Journal: Gastroenterology

Article Title: Neurotensin signaling activates microRNAs-21 and -155 and Akt, promotes tumor growth in mice, and is increased in human colon tumors.

doi: 10.1053/j.gastro.2011.07.038

Figure Lengend Snippet: Figure 4. NT regulates miR-21 and miR-155 signaling pathways in colon cancer cells. (A) Luciferase activity of the PTEN and SOCS1 3=UTRs after NT treatment (100 nmol/L) for 24 hours in HCT116 and DLD1 cells (untreated, as-miR-NC–treated, as-miR-21–treated, or as-miR-155–treated). (B) PTEN and SOCS1 mRNA levels, assessed by real-time PCR, in NT-treated (100 nmol/L, 6 h) HCT116 and DLD1 cells (untreated, as-miR-NC– treated, as-miR-21–treated, or as-miR-155–treated). (C) PTEN, SOCS1, and -actin protein levels, assessed by Western blot, in NT-treated (100 nmol/L, 6 h) HCT116 and DLD1 cells. (D) AKT phosphorylation (S473) levels, assessed by enzyme-linked immunosorbent assay, in HCT116 and DLD1 cells transfected with as-miR-NC (100 nmol/L), as-miR-21 (100 nmol/L), and/or as-miR-155 (100 nmol/L) for 24 hours and treated with NT (100 nmol/L) for 24 hours.

Article Snippet: AKT phosphorylation (S473) (DuoSet IC nzyme Linked Immunosorbent Assay, DYC887; R&D Systems, inneapolis, MN), phospho-GSK3 (S9) (DYC-1590; R&D Sysems), and phospho-p65(S536) (7834; Cell Signaling) were asessed by enzyme linked immunosorbent assay in cells transected with as-miR-21 (100 nmol/L) and/or as-miR-155 (100 mol/L) for 24 hours and treated with NT (100 nmol/L) for 24 ours.

Techniques: Protein-Protein interactions, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Gastroenterology

Article Title: Neurotensin signaling activates microRNAs-21 and -155 and Akt, promotes tumor growth in mice, and is increased in human colon tumors.

doi: 10.1053/j.gastro.2011.07.038

Figure Lengend Snippet: Figure 5. NT activates AKT through suppression of PPP2CA by direct interaction with miR-155. (A) miR-155 binding sites in 3=UTRs of PPP2CA predicted by Lever algorithm analysis. (B) Luciferase activity of the PPP2CA 3=UTRs in HCT116 and DLD1 cells transfected with as-miR-155 (100 nmol/L) or as-miR-155 (100 nmol/L) for 24 hours. (C) Luciferase activity of the PPP2CA 3=UTRs in HCT116 and DLD1 cells treated with NT (100 nmol/L). (D) PPP2CA and -actin protein levels, assessed by Western blot, in NT-treated (100 nmol/L, 6 h) HCT116 and DLD1 cells. (E) AKT phosphorylation (S473) levels, assessed by enzyme-linked immunosorbent assay, in HCT116 and DLD1 cells transfected with miR-155 (100 nmol/L) or si-PPP2CA (100 nmol/L) for 24 hours. (F) NF-B/p65 activity, assessed by enzyme-linked immunosorbent assay, in HCT cells transfected with NT (100 nmol/L) and as-miR-NC (100 nmol/L), as-miR-21 (100 nmol/L), as-miR-155 (100 nmol/L), or with 10 nmol/L of an AKT pharmacologic inhibitor (MK-2206).

Article Snippet: AKT phosphorylation (S473) (DuoSet IC nzyme Linked Immunosorbent Assay, DYC887; R&D Systems, inneapolis, MN), phospho-GSK3 (S9) (DYC-1590; R&D Sysems), and phospho-p65(S536) (7834; Cell Signaling) were asessed by enzyme linked immunosorbent assay in cells transected with as-miR-21 (100 nmol/L) and/or as-miR-155 (100 mol/L) for 24 hours and treated with NT (100 nmol/L) for 24 ours.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: Annals of Laboratory Medicine

Article Title: In vitro Stability of Heat Shock Protein 27 in Serum and Plasma Under Different Pre-analytical Conditions: Implications for Large-Scale Clinical Studies

doi: 10.3343/alm.2016.36.4.353

Figure Lengend Snippet: Stability results for HSP27: effects of immediate and delayed processing of blood samples with storage at 4℃

Article Snippet: Circulating HSP27 concentrations were measured by using the Human Total HSP27 DuoSet IC ELISA (Catalog No. DYC1580, R&D Systems, Minneapolis, MN, USA).

