Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: Responses of EGFR-driven cancer cells to EGFR inhibitors. A–C, A431 cells harboring genomic amplification of the EGFR gene were grown in monolayer cultures and exposed for 24 h to the indicated irreversible EGFR kinase inhibitors (EKIs: CI-1033 and PF-00299804) or neutralizing anti-EGFR antibody (Cetuximab) at increasing concentrations. The cells were tested for metabolic activity using the MTS assay. EKIs, but not Cetuximab, triggered marked and dose-dependent reduction in metabolic activity. D, effects of drug treatment on EGFR phosphorylation (P-EGFR; Western blotting); numerical values represent mean ± S.D. of several independent experiments; *, p > 0.05.

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Amplification, Activity Assay, MTS Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: Marked increase in the EV-mediated EGFR emission from EKI-treated cancer cells. A, unchanged levels of EGFR protein in lysates of A431 cells cultured for 24 h in the presence of control medium (control), CI-1033 (5 μm), PF-00299804 (5 μm), or Cetuximab (Cetux., 50 μg/ml), as measured by ELISA. B, unchanged levels of EGFR protein in lysates of A431 cells cultured in the presence of CI-1033 (5 μm), TGFα (50 ng/ml), or both (EGFR ELISA). C, dramatic increase in EGFR signal in the EV (P4) fraction of the conditioned medium corresponding to CI-1033 and PF-00299804 treatment; pooled data were from two independent experiments. D, increase in EV-mediated emission of EGFR upon treatment with CI-1033 and TGFα/CI-1033. The combined treatment highlights the ability of EGFR kinase inhibitor to trigger EV release; p values were as indicated. The results were independently reproduced using an antibody array (data not shown). NS, not significant.

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Ab Array

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: Differential EGFR phosphorylation profiles of cancer cells and their EVs in the presence or absence of PF-00299804 treatment. A431 cells and corresponding EV preparations were subjected to Western blotting for EGFR and for three different EGFR phosphosites (Tyr-845, Tyr-1068, and Tyr-1173). EVs differ in their EGFR phosphorylation profiles from their parental cells, including preponderance of 2 out of 3 phosphoisoforms of EGFR following drug treatment.

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: Differential phosphorylation profiles of EGFR, AKT, and ERK in cancer cells and their EVs following treatment with pan-Erb kinase inhibitor CI-1033. A431 cells were treated with the EGFR agonist (TGFα, 50 ng/ml), irreversible ErbB kinase inhibitor (CI-1033, 5 μm), or both. The cells and EVs were collected 24 h later and immunoblotted for total EGFR, three different EGFR phosphosites (P-EGFR Tyr-845, Tyr-1068, and Tyr-1173), AKT, P-AKT (Ser-473), ERK, and P-ERK (Thr-202/Tyr-Y204). As loading controls, β-actin and Ponceau red were used for cell and EV lysates, respectively. EVs differ in their EGFR phosphorylation profile from their parental cells. P-EGFR is less abundant in EVs than in corresponding cells. EVs accumulate Tyr-845 and Tyr-1068 P-EGFR isoforms, but not Tyr-1173, following treatment with CI-1033. EV-associated Tyr-1173 is increased following stimulation with TGFα. EVs contain no detectable P-AKT but are enriched for ERK and P-ERK following CI-1033 treatment (see text for details).

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: Pharmacological blockade of the oncogenic EGFR triggers emission of EVs with exosomal characteristics. A431-CD63/GFP cells were cultured for 24 h in the presence of control media or EGFR blocking concentrations of CI-1033 (5 μm). A, EVs were collected by ultracentrifugation at either 2500 × g (EV-P2) or 110,000 × g (EV-P4) and immunoblotted for EGFR or GFP, the latter to reveal the exogenous GFP/CD63 chimeric protein marker of exosomes. P4 fraction containing exosome-sized EVs was enriched in EGFR and GFP, especially after the CI-1033 treatment. B and C, A431-derived EVs were floated on the sucrose gradient, and the respective fractions were profiled for size and numerical EV distribution using nanoparticle tracking analysis system (NTA, Nanosight). Of note is the fact that CI-1033 treatment stimulated production of mainly small EVs (exosome-like), with sizes ranging between 51 and 150 nm and density between 1.11 and 1.21 g/ml, which corresponded to sucrose density fractions 3–8. NTA of individual fractions containing cancer cell-derived EVs suggests a highly heterogeneous size distribution. D and E, exosomal fractions (1.12–1.17 g/ml) of EVs purified from conditioned medium of control A431-CD63/GFP cells contain both EGFR and GFP (CD63) markers (immunoblotting). CI-1033 treatment triggered the increase in the EGFR and exosomal marker content (GFP-CD63 and CD9) across exosomal fractions 1.10–1.21 g/ml of A431-GFP/CD63-derived EVs. Data are representative of three independent experiments (see text for details).

