Journal: Frontiers in Physiology

Article Title: High Mobility Group Box 1 Promotes Aortic Calcification in Chronic Kidney Disease via the Wnt/β-Catenin Pathway

doi: 10.3389/fphys.2018.00665

Figure Lengend Snippet: High phosphate increases cytosolic HMGB1 levels and induces aortic calcification in a mouse model of CKD in vivo . (A) Serum HMGB1 levels of healthy controls and patients with CKD were analyzed by ELISA. *** p

Article Snippet: HMGB1 regulates the expression or release of TGF-β and BMP2, which activate osteogenic differentiation and lead to the induction of VC (Gao et al., ).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Physiology

Article Title: High Mobility Group Box 1 Promotes Aortic Calcification in Chronic Kidney Disease via the Wnt/β-Catenin Pathway

doi: 10.3389/fphys.2018.00665

Figure Lengend Snippet: Knockdown of HMGB1 inhibits CKD-induced aortic calcification and inflammation. CKD mice were administered with scramble or siHMGB1 lentivirus via tail vein injection and placed on high phosphate diet for 12 weeks. (A) Representative micrographs of Alizarin Red stained sections of the aortas. Scale bar = 50 μm. (B) Calcium content in the thoracic aortas. (C) . (D) Quantification of HMGB1 expression relative to that of β-actin. n = 8 per group. (E) . Quantification of TLR4 (F) and klotho (G) expression relative to that of β-actin. n = 8 per group. Data are presented as the mean ± SD. * p

Article Snippet: HMGB1 regulates the expression or release of TGF-β and BMP2, which activate osteogenic differentiation and lead to the induction of VC (Gao et al., ).

Techniques: Mouse Assay, Injection, Staining, Expressing

Journal: Frontiers in Physiology

Article Title: High Mobility Group Box 1 Promotes Aortic Calcification in Chronic Kidney Disease via the Wnt/β-Catenin Pathway

doi: 10.3389/fphys.2018.00665

Figure Lengend Snippet: Knockdown of HMGB1 attenuates renal dysfunction in CKD mice fed a high Pi diet. CKD mice were administered with scramble siRNA (scramble) or HMGB1 siRNA (siHMGB1) lentivirus via tail vein injection and placed on high phosphate diet for 12 weeks. Serum concentrations of HMGB1 (A) , blood urea nitrogen (B) , creatinine (C) , calcium (D) , phosphorus (E) , and FGF23 (F) were measured by ELISA. n = 8 per group. Data are presented as the mean ± SD. * p

Article Snippet: HMGB1 regulates the expression or release of TGF-β and BMP2, which activate osteogenic differentiation and lead to the induction of VC (Gao et al., ).

Techniques: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: Targeting sphingosine kinase 2 suppresses cell growth and synergizes with BCL2/BCL-XL inhibitors through NOXA-mediated MCL1 degradation in cholangiocarcinoma

doi:

Figure Lengend Snippet: SPHK2 inhibition suppresses cholangiocarcinoma cell growth, induces apoptosis and upregulates NOXA expression. A. RBE and HCCC9810 cells were treated with ABC294640 for 72 h and cell proliferation was quantified by BrdU ELISA assay. B. Cells were treated with ABC294640 at 50 μM for 72 h and cell viability was determined by CCK-8 assay. C. Cells were treated with ABC294640 at 50 μM for 72 h and cell apoptosis was then measured by Annexin V-FITC/PI labeling followed by flow cytometry. D. Real-time qPCR analysis of BCL2 family mRNA level in RBE and HCCC9810 cells treated with 50 μM ABC294640 or no drug control for 24 h. E. Western immunoblotting analysis of NOXA protein levels in RBE and HCCC9810 cells treated with different concentrations of ABC294640 for 24 h. Data shown represents 3 independent experiments. F. Real-time qPCR analysis of NOXA mRNA level in HuH28 and HuCCT1 cells treated with 50 μM ABC294640 for 24 h. G. Western immunoblotting analysis of NOXA protein levels in HuH28 and HuCCT1 cells treated with 50 μM ABC294640 or no drug control for 24 h. Data shown represents 3 independent experiments. H. Western immunoblotting analysis of NOXA protein levels in RBE and HCCC9810 cells treated with different concentrations of K145 for 24 h. Data shown represents 2 independent experiments. I. RBE and HCCC9810 cells were treated with different concentrations of K145 for 72 h and cell viability were determined by CCK-8 assay. Quantitative analysis from 3 independent experiments (Student’s t test; data are shown as mean ± SEM; *P

