Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Flavonoid Fraction of Orange and Bergamot Juices Protect Human Lung Epithelial Cells from Hydrogen Peroxide-Induced Oxidative Stress

doi: 10.1155/2015/957031

Figure Lengend Snippet: Cytofluorimetric evaluation of intracellular ROS. A549 cells treated for 18 h with FFBJ or FFOJ were oxidatively stressed by H 2 O 2 200 μ M for additional 2 h. Results from FFBJ (a) or FFOJ (b) treatments. The curves shifted rightward to higher emission values indicate the increase of ROS production. Data from (a) and (b) are expressed as percentage of reduction (%Δ) of DCF-DA emission values in FFBJ or FFOJ-pretreated cells and then exposed to H 2 O 2 compared to H 2 O 2 -stressed cells (c). The experiments were repeated at least three times. ∗∗ P < 0.01 versus H 2 O 2 -treated cells.

Article Snippet: The biological experiments were performed using a basal epithelial cell line A549 derived from human lung carcinoma (ATCC, Rockville, MD, USA).

Techniques:

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Flavonoid Fraction of Orange and Bergamot Juices Protect Human Lung Epithelial Cells from Hydrogen Peroxide-Induced Oxidative Stress

doi: 10.1155/2015/957031

Figure Lengend Snippet: Confocal laser scanning microscope images of DCF-DA-stained cells. A549 cells grown on cell slides were preincubated with FFBJ or FFOJ and after 18 h exposed to H 2 O 2 200 μ M. Green fluorescence represented the amounts of ROS. Images shown are representative of three independent experiments. 400x magnification.

Article Snippet: The biological experiments were performed using a basal epithelial cell line A549 derived from human lung carcinoma (ATCC, Rockville, MD, USA).

Techniques: Laser-Scanning Microscopy, Staining, Fluorescence

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Flavonoid Fraction of Orange and Bergamot Juices Protect Human Lung Epithelial Cells from Hydrogen Peroxide-Induced Oxidative Stress

doi: 10.1155/2015/957031

Figure Lengend Snippet: Cytofluorimetric evaluation of lipid hydroperoxides. The A549 cells incubated for 18 h with FFBJ or FFOJ were oxidatively stressed with H 2 O 2 200 μ M for 2 h and then loaded by DPPP probe. The graph reports the mean of fluorescence expressed in arbitrary fluorescence units (AFU) of three independent experiments. Data represent the mean ± SEM of at least three separate experiments. ∗∗∗ P < 0.001 versus control cells and ## P < 0.01 versus H 2 O 2 -treated cells.

Article Snippet: The biological experiments were performed using a basal epithelial cell line A549 derived from human lung carcinoma (ATCC, Rockville, MD, USA).

Techniques: Incubation, Fluorescence, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Flavonoid Fraction of Orange and Bergamot Juices Protect Human Lung Epithelial Cells from Hydrogen Peroxide-Induced Oxidative Stress

doi: 10.1155/2015/957031

Figure Lengend Snippet: Pearson's correlation. Plot of DCF-DA versus DPPP emission values in A549 cells pretreated or not with FFBJ or FFOJ for 18 h and then incubated with H 2 O 2 200 μ M for additional 2 h. r = 0.96; P < 0.001.

Article Snippet: The biological experiments were performed using a basal epithelial cell line A549 derived from human lung carcinoma (ATCC, Rockville, MD, USA).

Techniques: Incubation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Flavonoid Fraction of Orange and Bergamot Juices Protect Human Lung Epithelial Cells from Hydrogen Peroxide-Induced Oxidative Stress

doi: 10.1155/2015/957031

Figure Lengend Snippet: CLSM analysis of mitochondrial membrane potential. A549 cells were grown on cell slides and after treatment with FFBJ or FFOJ were incubated with H 2 O 2 . Mitochondrial membrane potential was detected by R123 staining. Red fluorescence indicates functional mitochondria. Images captured at 400x magnification are shown as representative from three independent experiments.

Article Snippet: The biological experiments were performed using a basal epithelial cell line A549 derived from human lung carcinoma (ATCC, Rockville, MD, USA).

