Journal: Iranian Journal of Pharmaceutical Research : IJPR

Article Title: Umbelliprenin Induces Apoptosis in CLL Cell Lines

doi:

Figure Lengend Snippet: Umbelliprenin (50 µM) retains its apoptotic activity in the presence of IL-4 after 48 h incubation. Data is shown as mean ± standard deviation. *p < 0.05 when compared IL-4 group with IL-4+ umbelliprenin group. Abbreviations: IL-4: Interleukin-4.

Article Snippet: Induction of apoptosis by umbelliprenin in the presence of IL-4 Jurkat T-CLL cells were cultured in a 24 well plate at a density of 10 6 cells/well in RPMI 1640 containing 10% FBS and incubated with 1 ng/mL human IL-4 (BD, Biosciences, Cat. No. 354068).

Techniques: Activity Assay, Incubation, Standard Deviation

Journal: Journal of Cell Science

Article Title: Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly

doi: 10.1242/jcs.198853

Figure Lengend Snippet: Caveolin-1 and cavin-1 are associated with RSV filaments. (A) Confocal micrographs of RSV-infected HeLa cells (22 hpi) stained with antibodies against caveolin-1 and RSV G protein. A1 and A2, close-up of boxed regions in A. (B) Average fluorescence intensity distribution of caveolin-1 and G protein in viral filaments ( n =10 line scans with a representative image shown in the inset, error bars are standard deviations). (C) Confocal micrographs of RSV-infected HeLa cells (22 hpi) stably transfected with cavin-1–EGFP and co-stained with antibodies against caveolin-1 and RSV G protein. (D) Indirect immunofluorescence and confocal microscopy of RSV-infected HeLa cells (22 hpi) using antibodies against cavin-1 and RSV G protein. (E) Isolation of RSV virions from HEp-2 cells. A schematic of the sucrose gradient is shown. Three interphase fractions (I1–I3) and the pellet (P) were subjected to immunoblotting using the indicated antibodies. The relative enrichment of caveolin-1, cavin-1 and flotillin-2 in I2 was determined by densitometry and plotted (representative of two independent experiments). Scale bars: 20 µm.

Article Snippet: HeLa cells were grown on fibronectin-coated glass-bottom dishes (MatTec Corp., Ashland, MA) and transfected with 1 µg caveolin-1–APEX2–EGFP plasmid DNA using FugeneHD (Promega, Singapore).

Techniques: Infection, Staining, Fluorescence, Stable Transfection, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Western Blot

Journal: Journal of Cell Science

Article Title: Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly

doi: 10.1242/jcs.198853

Figure Lengend Snippet: Caveolin-1 is incorporated into the RSV envelope. (A–C) Representative transmission electron micrographs of RSV-infected HeLa cells (22 hpi) transfected with caveolin-1–APEX2–EGFP. (A1–A3) Close-ups of regions boxed in A. Note the electron-dense stain on caveolar membranes (arrows) and the RSV envelope (arrowheads), and the presence of caveolae at the base of virus filaments (A1,A2,B,C). (D) Representative micrographs of control RSV filaments (osmium post-fixation alone, top) and RSV filaments stained with caveolin-1–APEX2–EGFP (bottom). (E) Quantification of staining intensity across the filament width (as indicated in D) in control (black line) and caveolin-1–APEX2–EGFP-stained RSV filaments (red line). Shown are the averages and standard deviations of 22 line scans each ( n =6 cells). Note the significant contrast increase in the RSV envelope in caveolin-1–APEX2–EGFP-expressing cells, and the presence of two discrete peaks (p, membrane-proximal; d, membrane-distal). (F) Representative tomographic slice through an RSV filament stained with caveolin-1–APEX2–EGFP. The cartoon depicts the slice position (red, viral envelope; light blue, viral matrix). F1 and F2 are representative close-up views; images were contrast-enhanced and gaussian-filtered. Line scans across the filament are shown. Scale bars are 500 nm unless otherwise stated.

Article Snippet: HeLa cells were grown on fibronectin-coated glass-bottom dishes (MatTec Corp., Ashland, MA) and transfected with 1 µg caveolin-1–APEX2–EGFP plasmid DNA using FugeneHD (Promega, Singapore).