Techniques: Clinical Proteomics

Journal: Annals of Laboratory Medicine

Article Title: In vitro Stability of Heat Shock Protein 27 in Serum and Plasma Under Different Pre-analytical Conditions: Implications for Large-Scale Clinical Studies

doi: 10.3343/alm.2016.36.4.353

Figure Lengend Snippet: Stability results for HSP27: effects of repeated freeze-thaw cycles with storage of serum/plasma samples at -80℃

Article Snippet: Circulating HSP27 concentrations were measured by using the Human Total HSP27 DuoSet IC ELISA (Catalog No. DYC1580, R&D Systems, Minneapolis, MN, USA).

Techniques: Clinical Proteomics

Journal: Annals of Laboratory Medicine

Article Title: In vitro Stability of Heat Shock Protein 27 in Serum and Plasma Under Different Pre-analytical Conditions: Implications for Large-Scale Clinical Studies

doi: 10.3343/alm.2016.36.4.353

Figure Lengend Snippet: In vitro stability of HSP27 under different pre-analytical conditions: (A) relative analyte stability in serum samples and (B) plasma samples stored for 2-6 hr at 4℃ with immediate and delayed sample processing; and (C) the effect of repeated freeze-thaw cycles on HSP27 serum and plasma concentrations. Graphs show relative analyte recoveries at distinct time points (each dot represents the mean analyte concentrations relative to the baseline values of 10 healthy individuals; whiskers indicate standard deviation).

Article Snippet: Circulating HSP27 concentrations were measured by using the Human Total HSP27 DuoSet IC ELISA (Catalog No. DYC1580, R&D Systems, Minneapolis, MN, USA).

Techniques: In Vitro, Clinical Proteomics, Standard Deviation

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Signature of chemokine ligands and (B) chemokine receptors in human ovarian cancer cell lines. After isolating total RNA from each cell line, PCR array was performed using a customized PCR array plate containing complementary sequences for human chemokine genes. Different colors indicate average cycle threshold with expression ranges from >35 to <25. (C) Protein expression of p53 and Mdm2 in ovarian cancer cell lines. Whole cell lysates were prepared and Western blot was carried out using antibodies specific to p53, Mdm2 and β-actin as loading control. Experiments were performed in duplicate and a representative result is shown. OV, OVCAR-3 cells; SK, SKOV-3 cells; A, A2780 cells; Ca, CaOV-3 cells; TOV, TOV-21G cells.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) TNF-induced chemokines in SKOV-3 cells. After isolating total RNA, PCR array was performed using a human chemokine PCR array plate. Dotted line indicates 2-fold increase; chemokines with a greater than 2-fold increase are recognized as TNF-induced chemokines. (B) Confirmation of p53 protein expression after transient transfection in SKOV-3 cells. After transfection of empty vector (EM) and p53 expression vector (p53), whole cell lysates were prepared and p53 expression was confirmed by Western blot. β-actin is used as a loading control. (C) Effect of p53 on TNF-induced chemokines. After overnight transfection of vectors, cells were treated with TNF (10 ng/ml) for 1 h and qRT-PCR was carried out using primers for CCL2, CXCL1, 2, 3 and 8. β-actin serves as normalization control. Different letters indicate significant differences (P≤0.05) within each chemokine group (ANOVA and Tukey's pairwise comparisons). Experiments were performed in triplicate and all data are shown as mean ± SE.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Control, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Nucleotide sequences of promoters for TNF-induced chemokines such as CCL20, CXCL1, 2, 3 and 8. These chemokine promoters contain one NF-κB site at the proximal region, except for CCL20, which has two NF-κB sites at the distal and proximal region. (B) Effect of p53 on NF-κB luciferase activity. After transfection of vectors or cotransfection with p65, cells were treated with TNF (10 ng/ml) for 4 h. (C) Effect of p53 on TNF-activated IκB. After transfection of empty vector or p53 in SKOV-3 (p53 null), OVCAR-3 (p53 mutant) and A2780 (p53 wild-type), cells were treated with TNF (10 ng/ml) for indicated times. β-actin serves as loading control. Experiments were performed in duplicate and a representative result is shown; numbers below are relative density values.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Mutagenesis, Control

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Accumulated effect of p53 on ubiquitylated proteins. After transient transfection of p53 in A2780, OVCAR-3 and SKOV-3 cells, whole cell lysates were prepared and Western blot was carried out using antibodies specific to ubiquitin, p21, IκB, p53 and β-actin (as loading control). Experiments were performed in duplicate and a representative result is shown. (B) Confirmation of p53 activity after transient transfection of p53. ELISA was performed in triplicate and data are shown as mean ± SE. Dark gray bars indicate significance (p<0.05, paired Student's t -test) within each cell line. (C) The effect of p53 on proteasome activity. Assays were performed in triplicate and data are shown as mean ± SE. Dark gray bars indicate significance (p<0.05, paired Student's t -test) within each cell line. (D) Effects of p53 on ubiquitination of IκB. Immunoprecipitated IκB was immunoblotted using ubiquitin antibody. Experiments were performed in duplicate and a representative result is shown.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Western Blot, Ubiquitin Proteomics, Control, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: Effect of p53 on ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3) obtained from comparison between empty vector and p53 vector transfected ovarian cancer cells.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Ubiquitin Proteomics, Comparison, Plasmid Preparation, Transfection

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Effect of p53 on Mdm2 expression. After transient transfection of p53 in A2780, OVCAR-3 and SKOV-3 cells, whole cell lysates were prepared and Western blot was carried out using antibodies specific to Mdm2; and β-actin served as loading control. (B) Effects of p53 expression on p53 binding to p65 and IκB. After transient transfection of p53, immunoprecipitated (IP) p53 was immunoblotted (IB) using p65 or IκB antibody. (C) Effect of p53 on expression of various IKK isoforms. After transient transfection of p53, whole cell lysates were prepared and Western blot was carried out using antibodies specific to IKKα, IKKβ, IKKγ, IKKε; β-actin served as loading control. Experiments were performed in duplicate and a representative result is shown.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transfection, Western Blot, Control, Binding Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: (A) Effect of nutlin-3 on NF-κB luciferase activity. After transfection of vectors or cotransfection with p53, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for 4 h. Different letters indicate significant differences (P≤0.05) within each group (ANOVA and Tukey's pairwise comparisons). Experiments were performed in triplicate and all data are shown as mean ± SE. (B) Effect of nutlin-3 on TNF-activated IκB. After transfection of empty vector or p53 in SKOV-3 cells, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for indicated times. β-actin serves as loading control. Experiments were performed in duplicate and a representative result is shown. (C) Effect of nutlin-3 on TNF-induced chemokines. After overnight transfection of vectors, cells were pretreated with nutlin-3 (10 µM) for 24 h followed by TNF (10 ng/ml) for 1 h and qRT-PCR was carried out using primers for CCL2, CXCL1, 2, 3 and 8. β-actin serves as normalization control. Asterisk indicates significant differences (P≤0.05, paired Student's t -test) when compared to the presence of nutlin-3. Experiments were performed in triplicate and all data are shown as mean ± SE.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Luciferase, Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Control, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

doi: 10.1371/journal.pone.0051116

Figure Lengend Snippet: Chronic inflammation promotes ovarian cancer progression via NF-κB signaling. Wild-type p53 reduces activity of the ubiquitin-proteasome system, resulting in low IκB degradation (blue line). This reduces NF-κB activity, inhibiting proinflammatory chemokine expression and attenuating the proinflammatory tumor microenvironment (blue arrow). On the other hand, p53 increases Mdm2 expression (dark arrow) in a feedback loop to compensate for the reduced activity of the ubiquitin-proteasome system. Loss of p53 observed frequently in advanced ovarian cancer triggers high proinflammatory chemokines by increasing NF-κB signaling which is composed of IκB and p65/p50 followed by a high IκB degradation (red arrow). Enhanced NF-κB activity results in potentiation of the proinflammatory tumor microenvironment for ovarian cancer progression such as peritoneal tumor dissemination and massive ascites. The imbalance between p53 and Mdm2 also contributes to increasing NF-κB signaling via the ubiquitin-proteasome system.

Article Snippet: Recombinant human TNF and human p53 DuoSet® IC ELISA kit were obtained from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Ubiquitin Proteomics, Expressing