Techniques: Membrane, Incubation, Staining, Fluorescence, Functional Assay

Journal: Oncotarget

Article Title: Increased expression and copy number amplification of LINE-1 and SINE B1 retrotransposable elements in murine mammary carcinoma progression.

doi: 10.18632/oncotarget.1188

Figure Lengend Snippet: Figure 1: Immunohistochemical detection of ORF2p during murine mammary cancer progression. A. Structure of a full-length LINE-1 (L1) element. Arrows mark the position of oligonucleotide pairs used for q-PCR and the vertical arrowhead identifies the protein domain recognized by ORF2p- specific antibody. B. Immunohistochemical analysis of LINE- 1ORF2p in normal breast (a) and in tumor tissues explanted from transgenic mice at sequential stages of breast cancer development from stage 1 to 6 (b to g). High magnification panels (a’ to g’) depict the intracellular distribution of LINE-1 ORF2p. Arrows point to positive nuclei for LINE-1 ORF2p.

Article Snippet: Confocal laser scanning microscopy (CLSM) CLSM analyses of LINE-1 ORF2p were carried out on paraffin- embedded tissues by indirect immunofluorescence using rabbit polyclonal antibody to LINE-1 ORF2p (1:100 dilution, Santa Cruz Biotechnology).

Techniques: Immunohistochemical staining, Transgenic Assay

Journal: Oncotarget

Article Title: Increased expression and copy number amplification of LINE-1 and SINE B1 retrotransposable elements in murine mammary carcinoma progression.

doi: 10.18632/oncotarget.1188

Figure Lengend Snippet: Figure 2: Confocal microscopy of ORF2p intracellular distribution during murine mammary cancer progression. The top row panels depict ORF2p-specific IF, the bottom rows panels show merged images of LINE-1 ORF2p (red channel) and Hoechst- counterstained nuclei (blue channel). The arrows point to LINE-1 ORF2p positive nuclei and arrowheads to perinuclear accumulation of LINE-1 ORF2p. Panels in rows 3 and 4 show high magnification image details. Bars, 10 micrometers.

Article Snippet: Confocal laser scanning microscopy (CLSM) CLSM analyses of LINE-1 ORF2p were carried out on paraffin- embedded tissues by indirect immunofluorescence using rabbit polyclonal antibody to LINE-1 ORF2p (1:100 dilution, Santa Cruz Biotechnology).

Techniques: Confocal Microscopy

Journal: Oncotarget

Article Title: Increased expression and copy number amplification of LINE-1 and SINE B1 retrotransposable elements in murine mammary carcinoma progression.

doi: 10.18632/oncotarget.1188

Figure Lengend Snippet: Figure 3: RT activity in murine breast cancer tissues. A. Western blot analysis of ORF2p (upper panel) and alpha- tubulin (lower panel) in tissue extracts from normal breast (NB) and breast cancer (stages 1+2, 3+4, and 5+6 were pooled). B. RT activity functional assay after incubation of MS2 phage RNA with extracts from breast carcinoma tissues. Histograms represent the retrotranscribed cDNA yield from each reaction; means and SD values from three independent assays are expressed in arbitrary units.

Article Snippet: Confocal laser scanning microscopy (CLSM) CLSM analyses of LINE-1 ORF2p were carried out on paraffin- embedded tissues by indirect immunofluorescence using rabbit polyclonal antibody to LINE-1 ORF2p (1:100 dilution, Santa Cruz Biotechnology).

Techniques: Activity Assay, Western Blot, Functional Assay, Incubation

Journal: BMC Cancer

Article Title: Co-expression of S100A14 and S100A16 correlates with a poor prognosis in human breast cancer and promotes cancer cell invasion

doi: 10.1186/s12885-015-1059-6

Figure Lengend Snippet: Expression and localization of S100A14 and S100A16 in human breast cancer cell lines. A , Relative mRNA expression levels measured by real-time RT-PCR. Expression levels were normalized to β-actin levels within the same sample. B , Protein expression of S100A14 and S100A16. Expression levels and subcellular localization were visualized using immunofluorescence staining. Scale bar; 50 μm. C , Subcellular localization of the S100A14 protein on the cell membrane. Z-axis images of confluent MCF7 cells were constructed using confocal laser scanning microscopy. Scale bar; 10 μm. D , Effect of omission of cell permeabilization on the immunofluorescent staining of S100A14. Following fixation with 4% paraformaldehyde, the cells were treated with or without 0.1% Triton X-100 in PBS prior to staining. Scale bar; 50 μm. E , Ca 2 -independent localization of S100A14 on the cell membrane. MCF7 cells transfected with the S100A14-GFP expression vector were observed by fluorescence microscopy over 180 min after addition of 10 mM EGTA in PBS. Scale bar; 50 μm.

Article Snippet: The human breast cancer cell lines MCF7, ZR75-1, SK-BR-3 and MDA-MB-231 were purchased from the American Type Culture Collection.

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Membrane, Construct, Confocal Laser Scanning Microscopy, Transfection, Plasmid Preparation, Fluorescence, Microscopy

Journal: BMC Cancer

Article Title: Co-expression of S100A14 and S100A16 correlates with a poor prognosis in human breast cancer and promotes cancer cell invasion

doi: 10.1186/s12885-015-1059-6

Figure Lengend Snippet: Interaction of the S100A14 protein with actin and analyses of its functional interaction with S100A16. A , An extract of MCF7 cells was immunoprecipitated (IP) with control rabbit IgG, anti-S100A14 or anti-actin antibodies. Precipitated and co-precipitated proteins were detected by immunoblotting with anti-S100A14 and anti-actin antibodies. The following were added to the extracts prior to precipitation. CaCl2; 0 mM (lane 1), 0.5 mM (lane 2) or 2 mM (lane 3), or, 2 mM CaCl2 and 10 mM EDTA (lane 4). Arrows indicate actin and S100A14. B , Co-localization of S100A14 with polymerized actin and S100A16 in MCF7 cells. Co-immunostaining with the anti-S100A14 antibody and a FITC-labeled second antibody (green), and with rhodamine-conjugated phalloidin (red) showed colocalization of both S100A14 and actin along the cell membrane of MCF7 cells (top). Co-localization of S100A14 and S100A16 was also confirmed in MCF7 cells (bottom). Scale bars; 20 μm. C , Analysis of the mRNA expression of S100A14 and S100A16 in MCF7 cells transfected with siRNA for S100A14 or S100A16. Gene knockdown of S100A14 or S100A16 does not affect the mRNA expression level of the other S100 protein in MCF7 cells. The relative mRNA expression levels were normalized to the GAPDH mRNA expression level. Error bars, + SD, n=3. D , Effect of siRNA targeted against S100A14 or S100A16 on the expression of each protein. Western blots of extracts of MCF7 cells transfected with siRNA targeted against S100A14 or S100A16 were probed with antibodies against S100A14, S100A16 and b-actin. E, Expression of S100A14 or S100A16 protein following knockdown of the counterpart molecule. Immunofluorescence analysis of the protein expression and localization of S100A14 and S100A16 in MCF7 cells 48 h after transfection with siRNA targeted against each protein. Scale bars; 10 μm.

Article Snippet: The human breast cancer cell lines MCF7, ZR75-1, SK-BR-3 and MDA-MB-231 were purchased from the American Type Culture Collection.

Techniques: Functional Assay, Immunoprecipitation, Control, Western Blot, Immunostaining, Labeling, Membrane, Expressing, Transfection, Knockdown, Immunofluorescence

Journal: BMC Cancer

Article Title: Co-expression of S100A14 and S100A16 correlates with a poor prognosis in human breast cancer and promotes cancer cell invasion

doi: 10.1186/s12885-015-1059-6

Figure Lengend Snippet: The effects of S100A14 and S100A16 knockdown on cancer cell invasion and motility. A , In vitro invasion assay using a Matrigel-coated transmembrane. Invading MCF7 and SK-BR-7 cells were counted 24 h after seeding of knockdown cells (the error bar represent S.D., n=3). B , Wound healing assay using MCF7 cells transfected with siRNA for S100A14or S100A16. The wound width was measured, and the distance over which the cells had migrated 48 h after scratching was calculated (the error bars represent S.D., n=3). C , A dual-color wound healing assay using MCF7 cells that were stably transfected with GFP or Tomato expression vectors that were further transfected with siRNA for S100A14 or control, respectively. The white lines represent the migration fronts at 48 h after scratching of a mixed culture of cells on a confluent monolayer. Scale bars; 50 μm. D , Confocal images of representative spheroids of MCF7 cells grown in a collagen gel for 48 h after transfection of S100A14 or control siRNA. Scale bar; 50 μm.

Article Snippet: The human breast cancer cell lines MCF7, ZR75-1, SK-BR-3 and MDA-MB-231 were purchased from the American Type Culture Collection.

Techniques: Knockdown, In Vitro, Invasion Assay, Wound Healing Assay, Transfection, Stable Transfection, Expressing, Control, Migration

Journal: Oncoimmunology

Article Title: pH regulators to target the tumor immune microenvironment in human hepatocellular carcinoma

doi: 10.1080/2162402X.2018.1445452

Figure Lengend Snippet: CAIX and CAXII expression and functional inhibition in HCC cell lines. The indicated HCC cell lines were cultured under either normoxic (N = 21% O 2 ) or hypoxic (H = 1% O 2 ) conditions for 72 h and were analyzed as indicated below. (A) The protein expression of CAIX, CAXII and vinculin was assessed by western blotting in cell lysates of the C3A, PLC/PRF/5 and SNU-449 cell lines. The cell surface expression of CAIX (B) and CAXII (C) was evaluated by flow cytometry. The number in each histogram plot shows the percentage of cells positive for the indicated markers (filled histograms) evaluated with respect to the corresponding secondary antibody (black line). Representative histograms from three independent experiments are shown. (D) The cellular distribution of CAXII was assessed by confocal laser scanning microscopy under the indicated conditions. Representative micrographs of triple immunofluorescence staining with anti-CAXII (green), anti-WGA (red, detecting the cell membrane) and anti-calnexin (blue, detecting the ER compartment) are reported. Scale bars = 50 μm and = 5 μm for the lower and higher magnification respectively. The cell viability of the HCC cell lines treated with different doses of S4 (µM) (E) and compound 25 (µM) (F) under normoxia (N = 21% O 2 , black triangles) or hypoxia (H = 1% O 2 , white triangles) for 72 h was evaluated using the MTT assay. The data show the percentage of viable cells of the untreated control and represent the mean of six replicate reactions from 3 independent experiments. IC 50 values for S4 were >100 μM for all HCC cell lines grown under normoxic conditions and 57.4, 53.9 and >100 μM for C3 A, PLC/PRF/5 and SNU-449 exposed to hypoxia respectively. For compound 25, C3 A, PLC/PRF/5 and SNU-449 cells grown under normoxia displayed IC 50 values of 198.5, 142.9 and >200 μM respectively. All the HCC cell lines exposed to hypoxia displayed IC 50 values >200 μM for compound 25.

Article Snippet: The human HCC cell lines C3A, PLC/PRF/5 and SNU-449 and the breast cancer cell line T-47D (ATCC, Manassas, Virginia, USA) were cultured in complete culture medium (RPMI-1640, Lonza) supplemented with 10% heat-inactivated fetal calf serum (FCS, Lonza), 100 U/ml penicillin and 100 U/ml streptomycin under normoxia (N = 21% O 2 ) or hypoxia (H = 1% O 2 ) as indicated.

Techniques: Expressing, Functional Assay, Inhibition, Cell Culture, Western Blot, Flow Cytometry, Confocal Laser Scanning Microscopy, Immunofluorescence, Staining, Membrane, MTT Assay, Control

Journal: Oncoimmunology

Article Title: pH regulators to target the tumor immune microenvironment in human hepatocellular carcinoma

doi: 10.1080/2162402X.2018.1445452

Figure Lengend Snippet: Effects of omeprazole on HCC cell line viability and on the tumor microenvironment of HCC tissue explants. (A) The protein expression of a1, C1, β-actin and α-tubulin was assessed by western blotting in cell lysates of the C3A, PLC/PRF/5 and SNU-449 cell lines. (B) The HCC cell lines were treated with different doses of omeprazole, and cell viability was evaluated using the MTT assay. The data on the y-axes report the percentage of viable cells of the untreated control and represent the mean of six replicate reactions from 3 independent experiments. The calculated IC 50 values for omeprazole are 100.9, 39.4, and 128.4 μg/ml for C3A, PLC/PRF/5 and SNU-449 respectively. (C) Expression levels of the indicated genes in HCC tissue explants ( MYC, VIM, CDH1, CCL22, IFNG N = 12; TNF , N = 8) cultured ex vivo in the presence of 100 μg/ml omeprazole or the vehicle of the drug for 24 h. The gene expression data are reported as 2 −ΔCt values. The p-values are calculated by the paired t test comparing cells treated with omeprazole or the vehicle.

Article Snippet: The human HCC cell lines C3A, PLC/PRF/5 and SNU-449 and the breast cancer cell line T-47D (ATCC, Manassas, Virginia, USA) were cultured in complete culture medium (RPMI-1640, Lonza) supplemented with 10% heat-inactivated fetal calf serum (FCS, Lonza), 100 U/ml penicillin and 100 U/ml streptomycin under normoxia (N = 21% O 2 ) or hypoxia (H = 1% O 2 ) as indicated.

Techniques: Expressing, Western Blot, MTT Assay, Control, Cell Culture, Ex Vivo, Gene Expression