Techniques: Transmission Assay, Infection, Transfection, Staining, Expressing

Journal: Journal of Cell Science

Article Title: Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly

doi: 10.1242/jcs.198853

Figure Lengend Snippet: Caveolin-1 is recruited to RSV filaments in an actin-dependent manner. (A,B) RSV-infected HeLa cells were treated at 14 hpi with 500 nM cytochalasin D (A2,B) or left untreated (A1). Cells were fixed at 20 hpi and stained with antibodies against caveolin-1 and G protein, and phalloidin–FITC. Confocal micrographs are shown. (A1) RSV filaments are aligned along actin fibers. (A2) Partial disruption of the actin network with cytochalasin D causes distortion and aggregation of RSV filaments. Arrows indicate virus filaments (B) RSV-infected and cytochalasin-treated HeLa cells showing complete disruption of the actin cytoskeleton. Note the lack of RSV filaments and the loss of colocalization between caveolin-1 (red arrows) and G protein (green arrows). Representative data of two independent experiments are shown. Scale bars: 10 µm.

Article Snippet: HeLa cells were grown on fibronectin-coated glass-bottom dishes (MatTec Corp., Ashland, MA) and transfected with 1 µg caveolin-1–APEX2–EGFP plasmid DNA using FugeneHD (Promega, Singapore).

Techniques: Infection, Staining

Journal: Journal of Cell Science

Article Title: Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly

doi: 10.1242/jcs.198853

Figure Lengend Snippet: RSV infection alters the stoichiometry of the caveolar coat complex by stabilizing cavin-1 protein. (A) Immunoblotting of whole-cell lysates of mock-infected and RSV-infected HeLa cells (22 hpi) using the indicated antibodies. Increasing amounts (1×, 2×, 3×) of total cell lysates were loaded. (B) Quantification of the data shown in A ( n =4; error bars indicate standard deviation) P <0.02; ns, not significant (Student's t -test). (C) HeLa cells stably expressing cavin-1–EGFP were treated at 16 hpi with 100 µg/ml cycloheximide (CHX), or were left untreated (Ctrl), and analyzed by immunoblotting at 22 hpi. Data was quantified by densitometry ( n =3; error bars indicate standard deviation). * P <0.05 (Student's t -test). (D) Immunoblots of 10–40% sucrose gradient fractions prepared from mock-infected and RSV-infected HeLa cells stably expressing cavin-1–EGFP. Live cells were crosslinked at 22 hpi with 2 mM DSP. Cells were detergent extracted and the lysate fractionated. The 80S-CCC is boxed. The graph below shows the mean distribution of caveolin-1 in the gradients ( n =3 separate experiments). (E) Silver stain gel of the affinity-purified 80S-CCC from mock-infected and RSV-infected cells. Immunoprecipitation was carried out from the boxed gradient fractions shown in D using anti-GFP or anti-RFP antibodies. (F) Immunoblotting of the affinity-purified 80S-CCC using the indicated antibodies. (G) Quantification of the data shown in F ( n =3; error bars indicate standard deviation). P <0.05 (Student's t -test). M, mock infected; I, RSV infected.

Article Snippet: HeLa cells were grown on fibronectin-coated glass-bottom dishes (MatTec Corp., Ashland, MA) and transfected with 1 µg caveolin-1–APEX2–EGFP plasmid DNA using FugeneHD (Promega, Singapore).

Techniques: Infection, Western Blot, Standard Deviation, Stable Transfection, Expressing, Silver Staining, Affinity Purification, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly

doi: 10.1242/jcs.198853

Figure Lengend Snippet: Caveolin-1 interacts with the RSV G and M protein complex on the surface of infected cells. (A) Immunoprecipitations of RSV G, M and M2-1 proteins from HeLa whole-cell lysates of mock-infected or RSV-infected cells (22 hpi). Immunoprecipitates were probed for G protein (top panel), M protein (middle panel) and caveolin-1 (bottom panel). Caveolin-1 protein was measured by densitometry (bottom graph). (B) Streptavidin–HRP blot of lysates of surface-biotinylated HeLa cells. M, mock infected; I, RSV infected. (C) Streptavidin–HRP blot of immunoprecipitates from lysates shown in B, using antibodies against RSV G protein or caveolin-1. Representative data of two independent experiments are shown.

Article Snippet: HeLa cells were grown on fibronectin-coated glass-bottom dishes (MatTec Corp., Ashland, MA) and transfected with 1 µg caveolin-1–APEX2–EGFP plasmid DNA using FugeneHD (Promega, Singapore).