Article Snippet: Pharmacology and antitumor activity of ABC294640, a selective inhibitor of sphingosine kinase-2.

Techniques: Inhibition, Expressing, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Labeling, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Western Blot

Journal: American Journal of Cancer Research

Article Title: Targeting sphingosine kinase 2 suppresses cell growth and synergizes with BCL2/BCL-XL inhibitors through NOXA-mediated MCL1 degradation in cholangiocarcinoma

doi:

Figure Lengend Snippet: ABC294640 potentiates apoptosis induced by BH3-mimetics in cholangiocarcinoma cells. A, B. RBE, HCCC9810 and HuH28 cells were treated with ABC294640 (ABC) alone or in combination with ABT-263 or Obatoclax at indicated concentrations for 72 h. Cell apoptosis was measured by Annexin V-FITC/PI staining followed by flow cytometry. C. RBE, HCCC9810 and HuH28 cells were treated with ABC294640 alone or in combination with ABT-263 at indicated concentration for 48 h. Cell apoptosis was determined by the Caspase 3/7 activity assay. Quantitative analysis from 3 independent experiments (one-way ANOVA with a Turkey post hoc test; data are shown as mean ± SEM; *P

Article Snippet: Pharmacology and antitumor activity of ABC294640, a selective inhibitor of sphingosine kinase-2.

Techniques: Staining, Flow Cytometry, Cytometry, Concentration Assay, Activity Assay

Journal: American Journal of Cancer Research

Article Title: Targeting sphingosine kinase 2 suppresses cell growth and synergizes with BCL2/BCL-XL inhibitors through NOXA-mediated MCL1 degradation in cholangiocarcinoma

doi:

Figure Lengend Snippet: ABC294640 acts synergistically with BH3-mimetics in inhibiting cholangiocarcinoma cell growth. A. RBE, HCCC9810 and HuH28 cells were treated with various concentrations of ABC294640 or ABT-263 alone or their combination for 72 h, then the cell viability was analyzed by CCK-8 assay. The results are presented as mean ± SEM from 3-4 independent experiments. The combination index (CI) was determined using the Chou-Talalay Method. CI

Article Snippet: Pharmacology and antitumor activity of ABC294640, a selective inhibitor of sphingosine kinase-2.

Techniques: CCK-8 Assay

Journal: American Journal of Cancer Research

Article Title: Targeting sphingosine kinase 2 suppresses cell growth and synergizes with BCL2/BCL-XL inhibitors through NOXA-mediated MCL1 degradation in cholangiocarcinoma

doi:

Figure Lengend Snippet: ABC294640 induces degradation of pro-survival protein MCL1. A, B. Western immunoblotting analysis of MCL1, BCL-XL and BCL2 protein levels in RBE and HCCC9810 cells treated with different concentrations of ABC294640 for 24 h. MCL1 protein level following ABC294640 treatment for 24 h was quantified using Image J. Quantitative analysis from three independent experiments (one-way ANOVA with a Turkey post hoc test; data are shown as mean ± SEM; *P

Article Snippet: Pharmacology and antitumor activity of ABC294640, a selective inhibitor of sphingosine kinase-2.

Techniques: Western Blot

Journal: Oncotarget

Article Title: Identification, characterization and application of a new peptide against anterior gradient homolog 2 (AGR2)

doi: 10.18632/oncotarget.25221

Figure Lengend Snippet: Surface plasmon resonance (SPR) analysis of H10/AGR2 binding (A) Schematic of SPR censor chip used to measure binding affinity of Δ27 AGR2 to H10 peptide. (B) Binding curve of Δ27 AGR2 to immobilized H10 peptide. AGR2 was injected at the indicated concentrations. (C) Pre-incubation of H10-peptide (500 nM) blocks AGR2 (500 nM) binding to immobilized H10 peptide. (D) AGR3 does not show significant binding to immobilized H10 peptide. Both AGR2 and AGR3 were used at 200 nM.

Article Snippet: Bladder cancer cells secrete while normal bladder cells express but do not secrete AGR2.

Techniques: SPR Assay, Binding Assay, Chromatin Immunoprecipitation, Injection, Incubation

Journal: Oncotarget

Article Title: Identification, characterization and application of a new peptide against anterior gradient homolog 2 (AGR2)

doi: 10.18632/oncotarget.25221

Figure Lengend Snippet: Characterization of a novel in vitro AGR2 ELISA (A) Colorimetric detection of AGR2 by H10 ELISA with a commercial antibody in PBS or DMEM with 10% FBS. (B) Quantification of the dynamic range of the AGR2 ELISA in PBS or DMEM with 10% FBS. (C) Detection of eAGR2 from spent media derived from MCF-7 and 22Rv1 cell lines. Comparison of H10 based ELISA and commercial AGR2 ELISA (USCN SEC285Hu) to compare performance of each assay. All data represent three independent biological replicates.

Article Snippet: Bladder cancer cells secrete while normal bladder cells express but do not secrete AGR2.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Oncotarget

Article Title: Identification, characterization and application of a new peptide against anterior gradient homolog 2 (AGR2)

doi: 10.18632/oncotarget.25221

Figure Lengend Snippet: H10 binds AGR2 at two distinct sites (A) Schematic of AGR2 construct with location of mutations or truncations that were used to identify the binding interface of H10. (B) H10 ELISA with various AGR2 constructs to measure the relative binding. (C) Two distinct regions of AGR2 show protection from trypsin treatment upon pre-incubation with H10 followed by lysine-based cross-linking. Number of peptides score matches (PSMs) were determinate by bottom-up proteomics. Amino acid regions with loss of PSMs are highlighted in red boxes. (D) Structure of AGR2 dimer superimposed with the proposed H10 binding site by performing flexible docking simulations with the CABS algorithm. AGR2 molecules are colored green and blue. H10 space fill is colored red. (E) Specific amino acid contacts between H10 (red) and AGR2 (blue, green) highlight the potential binding interface as determined by mutagenesis, cross-linking, and docking.

Article Snippet: Bladder cancer cells secrete while normal bladder cells express but do not secrete AGR2.

Techniques: Construct, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Mutagenesis

Journal: Oncotarget

Article Title: Identification, characterization and application of a new peptide against anterior gradient homolog 2 (AGR2)

doi: 10.18632/oncotarget.25221

Figure Lengend Snippet: H10 inhibits the migration of PC-3 and MDA-MB-231 cancer cell lines (A, B) Fluorescent images of cells treated with 5 nM Taxol, 10 μg/ml H10 and 10 μg/ml control peptide. Images taken after 48 hours of treatments on the Oris cell migration plates. PC-3 cells with AGR2 neutralized antibody, IgG antibody. AGR2 CRISPR/Cas9 knockouts as control. The cells were stained with Hoechst (blue) for nuclei and rhodamine phalloidin (red) for actin. One representative image is shown (4 repeats). (C, D) Quantification of cell migration by microscopy images, analysis of fluorescent signal in the cell-free zone (methods). Error bars represent standard deviations. All data represent at least three independent biological replicates, statistical significance was assessed with the students t -test. Asterisks indicate statistical significance with greater than 95% confidence (p

Article Snippet: Bladder cancer cells secrete while normal bladder cells express but do not secrete AGR2.

Techniques: Migration, Multiple Displacement Amplification, CRISPR, Staining, Microscopy

Journal: Oncotarget

Article Title: Identification, characterization and application of a new peptide against anterior gradient homolog 2 (AGR2)

doi: 10.18632/oncotarget.25221

Figure Lengend Snippet: Characterization of recombinant protein activity and selection of AGR2 binding peptides by mRNA display (A) Homology between amino acid sequences from NCBI database. Homo sapiens: AGR2 CCDS5364.1 and AGR3 CCDS5365, were aligned by ClustalW. Asterisks indicate conserved amino-acids between the two proteins (65% identity). (B) Δ27 AGR2 enhances soft-agar colony formation. LNCaP and PC-3 cell lines were treated with recombinant AGR2 (100 ng/mL) for 72 hours. Formation of colonies was counted by microscopy. (C) Cell migration assay was performed for 48 hours by traditional Boyden Chamber method with recombinant Δ27 AGR2 and Δ27 AGR2-BAP. (D) Cartoon of the selection process for AGR2 binding peptides by mRNA display. All data represent at least three independent biological replicates. Asterisks indicate statistical significance with greater than 95% confidence (p

Article Snippet: Bladder cancer cells secrete while normal bladder cells express but do not secrete AGR2.

Techniques: Recombinant, Activity Assay, Selection, Binding Assay, Microscopy, Cell Migration Assay

Journal: EMBO Molecular Medicine

Article Title: Pyrin dephosphorylation is sufficient to trigger inflammasome activation in familial Mediterranean fever patients

doi: 10.15252/emmm.201910547

Figure Lengend Snippet: Expression of p.M694V Pyrin is necessary and sufficient to confer to PKC inhibitors the ability to activate the inflammasome U937 cells of the indicated genotype and expressing the indicated plasmids were treated with (A, C) UCN‐01 or LPS + nigericin (Nig). Propidium iodide (PI) influx/fluorescence was monitored every 5 min for 3 h. PMA‐differentiated U937 cells of the indicated genotype and expressing the indicated plasmids were primed with LPS for 3 h and treated with UCN‐01, staurosporine (Stauro) or nigericin as indicated. IL‐1β level in the supernatant was quantified by ELISA at 3 h post‐treatment. Pyrin S242 phosphorylation was assessed by Western blot in the indicated cell lines with and without UCN‐01 treatment for 15 min. Data information: (A–C) One experiment representative of three independent experiments is shown. Mean and standard deviations from two biological replicates are shown. (D, E) Mean and standard deviations from three independent experiments are shown. Source data are available online for this figure.

Article Snippet: Antibodies used were mouse monoclonal anti‐FLAG® (Sigma‐Aldrich, clone M2; 1:1,000 dilution), anti‐Pyrin (Adipogen, AL196, 1: 1,000 dilution), anti‐phospho S242 Pyrin (Abcam, ab200420; 1:1,000 dilution; Gao et al , ), and anti‐PKN1 (Becton Dickinson, BD 610687; 1:1,000 dilution).

Techniques: Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Pyrin dephosphorylation is sufficient to trigger inflammasome activation in familial Mediterranean fever patients

doi: 10.15252/emmm.201910547

Figure Lengend Snippet: Doxycycline‐mediated expression of p.[M694V] MEFV is necessary and sufficient to confer to PKC inhibitors the ability to trigger IL ‐18 release U937 MEFV KO The expression of Flag‐Pyrin was revealed by Western blotting analysis against Flag (A) or against Pyrin (B). IL‐18 levels were quantified in the supernatant of PMA‐differentiated U937 cells by ELISA at 3 h post‐treatment with UCN‐01 or staurosporine (Stauro). One experiment representative of two independent experiments is shown. The bar represents the mean of a biological triplicate. Source data are available online for this figure.

Article Snippet: Antibodies used were mouse monoclonal anti‐FLAG® (Sigma‐Aldrich, clone M2; 1:1,000 dilution), anti‐Pyrin (Adipogen, AL196, 1: 1,000 dilution), anti‐phospho S242 Pyrin (Abcam, ab200420; 1:1,000 dilution; Gao et al , ), and anti‐PKN1 (Becton Dickinson, BD 610687; 1:1,000 dilution).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay