Journal: BMC cancer

Article Title: Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer.

doi: 10.1186/1471-2407-13-518

Figure Lengend Snippet: Figure 5 EGF receptor is autophosphorylated in Caco-2 and activates downstream signalling pathways. Caco-2 cells were stimulated with 20 ng/mL EGF for the time periods indicated. Western blotting for (a) phosphorylated EGFR or total EGFR and (b) antibodies recognising signal- ling enzymes is shown. Cell lysate of EGF-treated A431 cells was used as positive control. α-tubulin is shown as a loading control. Densitometry was performed using Phoretix 1D analysis software against α-tubulin (for ERK, data for p42 and p44 are shown).

Article Snippet: Whole cell lysate of EGF-treated A431 epithelial carcinoma cells used as positive control was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Positive Control, Control, Software

Journal: Bioconjugate chemistry

Article Title: Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells.

doi: 10.1021/acs.bioconjchem.5b00613

Figure Lengend Snippet: Fig. 6. Detection of proteins in cell lysates. A panel of 24-plex antibody binders was used to detect their respective target proteins in MCF7, K562, A549, and A431 cell lysates. The cell lysates were diluted to different concentrations and reacted with a mixture of antibody binders. Only selected examples of protein targets CSTB (A), CASP3 (B), Ki-67(C), and GATA3 (D) are shown. Ct values were shown as the means ± SD. Ct: cycle threshold.

Article Snippet: MCF7, K562, A549, and A431 cell lysates were obtained from Novus (Littleton, CO).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Oxidized Mitochondrial DNA Engages TLR9 to Activate the NLRP3 Inflammasome in Myelodysplastic Syndromes

doi: 10.3390/ijms24043896

Figure Lengend Snippet: Ox-mtDNA activates pyroptosis. ( A ) Log10-transformed, glucose-adjusted ox-mtDNA levels from Low Risk (LR) MDS PB (n = 100) and BM plasma (n = 70), and Normal PB (n = 30). ( B ) Western blot of U937 and SKM1 cells treated with 10-fold increasing isolated mtDNA of the ND1 gene region amplified with oxidized guanosine (ox-mtDNA) for 2 h to induce Caspase-1 (arrow depicts cleaved fragment) and phosphorylated NFκB to establish dosage (representative blot of n = 3). Remaining figures, ox-mtDNA treatment is 50 ng/mL ox-mtDNA for 2 h unless otherwise stated. ( C ) Western blot of SKM1 and U937 cells treated with ox-mtDNA showing, cleavage of caspase-1 (arrow depicts cleaved fragment), and IL-1β (arrow depicts cleaved fragment) demonstrating inflammasome activation (representative blot of n = 3). ( D ) Fold change of caspase-1 activity quantified by Caspase-1 Glo ® assay in SKM1 and U937 cells treated with ox-mtDNA, (mean ± SEM of n = 5). ( E ) Fold change of LDH media release (measurement of cell death), quantified by LDH-Glo™ Cytotoxicity Assay, of SKM1 and U937 cells treated with ox-mtDNA (mean ± SEM of n = 5). ( F ) Fold change of caspase-1 activity quantified by Caspase-1 Glo ® assay in SKM1 cells pretreated with either CRISPR KO (pooled guides) for NLRP3 or 10 uM MCC950 for 48 h prior to treatment with ox-mtDNA (mean ± SEM of n = 4). ( G ) Representative confocal IF micrographs showing increased ASC specks in ox-mtDNA stimulated cells compared to untreated controls [DAPI (blue), ASC (green) (×2520)]. ( H ) Quantification of 1-to-2 μm ASC speck IF, at least 200 cells counted per group, (mean ± SEM of n = 3). ( I ) Immunoblot of ASC following chemical crosslinking, cells treated with ox-mtDNA or positive control LAN (LPS + ATP + Nigericin), arrow indicates oligomers. ( J ) Western Blot for PARP and Caspase-3. Western blot of SKM1 and U937 cells treated with ox-mtDNA, and an apoptosis positive control (A431 EGF Stimulated) showing induction of PARP1, cleavage of caspase-3 (arrow depicts cleaved fragments, representative blots of n = 3). ( K ) Fold change of caspase-1 activity quantified by Caspase-1 Glo ® assay and LDH media release quantified by LDH-Glo™ Cytotoxicity Assay in primary Normal BM-MNC cells treated with ox-mtDNA (mean ± SEM of n = 3). ( L ) Colony formation assay for hematopoiesis in Healthy BM-MNCs treated with 50 ng/mL ox-mtDNA for 14 days (representative picture). ( M ) Quantification of various hematopoietic progenitor colonies in response to treatment with 50 ng/mL of ox-mtDNA for 14 days (mean ± SEM of n = 4). In this figure, significance was assessed by either paired t -test (comparison between two groups only) or ordinary one-way ANOVA with multiple comparison analysis in GraphPad Prism. p values are shown as asterisk: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: Apoptosis positive control was A431 Whole Cell Lysate EGF Stimulated (Rockland Immunochemicals, Inc., Limerick, PA, USA).

Techniques: Transformation Assay, Clinical Proteomics, Western Blot, Isolation, Amplification, Activation Assay, Activity Assay, Glo Assay, Cytotoxicity Assay, CRISPR, Positive Control, Colony Assay, Comparison

Journal: Hepatology (Baltimore, Md.)

Article Title: Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats.

doi: 10.1053/jhep.2002.31355

Figure Lengend Snippet: Fig. 3. Group phenotyping of LPC strains and clonal lines cultured on STO feeder layers. Cellular extracts from 22 sources were made 3 days postplating and analyzed (lanes 4-25) for AFP, albumin, CK14, c-kit, CD34, and desmin by Western blotting. The names of individual cell strains and cell lines are shown above each lane; the larger single numbers above the names designate group assignments (see Table 2). Extracts used as internal controls were obtained from neonatal liver (BRL) and, as indicated, from bone marrow and skeletal muscle (lane 1), STO fibroblasts (lane 2), freshly isolated adult rat hepatocytes and, as annotated, human A431 cells (lane 3). The 6 positive control markers included AFP, albumin, CK14, and c-kit (BRL), CD34 (bone marrow), and desmin (skeletal muscle) in lane 1; and CK14 (A431 cells) and albumin (adult hepatocytes) in lane 3. Negative controls were provided by STO cells for all markers (lane 2), by adult rat hepatocytes for AFP and c-kit (lane 3), and for CK14 (not shown). Molecular weights of the 6 markers are listed to the right of lane 25.

Article Snippet: The following materials were obtained commercially: dexamethasone and bFGF (Sigma); Oncostatin M (R&D Systems, Minneapolis, MN); Matrigel (Collaborative Research, Bedford, MA); human epidermoid carcinoma cell line A431 lysates (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Cell Culture, Western Blot, Isolation, Positive Control

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: Molecular Medicine Reports

Article Title: AQP3 small interfering RNA and PLD2 small interfering RNA inhibit the proliferation and promote the apoptosis of squamous cell carcinoma

doi: 10.3892/mmr.2017.6847

Figure Lengend Snippet: Inhibitory effects of AQP3 and PLD2 siRNA sequences on the target gene. siRNA transfection and reverse transcription-quantitative polymerase chain reaction analyses were performed. The groups were as follows: CK group, normal cultured A431 cells; NC group, A431 cells were transfected with NC siRNA; 1, AQP3-homo-612 siRNA group; 2, AQP3-homo-363 siRNA group; 3, AQP3-homo-360 group; 4, PLD2-homo-602 group; 5, PLD2-homo-1352 group; 6, PLD2-homo-5338 group. siRNA, small interfering RNA; AQP3, aquaporin 3; PLD2, phospholipase D2; CK, control check; NC, negative control.

Article Snippet: The human A431 SCC cell line was purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Culture, Small Interfering RNA, Control, Negative Control

Journal: Molecular Medicine Reports

Article Title: AQP3 small interfering RNA and PLD2 small interfering RNA inhibit the proliferation and promote the apoptosis of squamous cell carcinoma

doi: 10.3892/mmr.2017.6847

Figure Lengend Snippet: Inhibitory effect of siRNA transfection on the proliferation of A431 cells. Cell proliferation was analyzed and the results are representative of three independent experiments. The statistical significance between two groups was determined using a paired-samples t-test. **P<0.01, compared with the NC group. siRNA, small interfering RNA; AQP3, aquaporin 3; PLD2, phospholipase D2; CK, control check; NC, negative control.

Article Snippet: The human A431 SCC cell line was purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Transfection, Small Interfering RNA, Control, Negative Control

Journal: Molecular Medicine Reports

Article Title: AQP3 small interfering RNA and PLD2 small interfering RNA inhibit the proliferation and promote the apoptosis of squamous cell carcinoma

doi: 10.3892/mmr.2017.6847

Figure Lengend Snippet: AQP3 siRNA and PLD2 siRNA increase the apoptosis of A431 cells. Cell apoptosis was determined; results are representative of three independent experiments. The statistical significance between two groups was determined using a paired-samples t-test. **P<0.01, compared with the NC group. siRNA, small interfering RNA; AQP3, aquaporin 3; PLD2, phospholipase D2; CK, control check; NC, negative control.

Article Snippet: The human A431 SCC cell line was purchased from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Small Interfering RNA, Control, Negative Control