Techniques: Infection

Journal: Journal of Cell Science

Article Title: Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly

doi: 10.1242/jcs.198853

Figure Lengend Snippet: Caveolae are not required for RSV filament morphogenesis. (A) Immunoblots of HeLa cell lysates transfected with caveolin-1 siRNA or control siRNA. Lysates were prepared 4 days post transfection and probed with the indicated antibodies. Shown is a representative of five independent experiments. (B) Confocal micrographs of control and caveolin-1-siRNA-treated HeLa cells stained with antibodies against caveolin-1. (C) Maximum intensity projection (left) and 3D reconstruction of confocal stacks (right) of HeLa cells treated with 80 nM caveolin-1 siRNA for 3 days, infected with RSV (MOI 3) for 22 h, and fixed and stained with anti-G protein antibodies. Note the abundance of RSV filaments (arrowheads). (D) Immunoblots of HEp-2 cell lysates. Cells were transfected with 80 nM caveolin-1 siRNA or control siRNA for 3 days and either infected (I) with RSV (MOI 0.0001) or mock infected (M). Cell lysates were prepared at 2 dpi and 3 dpi, and probed with the indicated antibodies. (E) Representative micrographs of HEp-2 cells transfected with caveolin-1 siRNA and infected with RSV (MOI 0.0001). Cells were fixed 2 dpi and stained with anti-G and anti-caveolin-1 antibodies. The outline of the plaque is shown. Images on the right show the boxed region on the left. Note the presence of RSV filaments on the apical surface of the plaques (arrowheads). (F) Representative fluorescence micrographs of RSV-infected (MOI 0.0001) HEp-2 cells transfected with caveolin-1 siRNA or control siRNA. Cells were fixed 2 dpi and stained with anti-RSV antibody. (G) Quantification of plaque size at 2 dpi. Bar graphs show the mean plaque area, error bars indicate standard deviation ( n =132 plaques each). (H) Virus titers determined at 2 dpi and 3 dpi. D–H show representative data from two independent experiments.

Article Snippet: HeLa cells were grown on fibronectin-coated glass-bottom dishes (MatTec Corp., Ashland, MA) and transfected with 1 µg caveolin-1–APEX2–EGFP plasmid DNA using FugeneHD (Promega, Singapore).

Techniques: Western Blot, Transfection, Staining, Infection, Fluorescence, Standard Deviation

Journal: Oncology Letters

Article Title: Efficacy of pEgr-1-endostatin combined with ionizing radiation on hypoxic conditions in nude mice bearing SKOV3 ovarian carcinoma

doi: 10.3892/ol.2017.5559

Figure Lengend Snippet: Primers for polymerase chain reaction amplification.

Article Snippet: The membranes were blocked with PBS containing 1% Tween 20 at 4°C overnight, washed and incubated overnight at 4°C with anti-HIF-1α (#AH339-2) and anti-endostatin (#BAF1098) primary antibodies (1:1,000 dilution in TBS-T; Bioworld Technology, Inc., St. Louis Park, MN, USA).

Techniques: Polymerase Chain Reaction, Amplification, Sequencing

Journal: Oncology Letters

Article Title: Efficacy of pEgr-1-endostatin combined with ionizing radiation on hypoxic conditions in nude mice bearing SKOV3 ovarian carcinoma

doi: 10.3892/ol.2017.5559

Figure Lengend Snippet: Expression of endostatin in the three groups. (A) Agarose gel electrophoresis results of endostatin mRNA expression, as detected by reverse transcription-polymerase chain reaction in the different groups. (B) The relative mRNA level of endostatin was examined after group C had been injected with pEgr-1-endostatin plasmid for 12 h and exposed to 300 cGy/min X-ray for 48 h. Endostatin was successfully increased in the group C compared with that in groups A and B (F=380.078, P<0.001). (C) The protein level of endostatin was evaluated by western blotting. The results indicated that the expression of endostatin was significantly increased in group C compared with groups A and B (P=0.039). (D) ELISA was used to determine the plasma endostatin level, and the expression of endostatin in group C was observed to be higher than that in groups A (t=44.770, P<0.001) and B (t=42.480, P<0.001). A, control group; B, IR-treated group; C, endostatin-IR-treated group; IR, ionizing radiation; M, marker; mRNA, messenger RNA. ***P<0.001.

Article Snippet: The membranes were blocked with PBS containing 1% Tween 20 at 4°C overnight, washed and incubated overnight at 4°C with anti-HIF-1α (#AH339-2) and anti-endostatin (#BAF1098) primary antibodies (1:1,000 dilution in TBS-T; Bioworld Technology, Inc., St. Louis Park, MN, USA).