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Cell Culture, Blocking Assay, Marker, Derivative Assay, Purification, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: EKI-induced cellular vesiculation depends on the neutral sphingomyelinase pathway of exosomal biogenesis. A, NTA analysis of EVs emitted from A431 cells treated with EKI (CI-1033 (CI)), inhibitor of neutral sphingomyelinase (GW4869), inhibitor of acidic sphingomyelinase (FTY720), or their indicated combinations. Of note is a shift to the right in the median size peak of EVs in presence of GW4869 indicative of reduced numbers of exosome-seized vesicles and the preponderance of larger EV sizes. B, EV output into the A431 conditioned medium as measured within the median exosome size (range 150–200 nm) of vesicles. CI-1033-induced increase in EV numbers is attenuated by GW4869 pretreatment but not by FTY720 pretreatment, which increases the EV production; compilation is of three independent experiments, and p value is as indicated. NS, nonsignificant.

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: EKI-triggered vesiculation involves caspase activity. A, activation of caspase 3 in A431 cells treated with CI-1033 and etoposide. Floating (P1) cells reveal the cleaved caspase 3 (lower) band. Cell cultures were treated with growth inhibitory (5 μm) and subthreshold (1 μm) concentrations of CI-1033, and with pro-apoptotic concentrations of etoposide. In all cases caspase 3 cleavage was inhibited by ZVAD peptide. No cleaved caspase was detectable in adherent cells. Caspase 3 was undetectable in microvesicle-like (P2) and exosome-like (P4) EV fractions. B–E, NTA profile reveals that growth inhibitory dosing of CI-1033 (5 μm), but not of etoposide, evoked vesicular emissions from A431 cells, despite caspase 3 activation in both cases. CI-1033-induced EV release was selectively inhibited by ZVAD pretreatment, as demonstrated by NTA of total (C), and exosome-like (150–200 nm (D)) vesicle populations, but this effect was not observed in larger microvesicle-like EV subsets (350–400 nm; E); data are representative of three independent experiments, p value is as indicated. NS, nonsignificant.

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Activity Assay, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: EKI treatment leads to cellular emission of genomic DNA within exosome-like fraction of EVs. A, A431 cells were cultured and treated with CI-1033 (5 μm), and sucrose fractions of their EVs were tested for extracellular genomic DNA using QC Analyzer (A) or PCR (B) assays. EV fractions are as follows: 3 (below exosomal density), 6 (exosomal like), and 9 (above exosomal density) were analyzed for DNA content. DNA was detected (arrowhead) only in fractions 6 and 9, and upon treatment with CI-1033. B, PCR amplification of the EGFR (exon 18) genomic sequence in A431-derived EV fractions obtained with and without CI-1033 treatment. These profiles suggest the emission of DNA within exosome-like fractions of EVs following the EGFR blockade. EVs at a higher density contain gDNA regardless of treatment (see text for details).

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Cell Culture, Amplification, Sequencing, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Oncogenic Epidermal Growth Factor Receptor Kinase Triggers Release of Exosome-like Extracellular Vesicles and Impacts Their Phosphoprotein and DNA Content *

doi: 10.1074/jbc.M115.679217

Figure Lengend Snippet: EV-mediated extracellular co-emission of EGFR and DNA following cancer cell exposure to EKIs. A, experimental design: ImmunoCapture of pre-filtered, EGFR-containing EVs produced by A431 cells either in the absence of in the presence of EKIs, followed by detection of extracellular genomic DNA (exo-gDNA; PCR). B, analysis of EGFR genomic sequences in EV fractions captured on plates coated with anti-EGFR antibody. This assay reveals that the same population of small EGFR-positive exosome-like EVs, which are able to pass through both 0.8- and 0.45-μm filters, is enriched in exo-gDNA, but only if the cells were pretreated with growth inhibitory doses of EKIs, CI-1033 or PF-00299804 (see text for details). UC, ultracentrifugation; -, possible but not obligatory step in the procedure.

Article Snippet: The clonal A431-CD63/GFP cell line was prepared by transfecting A431 parental cells with the plasmid pCMV6-AC-GFP (OriGene, Rockville, MD), clone RG 201733, encoding the CD63-GFP fusion protein.

Techniques: Produced, Genomic Sequencing

Journal: NPJ Breast Cancer

Article Title: NDRG4 promoter hypermethylation is a mechanistic biomarker associated with metastatic progression in breast cancer patients

doi: 10.1038/s41523-019-0106-x

Figure Lengend Snippet: NDRG4 depletion in breast tumor cells increases lymph node adhesion and cell migration toward VN. a NDRG4 expression meta-analysis in human breast cancer cell lines using GOBO application . Breast cancer subtypes can be identified by different colors: red = Basal A, gray = Basal B and blue = Luminal cells. b Analysis of NDRG4 mRNA expression levels in MCF-7 cells transfected with NDRG4-shRNAs or scramble control (shSCR). Expression levels are relative to wild type cells and normalized to hydroxymethylbilane synthase (HMBS) gene. Error bars represent SEM of biological replicates ( n = 5). *** p < 0.001, ns = not significant, by one-way ANOVA. c Western blot analysis of NDRG4 expression in cytoplasmic extracts of MCF-7 shNDRG4s or shSCRs cells, using antibodies against NDRG4 and Actin. MCF-7 cells express all the three isoforms of NDRG4: 37 kDa (NDRG4-B), 39 kDa (NDRG4-B var ) and 41 kDa isoform (NDRG4-H). For simplicity, results obtained for two independent shNDRG4 clones were grouped and presented as the shNDRG4 group. ( d , left) Representative images of adherent red fluorescent MCF-7 cells on frozen rat lymph node sections. d , e Quantification of adherent MCF7 ( d ) and T47D ( e ) breast tumor cells in lymph nodes sections (right, n = 4 independent experiments for MCF-7 and 3 for T47D, ** p < 0.01 by two-tailed t test). f Stationary adhesion assays showed that NDRG4 knockdown promotes ‘adhesive switch’ between FN and VN. Error bars represent SEM of biological replicates ( n = 4). Cells do not adhere to albumin control (BSA, data not shown). * p < 0.01, ** p < 0.01, ns = not significant by two-tailed t tests. g , h Haptotactic cell migration toward VN was analyzed by transwell migration assay in MCF-7 ( g ) and T47D ( h ) cell lines. Error bars represent SEM of biological replicates ( n = 4). *** p < 0.001 by two-tailed t tests

Article Snippet: For stable transfection of NDRG4 shRNA into MCF-7 and T47D breast tumor cells, the pGFP-V-RS vector containing shRNA inserts which targeted the 3′-unttranslated region of NDRG4 (TRCN0000134583 and TRCN0000137216) was purchased (Origene).

Techniques: Migration, Expressing, Transfection, Western Blot, Clone Assay, Two Tailed Test, Transwell Migration Assay

Journal: NPJ Breast Cancer

Article Title: NDRG4 promoter hypermethylation is a mechanistic biomarker associated with metastatic progression in breast cancer patients

doi: 10.1038/s41523-019-0106-x

Figure Lengend Snippet: NDRG4 knockdown promotes clustering of β1-integrin at the leading edge of T47D cells. Representative confocal images of β1 integrin subunit (MAB1965, green) at the ventral cell surface of VN-adherent T47D shNDRG4 or shSCR cells

Article Snippet: For stable transfection of NDRG4 shRNA into MCF-7 and T47D breast tumor cells, the pGFP-V-RS vector containing shRNA inserts which targeted the 3′-unttranslated region of NDRG4 (TRCN0000134583 and TRCN0000137216) was purchased (Origene).

Techniques:

Journal: Oncotarget

Article Title: The metastasis suppressor CD82/KAI1 inhibits fibronectin adhesion-induced epithelial-to-mesenchymal transition in prostate cancer cells by repressing the associated integrin signaling

doi: 10.18632/oncotarget.13767

Figure Lengend Snippet: A. Human prostate epithelial (PZ-HPV-7) and cancer (DU145, LNCaP, and PC3) cell lines and stably CD82-transfected DU145 and antisense CD82 fragment-transfected LNCaP cell clones were assessed for CD82 expression levels through immunoblotting analysis. B. Stably CD82-transfected DU145 and antisense CD82 fragment-transfected LNCaP cell clones grown on fibronectin (FN) were viewed under a phase-contrast microscope. Scale bar, 10 μm. C. The cells were seeded onto plates precoated with poly-L(+)-lysine (p-Lys) or FN and cultured for the indicated time periods. Expression of E-cadherin and mesenchymal marker proteins was examined through immunoblotting analysis using antibodies specific to each protein.

Article Snippet: PZ-HPV-7 (ATCC), a human prostate epithelial cell line transformed by human papillomavirus-18, was cultured in K-SFM medium (Invitrogen, Carlsbad, CA) supplemented with 0.05 mg/ml bovine pituitary extract and 5 ng/ml EGF.

Techniques: Stable Transfection, Transfection, Clone Assay, Expressing, Western Blot, Microscopy, Cell Culture, Marker

Journal: Oncotarget

Article Title: The metastasis suppressor CD82/KAI1 inhibits fibronectin adhesion-induced epithelial-to-mesenchymal transition in prostate cancer cells by repressing the associated integrin signaling

doi: 10.18632/oncotarget.13767

Figure Lengend Snippet: A. PZ-HPV-7 prostate epithelial cells were lysed with Brij 97 detergent, and immunoprecipitation (IP) was performed with normal mouse IgG or anti-CD82 antibody. The immunoprecipitates were analyzed by immnublotting using anti-integrin β 1 , α 3 , α 5 , or α 6 antibody. B. CD82 mutant cDNA, which encodes CD82 with a large extracellular loop (LEL) substituted with that of TM4SF2 as illustrated, was generated by PCR and subcloned into the pAdEasy-1 adenoviral vector to produce recombinant adenovirus. C. CD82-deficient PC3 prostate cancer cells grown on fibronectin (FN) were infected with adenovirus containing a wild-type (wt) or mutant (mt) CD82 expression construct, and Brij 97 detergent lysates were subjected to immunoprecipitation with an anti-β 1 integrin antibody followed by immunoblotting analysis using antibodies that recognize the C-terminus or LEL of CD82 and the LEL of TM4SF2. D. PC3 cells grown on poly-L(+)-lysine (p-Lys) or FN were infected with adenovirus containing a wt- or mt-CD82 expression construct and then assessed for the protein levels of E-cadherin and Snail. E. PC3 cells grown on FN were infected with wt-CD82 construct-containing adenovirus either alone or together with mt-CD82 construct-containing adenovirus and examined for E-cadherin and Snail expression. Numbers in parentheses represent the MOI values of adenovirus.

Article Snippet: PZ-HPV-7 (ATCC), a human prostate epithelial cell line transformed by human papillomavirus-18, was cultured in K-SFM medium (Invitrogen, Carlsbad, CA) supplemented with 0.05 mg/ml bovine pituitary extract and 5 ng/ml EGF.

Techniques: Immunoprecipitation, Mutagenesis, Generated, Plasmid Preparation, Recombinant, Infection, Expressing, Construct, Western Blot

Journal: Translational Oncology

Article Title: Anticancer Activity of a Broccoli Derivative, Sulforaphane, in Barrett Adenocarcinoma: Potential Use in Chemoprevention and as Adjuvant in Chemotherapy 1

doi:

Figure Lengend Snippet: Effect of SFN on BEAC cell survival. BEAC cells were cultured in the medium containing no SFN or various concentrations of SFN. Cells were harvested at different time points as indicated and proliferative potential was assessed by trypan blue exclusion and/or proliferation assay, based on the production of a yellow product (formazan) after reduction of a highly water-soluble tetrazolium salt by dehydrogenases in viable cells. The growth curves show the mean of three independent experiments, with SEM. (A) Barrett adenocarcinoma (FLO-1) cells treated with various concentrations of SFN. (B) BEAC (OE33) cells treated with various concentrations of SFN. (C) Photomicrograph of BEAC (FLO-1 and OE33) cells treated with 3 µM SFN for 72 hours. (D) Photomicrograph of normal diploid fibroblasts and primary normal esophageal epithelial cells (ScienCell Research Laboratories) treated with 3 µM SFN for 72 hours. (E) FLO-1 cells were treated with SFN for 48 hours, detached floating cells from the medium and the attached cells (by trypsinization) were collected separately and evaluated for number and viability using trypan blue exclusion. The number of cells detached after treatment with various concentrations of SFN is expressed as percent of untreated FLO-1 cells. “Total” represents the total number of detached cells whereas “Dead” reflects the fraction of dead cells in detached cell population. (F) Panel (I): FLO-1 cells were incubated with various concentrations of SFN for 48 hours, and the expression of caspase 8 was detected by Western blot analysis, using anti-caspase 8 mouse monoclonal antibody (Cell Signaling, Danvers, MA). Panel (II): Bar graph showing caspase 8 expression relative to β-actin.

Article Snippet: Normal primary human esophageal epithelial cells (HEEC) were purchased from ScienCell Research Laboratories (Carlsbad, CA) and have been described previously [ 30 ].

Techniques: Cell Culture, Proliferation Assay, Incubation, Expressing, Western Blot

Journal: Oncology Letters

Article Title: Anticancer effect of arsenite on cell migration, cell cycle and apoptosis in human pancreatic cancer cells

doi: 10.3892/ol.2016.4564

Figure Lengend Snippet: Effects of PDGF-BB on the migration of AsPC-1 and BxPC-3 pancreatic cancer cells, compared with normal PE cells. (A) AsPC-1, (B) BxPC-3 and (C) PE cells (5×104 cells/well) were treated with the indicated doses of arsenite for 24 h, prior to be seeded in the upper Boyden chamber. Following incubation for 16 h, cells were exposed to 30 ng/ml PDGF-BB for 36 h at 37°C. Cells were then fixed, stained and visualized under a microscope. The average number of migrated cells from five randomly selected fields on the lower surface of the membrane was counted. Data were obtained from ≥3 independent experiments. *P<0.05 vs. controls. Right panels show representative images of the migrated cells stained with clonogenic reagent. PDGF, platelet-derived growth factor; PE, pancreatic epithelial.

Article Snippet: Primary normal human pancreatic epithelial (PE) cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan) and maintained in CSC medium (Cell Systems Corporation, Kirkland, WA, USA).

Techniques: Migration, Incubation, Staining, Microscopy, Membrane, Derivative Assay

Journal: Oncology Letters

Article Title: Anticancer effect of arsenite on cell migration, cell cycle and apoptosis in human pancreatic cancer cells

doi: 10.3892/ol.2016.4564

Figure Lengend Snippet: Effects of arsenite on PARP cleavage in AsPC-1, BxPC-3 and PE cells. (A) AsPC-1, (B) BxPC-3 and (C) PE cells were exposed to arsenite (30 µM) for the indicated time periods. Protein extracts were then harvested and examined by western blotting using anti-PARP and anti-glyceraldehyde-3-phosphate dehydrogenase antibodies. Background-subtracted signal intensity of each protein band was normalized to GAPDH. Data are presented as the mean ± standard deviation of triplicate assay. *P<0.05. PARP, poly(adenosine diphosphate-ribose) polymerase; PE, pancreatic epithelial; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Primary normal human pancreatic epithelial (PE) cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan) and maintained in CSC medium (Cell Systems Corporation, Kirkland, WA, USA).

Techniques: Western Blot, Standard Deviation

Journal: Oncology Letters

Article Title: Anticancer effect of arsenite on cell migration, cell cycle and apoptosis in human pancreatic cancer cells

doi: 10.3892/ol.2016.4564

Figure Lengend Snippet: Effects of arsenite on the phosphorylation of p44/p42 MAPK and Akt in AsPC-1, BxPC-3 and PE cells. (A) AsPC-1, (B) BxPC-3 and (C) PE cells were exposed to arsenite (30 µM) and incubated with 30 ng/ml platelet-derived growth factor-BB for the indicated time periods. Protein extracts were then harvested and examined by western blotting using specific antibodies against phospho-p44/p42 MAPK, p44/p42 MAPK, phospho-Akt and Akt. Background-subtracted signal intensity of each protein band was normalized to Akt. Data are presented as the mean ± standard deviation of triplicate assay. *P<0.05 vs. cells without arsenite exposure. PE, pancreatic epithelial; PDGF, platelet-derived growth factor; phospho, phosphorylated; MAPK, mitogen-activated protein kinase; M, marker.

Article Snippet: Primary normal human pancreatic epithelial (PE) cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan) and maintained in CSC medium (Cell Systems Corporation, Kirkland, WA, USA).

Techniques: Phospho-proteomics, Incubation, Derivative Assay, Western Blot, Standard Deviation, Marker

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Long Non-coding RNA HOXA11 Antisense Promotes Cell Proliferation and Invasion and Predicts Patient Prognosis in Serous Ovarian Cancer

doi: 10.4143/crt.2016.263

Figure Lengend Snippet: Knockdown of HOXA11 antisense ( HOXA11as ) inhibits serous ovarian cancer cell proliferation. (A) Expression of HOXA11as in human ovarian surface epithelial cell line (HOSE) and six ovarian cancer cell lines determined by quantitative real time polymerase chain reaction (qRT-PCR). (B) Knockdown efficiency was determined by qRT-PCR analysis in OVCA429 and SKOV3 cells. Cells were transfected with HOXA11as -specific siRNA (siHOXA11as) and negative control siRNA (siNC). (C, D) Knockdown of HOXA11as significantly reduced cell proliferation in OVCA429 and SKOV3 cells as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Bars indicate mean±standard deviation of three independent experiments performed in triplicate. * p < 0.05 vs. siNC. siHOXA11as, HOXA11as -specific siRNA.

Article Snippet: Normal human ovarian surface epithelial (HOSE) cells were purchased from ScienCell Research Laboratories (San Diego, CA).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Negative Control, MTT Assay, Standard Deviation