfection and assayed for (a) FL activity by luminometer and (b) TRAIL expression by Eliza. The error bars represent SEM for triplicate measurements. All values have been normalized for transfection efficiency. * indicates p<0.05 comparing one-step systems vs unidirectional TSTA systems.

Article Snippet: Cell lines and media Colorectal cancer cell lines HCT116 and HT29 were obtained from ATCC while WiDr cells are a kind gift from Dr. Anna Wu (School of Medicine, UCLA).

Techniques: Transfection, Activity Assay, Expressing

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Noninvasive Imaging of Therapeutic Gene Expression Using a Bi-directional Transcriptional Amplification Strategy

doi: 10.1038/mt.2008.180

Figure Lengend Snippet: To assess the optimum number of total Gal4-binding sites, 0(G5), 2(G7), 3 (G8) or 4 (G9) additional Gal4-binding sites were inserted in the vicinity of the E4TATA promoter (on the –ve strand) in the bi-directional TSTA system. HCT116 cells transfected with either the different bi-directional TSTA system or the one-step system were harvested, lysed and assayed for (a) FL activity or (b) TRAIL concentration. The error bars represent SEM for triplicate measurements. All values have been normalized for transfection efficiency. * indicates p<0.05 comparing all TSTA systems vs one-step systems.

Article Snippet: Cell lines and media Colorectal cancer cell lines HCT116 and HT29 were obtained from ATCC while WiDr cells are a kind gift from Dr. Anna Wu (School of Medicine, UCLA).

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

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Noninvasive Imaging of Therapeutic Gene Expression Using a Bi-directional Transcriptional Amplification Strategy

doi: 10.1038/mt.2008.180

Figure Lengend Snippet: Two different TSTA systems were examined for any directional bias that might affect the amplification of genes. Accordingly, FL and TR gene positions were swapped in case of both G5- and G8-based bi-directional TSTA systems and compared with the original orientation. HCT116 cells were then transfected with i) TE buffer, ii) one-step system (pSurv-FL or pSurv-TR, G5-based bi-directional TSTA systems iii) pSurv-FL-G5-TRand iv) pSurv-TR-G5-FL (switched iii), G8-based bi-directional TSTA systems v) pSurv-TR-G8-FL and vi) pSurv-FL-G8-TR (switched v). (a) FL activity or (b) TRAIL concentration. The error bars represent SEM for triplicate measurements. All values have been normalized for transfection efficiency. * indicates p<0.05 comparing all TSTA systems vs one-step systems.

Article Snippet: Cell lines and media Colorectal cancer cell lines HCT116 and HT29 were obtained from ATCC while WiDr cells are a kind gift from Dr. Anna Wu (School of Medicine, UCLA).

Techniques: Amplification, Transfection, Activity Assay, Concentration Assay

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Noninvasive Imaging of Therapeutic Gene Expression Using a Bi-directional Transcriptional Amplification Strategy

doi: 10.1038/mt.2008.180

Figure Lengend Snippet: HCT116 cells infected with i) Media only (mock), ii) Ad-pSurv-FL, iii) Ad-pSurv-TR or iv) Ad-pSurv-TR-G8-FL at MOI 15 were harvested after 48hrs and cells lysates were assayed for TRAIL production (a) or FL activity (b). The error bars represent SEM for triplicate measurements. * indicates p<0.05 comparing TSTA virus vs one-step virus. (c) Linear increase in expression of the FL (line) and TR (bar) genes with increasing dose of bi-directional TSTA adenovirus (Ad-pSurv-TR-G8-FL) (MOI 0–10). The results were obtained 48hrs after infection in HCT116 cells. The error bars indicate SEM for triplicate measurements. (d) Correlation between FL and TR expression in HCT116 cells infected with different doses of Ad-pSurv-TR-G8-FL. The correlation is R2=0.99.

Article Snippet: Cell lines and media Colorectal cancer cell lines HCT116 and HT29 were obtained from ATCC while WiDr cells are a kind gift from Dr. Anna Wu (School of Medicine, UCLA).

Techniques: Infection, Activity Assay, Virus, Expressing

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Noninvasive Imaging of Therapeutic Gene Expression Using a Bi-directional Transcriptional Amplification Strategy

doi: 10.1038/mt.2008.180

Figure Lengend Snippet: (a) Optical CCD imaging of a mouse carrying HCT116 xenografts infected with i) Ad-pCMV-FL (top, n=5), ii) Ad-pSurv-TR-G8-FL (middle, n=6) and iii) Ad-pSurv-FL (bottom, n=4). HCT116 xenografts grown on nude mice were injected intratumorally with 2×107 pfu of the different adenoviruses and the animals were imaged for a week. Shown here are images of a representative animal from each group. All images have been adjusted to the same scale. (b) ROIs were drawn around the tumor in these images and photon emission was quantified using the Living Image software. Plot shows a comparison of the intratumoral luciferase signal across the three groups. The error bars represent SEM. (c) Plot shows dose dependent expression of both genes following intratumoral injections of the bi-directional TSTA adenoviral vector. Tumors were injected with either 1.2 or 3.0×107 pfu of the adenovirus (n=3 per group). Firefly expression was detected in live animals by bioluminescence imaging while intratumoral TRAIL concentration was determined by ELIZA ex-vivo. (d) Correlation of FL and TR gene expression from 7 mice injected intratumorally with different doses of Ad-pSurv-TR-G8-FL. Plot of TRAIL expression (picogram/microgram of tumor lysate) measured on tumor lysates ex-vivo versus FL activity expressed as maximum (photons/cm2/sec/sr) obtained from ROIs drawn over the tumors on mouse images non-invasively (R2=0.67, n=7 mice).

Article Snippet: Cell lines and media Colorectal cancer cell lines HCT116 and HT29 were obtained from ATCC while WiDr cells are a kind gift from Dr. Anna Wu (School of Medicine, UCLA).

Techniques: Imaging, Infection, Injection, Software, Comparison, Luciferase, Expressing, Plasmid Preparation, Concentration Assay, Ex Vivo, Gene Expression, Activity Assay

Journal: Nature Communications

Article Title: Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

doi: 10.1038/ncomms15090

Figure Lengend Snippet: ( a ) Cellular internalization and cytosol localization of GFP11-SBP2-fused RT11 and TMab4 antibodies, as assessed by confocal microscopy measuring complemented GFP signals (green) in HeLa-SA-GFP1-10 cells after treatment with 1 μM of the antibodies for 6 h. Scale bar, 20 μm. ( b ) Cellular internalization and co-localization of RT11 (green), but not TMab4 (green), with the inner plasma membrane-anchored active Ras (red) in mCherry-KRas G12V -transformed NIH3T3 and KRas G12V -harbouring SW480 cells, analysed by confocal microscopy. The Ras WT -harbouring HT29 cells were also analysed as a control. The areas in the white boxes are shown at a higher magnification for better visualization. The arrow indicates the co-localization of RT11 with activated Ras. The cells were treated with 2 μM of antibody for 12 h. Scale bar, 5 μm. In a , b , nuclei are counterstained with Hoechst33342 (blue). ( c ) Immunoprecipitation (IP) of KRas mutant with RT11, but not TMab4, from endosome-depleted cell lysates of HA-KRas G12V -transformed NIH3T3 and SW480 cells, treated with 2 μM of antibody for 12 h before analysis. The endosome-depleted cell lysates were assessed by the absence of Rab5, an early endosome marker . In a – c , images are representative of at least two independent experiments.

Article Snippet: The human cell lines, cervix carcinoma HeLa cells, colorectal carcinoma SW480, LoVo, HT29, and Colo320DM cells, pancreatic carcinoma AsPC-1 and PANC-1 cells, soft tissue sarcoma HT1080 cells, lung carcinoma H1299, acute myeloid leukemia HL60 cells, chronic myeloid leukemia K562 cells, breast carcinoma MCF-7 cells and mouse embryonic fibroblast NIH3T3 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Confocal Microscopy, Clinical Proteomics, Membrane, Transformation Assay, Control, Immunoprecipitation, Mutagenesis, Marker

Journal: Nature Communications

Article Title: Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

doi: 10.1038/ncomms15090

Figure Lengend Snippet: ( a ) Cellular proliferation assay, after cells were treated twice at 0 and 72 h with antibody at the indicated concentrations for 6 d. Error bars±s.d. ( n =3). * P <0.05, ** P <0.01, *** P <0.001 versus TMab4-treated cells; NS, not significant. ( b ) Intracellular distribution of eGFP-fused cRaf RBD protein (green) in eGFP-cRaf RBD -transformed SW480 cells, treated with antibody (2 μM) for 12 h before microscopic confocal analysis. Scale bar, 20 μm. ( c , d ) IP of endogenous Raf proteins (bRaf and cRaf) with HA-tagged KRas G12V from the endosome-depleted cell lysates of HA-KRas G12V -transformed NIH3T3 cells ( c ) and IP of endogenous KRas G12V with cRaf RBD from the endosome-depleted cell lysates of SW480 cells ( d ). The cells were treated with 2 μM of RT11 and TMab4 for 12 h before analysis. ( e , f ) Inhibitory effect of RT11 on the downstream signalling of KRas-effector PPIs in HA-KRas G12V -transformed NIH3T3 cells ( e ) and SW480 cells ( f ), analysed by western blotting. The cells were serum-starved for 6 h before treatment with antibody, Raf kinase inhibitor sorafenib, or PI3K-Akt inhibitor LY294002 for 6 h in serum-free growth medium. Cells were washed and then stimulated with 10% FBS ( e ) and EGF (50 ng ml −1 in serum free-media) ( f ) for 10 min before cell lysis. The number below the panel indicates relative value of band intensity of phosphorylated proteins compared to that in the PBS-treated control after normalization to the band intensity of respective total protein for each sample. * P <0.05, ** P <0.01, *** P <0.001 versus PBS-treated control cells. In b – f , images are representative of at least two independent experiments.

Article Snippet: The human cell lines, cervix carcinoma HeLa cells, colorectal carcinoma SW480, LoVo, HT29, and Colo320DM cells, pancreatic carcinoma AsPC-1 and PANC-1 cells, soft tissue sarcoma HT1080 cells, lung carcinoma H1299, acute myeloid leukemia HL60 cells, chronic myeloid leukemia K562 cells, breast carcinoma MCF-7 cells and mouse embryonic fibroblast NIH3T3 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Proliferation Assay, Transformation Assay, Western Blot, Lysis, Control

Journal: Nature Communications

Article Title: Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

doi: 10.1038/ncomms15090

Figure Lengend Snippet: ( a ) Generation of integrin αvβ3/αvβ5-targeting RT11-i by genetic fusion of RGD10 peptide, using a (G 4 S) 2 linker, to the N-terminus of the LC of RT11. ( b , c ) RT11-i and TMab4-i bind to cell surface-expressed integrin ανβ3 and ανβ5. In b , flow cytometric analysis of the cell surface expression levels of integrin ανβ3 and ανβ5 on WT K562, integrin ανβ3-transformed K562, and human tumour cells, analysed by PE-conjugated anti-human integrin ανβ3 and ανβ5 antibodies. In c , flow cytometric analysis of cell surface binding levels of the indicated antibodies, co-incubated at 100 nM with 300 IU ml −1 heparin for 1 h at 4 °C with the indicated cells before analysis. ( d ) Cellular internalization and co-localization of RT11-i, but not TMab4-i, with the inner plasma membrane-anchored active Ras·GTP in KRas G12V -harbouring SW480 cells. The Ras WT -harbouring HT29 cells were also analysed as a control. The areas in the white boxes are shown at increased magnification for better visualization. The arrow indicates the co-localization of RT11-i with activated Ras. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 5 μm. ( e ) IP of endogenous KRas G12V with RT11 or RT11-i, but not TMab4 and TMab4-i, from endosome-depleted cell lysates of SW480 cells. Images are representative of two independent experiments. In d , e , the cells were treated with 1 μM of antibodies for 12 h before analysis. ( f ) Inhibition of tumour cell soft agar colony formation by RT11-i compared to that with TMab4-i. Following treatment of cells with PBS, TMab4-i (2 μM), or RT11-i (2 μM) every 72 h for 2–3 weeks, the number of colonies (diameter>200 μm) was counted by BCIP/NBT staining, as shown in the pictures of representative soft agar plates . The results are presented as percentages compared to the PBS-treated control. Error bars represent the mean±s.d. ( n =3). **P <0.01, ***P <0.001; NS, not significant.

Article Snippet: The human cell lines, cervix carcinoma HeLa cells, colorectal carcinoma SW480, LoVo, HT29, and Colo320DM cells, pancreatic carcinoma AsPC-1 and PANC-1 cells, soft tissue sarcoma HT1080 cells, lung carcinoma H1299, acute myeloid leukemia HL60 cells, chronic myeloid leukemia K562 cells, breast carcinoma MCF-7 cells and mouse embryonic fibroblast NIH3T3 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Transformation Assay, Binding Assay, Incubation, Clinical Proteomics, Membrane, Control, Inhibition, Staining

Journal: Nature Communications

Article Title: Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

doi: 10.1038/ncomms15090

Figure Lengend Snippet: ( a ) Pharmacokinetic profiles of RT11-i and TMab4-i in non-tumour bearing mice. Serum concentrations of TMab-i and RT11-i were determined by ELISA in female BALB/c nude mice following a single intravenous injection of 20 mg kg −1 in a total volume of 200 μl. Error bars represent the mean±s.d. ( n =3 per time point). The solid lines represent the fit of a two-compartment pharmacokinetic model to the data to estimate the initial rapid clearance phase (T 1/2 α) and later terminal serum clearance phase (T 1/2 β). The inset table shows the pharmacokinetic parameters. ( b ) Tumour-targeting ability of RT11-i and TMab4-i, evaluated by intravenously injecting Dylight755-labelled antibodies (20 μg per mouse) into SW480 xenograft tumour-bearing mice, followed by in vivo fluorescence imaging. Representative images are shown, which were acquired at the indicated times post-injection. Fluorescence intensities in the tumour tissue (T), as indicated by arrows, and normal tissues (N) were quantified by radiant efficiency (photons s −1 cm −2 steradian −1 μW −1 cm −2 ) using Living Image software. Error bars,±s.d. ( n =5 per group).

Article Snippet: The human cell lines, cervix carcinoma HeLa cells, colorectal carcinoma SW480, LoVo, HT29, and Colo320DM cells, pancreatic carcinoma AsPC-1 and PANC-1 cells, soft tissue sarcoma HT1080 cells, lung carcinoma H1299, acute myeloid leukemia HL60 cells, chronic myeloid leukemia K562 cells, breast carcinoma MCF-7 cells and mouse embryonic fibroblast NIH3T3 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, In Vivo, Fluorescence, Imaging, Software

Journal: Nature Communications

Article Title: Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

doi: 10.1038/ncomms15090

Figure Lengend Snippet: ( a ) In vivo anti-tumour efficacy of RT11-i compared to that of vehicle and TMab4-i controls, analysed by measuring the tumour volume during treatment of female BALB/c nude mice harbouring the indicated tumour xenografts. Antibodies were intravenously dosed at 20 mg kg −1 every 2 d (indicated by the arrows). Error bars,±s.d. ( n =8 per group). ( b , c ) Immunohistochemical images showing the levels of p-ERK1/2 and p-Akt ( b ) or cellular penetration and co-localization of antibodies with the active Ras form ( c ) in SW480 tumour tissues excised from mice following treatment described in a . Images are representative of three independent experiments. Nuclei were counterstained with Hoechst33342 (blue). Scale bar, 100 μm in b or 10 μm in c . In b , the right panel shows the percent relative fluorescence intensity compared to that of vehicle-treated control. Error bars,±s.d. of five random fields for each tumour (two tumours per group). In c , the areas in the white boxes are shown at a higher magnification for better visualization. The arrows indicate the co-localization of RT11-i with activated Ras. In a , b , statistical analysis was performed using a one-way analysis of variance followed by the Newman–Keuls post-test. * P <0.05, ** P <0.01, *** P <0.001 versus TMab4-i; NS, not significant.

Article Snippet: The human cell lines, cervix carcinoma HeLa cells, colorectal carcinoma SW480, LoVo, HT29, and Colo320DM cells, pancreatic carcinoma AsPC-1 and PANC-1 cells, soft tissue sarcoma HT1080 cells, lung carcinoma H1299, acute myeloid leukemia HL60 cells, chronic myeloid leukemia K562 cells, breast carcinoma MCF-7 cells and mouse embryonic fibroblast NIH3T3 cells were purchased from the American Type Culture Collection (ATCC).

Techniques: In Vivo, Immunohistochemical staining, Fluorescence, Control

Journal: Investigational New Drugs

Article Title: Blood glutamate scavengers prolong the survival of rats and mice with brain-implanted gliomas

doi: 10.1007/s10637-012-9794-x

Figure Lengend Snippet: a . A representative example of MRI analysis of C6 glioma growth in vivo. At 7 days post-implantation and before the treatment, an MRI imaging was performed (left column, 1–2 pictures T2 imaging, 3–4 pictures T1 imaging). The rats were randomized and either treated with 0.2 M OxAc or 0.3 M NaCl for additional 14 days. The MRI imaging was performed again at the end of the treatment (21 days post glioma implantation, right column, 1–4 pictures T2 imaging). b . Tumor volume of the rats before and after the treatment was calculated in n = 14 for control group and in n = 15 for the treated group using MRIcro software. OxAc-treated rats show significantly reduced tumor growth compared to the control group. * p < 0.01 (repeated measures ANOVA test)

Article Snippet: Rat C6 glioma and human U-87 Glioma cells were obtained from ATCC.

Techniques: In Vivo, Imaging, Control, Software