Techniques: Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Injection, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Marker

Journal: Oncology Letters

Article Title: Efficacy of pEgr-1-endostatin combined with ionizing radiation on hypoxic conditions in nude mice bearing SKOV3 ovarian carcinoma

doi: 10.3892/ol.2017.5559

Figure Lengend Snippet: MVD in tumor tissues. (A) The MVD counts for each group were significantly different among the different groups (F=22.660, P<0.001). An obvious increase in MVD was observed in group B (20.970±1.410) compared with that in group A (15.890±1.476, t=7.040, P<0.001), while a significant decrease in MVD was observed in group C (6.366±0.155) compared with that in groups A (t=18.153, P<0.001) and B (t=29.120, P<0.001). (B) Tumor microvessels were identified by cluster of differentiation 31 immunohistochemistry (magnification, ×200). Comparison of the morphology of the tumor vasculature in the three groups revealed that the microvessels in the (a) endostatin-IR-treated group were more regularly distributed and had fewer giant branches than those in the (b) control and (c) IR-treated groups. A, control group; B, IR-treated group; C, endostatin-IR-treated group; IR, ionizing radiation; MVD, microvessel density. ***P<0.001.

Article Snippet: The membranes were blocked with PBS containing 1% Tween 20 at 4°C overnight, washed and incubated overnight at 4°C with anti-HIF-1α (#AH339-2) and anti-endostatin (#BAF1098) primary antibodies (1:1,000 dilution in TBS-T; Bioworld Technology, Inc., St. Louis Park, MN, USA).

Techniques: Immunohistochemistry

Journal: Oncology Letters

Article Title: Efficacy of pEgr-1-endostatin combined with ionizing radiation on hypoxic conditions in nude mice bearing SKOV3 ovarian carcinoma

doi: 10.3892/ol.2017.5559

Figure Lengend Snippet: Expression of HIF-1α in tumor tissues. (A) Agarose gel electrophoresis results of HIF-1α mRNA expression, as evaluated by reverse transcription-polymerase chain reaction in different groups. (B) Relative levels of HIF-1α mRNA in different groups. The difference was statistically significant in the three groups: Groups A and B (t=8.961, P<0.001); groups A and C (t=5.339, P=0.001); groups B and C (t=12.930, P<0.001). (C) The protein level of HIF-1α was analyzed by western blotting. A higher average optical density value for HIF-1α was observed in group B compared with that in groups A and C. The difference was statistically significant (P=0.046). (D) Immunohistochemistry assessment indicated that the expression of HIF-1α was (a) moderate in the control group; (b) strong in the IR-treated group; and (c) weak in the endostatin-IR-treated group (magnification, ×200). A, control group; B, IR-treated group; C, endostatin-IR-treated group; IR, ionizing radiation; M, marker; mRNA, messenger RNA; HIF, hypoxia-inducible factor. ***P<0.001.

Article Snippet: The membranes were blocked with PBS containing 1% Tween 20 at 4°C overnight, washed and incubated overnight at 4°C with anti-HIF-1α (#AH339-2) and anti-endostatin (#BAF1098) primary antibodies (1:1,000 dilution in TBS-T; Bioworld Technology, Inc., St. Louis Park, MN, USA).

Techniques: Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Marker

Journal: Oncology Letters

Article Title: Efficacy of pEgr-1-endostatin combined with ionizing radiation on hypoxic conditions in nude mice bearing SKOV3 ovarian carcinoma

doi: 10.3892/ol.2017.5559

Figure Lengend Snippet: Expression of VEGF in tumor tissues. (A) Agarose gel electrophoresis results of VEGF mRNA expression in tumor tissues, as detected by reverse transcription-polymerase chain reaction. (B) A significant difference was observed in the three groups (F=119.691, P<0.001). Compared with group A (0.530±0.0444), the relative VEGF mRNA expression was increased in group B (0.653±0.0727, P=0.001) but decreased in group C (0.250±0.0359, P<0.001). The expression of VEGF in group C was significantly lower than that in group B (t=14.050, P<0.001). A, control group; B, IR-treated group; C, endostatin-IR-treated group; VEGF, vascular endothelial growth factor; IR, ionizing radiation; mRNA, messenger RNA. **P≥0.001 and <0.05, ***P<0.001.

Article Snippet: The membranes were blocked with PBS containing 1% Tween 20 at 4°C overnight, washed and incubated overnight at 4°C with anti-HIF-1α (#AH339-2) and anti-endostatin (#BAF1098) primary antibodies (1:1,000 dilution in TBS-T; Bioworld Technology, Inc., St. Louis Park, MN, USA).

Techniques: Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction