Journal: OncoTargets and therapy

Article Title: Dynamic Changes in Serum Markers and Their Utility in the Early Diagnosis of All Stages of Hepatitis B-Associated Hepatocellular Carcinoma

doi: 10.2147/OTT.S229835

Figure Lengend Snippet: The Clinicopathological Characteristics of the Study Population

Article Snippet: The following antibodies were spotted on aldehyde-coated microscope slides (Shanghai Baiao, China) by a GeSiM Nano-Plotter TM Micropipetting System (Radeberg, Germany): mouse monoclonal AFP antibody (1.25 mg/mL, Frpon Botech Inc, Shenzhen, China), mouse monoclonal GPC3 antibody (1 mg/mL, R&D Bio-techne, Minneapolis, USA), mouse monoclonal Golgi membrane protein 1 (GOLM1) antibody (0.5 mg/mL, Novus, Centennial, USA) and mouse monoclonal prothrombin factor II antibody (2.5mg/mL, Fujirebio Inc, Tokyo, Japan).

Techniques:

Journal: OncoTargets and therapy

Article Title: Dynamic Changes in Serum Markers and Their Utility in the Early Diagnosis of All Stages of Hepatitis B-Associated Hepatocellular Carcinoma

doi: 10.2147/OTT.S229835

Figure Lengend Snippet: The levels of serum AFP, GPC3, GP73 and DCP in each subgroup. ( A ) AFP. ( B ) GPC3. ( C ) GP73. ( D ) DCP. ( E ) Representative image of each subgroup. * P <0.05, ** P <0.01. Abbreviations: HCC, hepatocellular carcinoma; LC, liver cirrhosis; CHB, chronic hepatitis B virus infection; HC, healthy controls; AFP, α-fetoprotein; GPC3, glypican 3; GP73, golgi protein 73; DCP, des-γ-carboxy prothrombin; ns, no significance; int, intensity.

Article Snippet: The following antibodies were spotted on aldehyde-coated microscope slides (Shanghai Baiao, China) by a GeSiM Nano-Plotter TM Micropipetting System (Radeberg, Germany): mouse monoclonal AFP antibody (1.25 mg/mL, Frpon Botech Inc, Shenzhen, China), mouse monoclonal GPC3 antibody (1 mg/mL, R&D Bio-techne, Minneapolis, USA), mouse monoclonal Golgi membrane protein 1 (GOLM1) antibody (0.5 mg/mL, Novus, Centennial, USA) and mouse monoclonal prothrombin factor II antibody (2.5mg/mL, Fujirebio Inc, Tokyo, Japan).

Techniques: Virus, Infection

Journal: OncoTargets and therapy

Article Title: Dynamic Changes in Serum Markers and Their Utility in the Early Diagnosis of All Stages of Hepatitis B-Associated Hepatocellular Carcinoma

doi: 10.2147/OTT.S229835

Figure Lengend Snippet: The Value of Serum AFP, GPC3, GP73 and DCP in the Diagnosis of HCC (Including All HCC Patients)

Article Snippet: The following antibodies were spotted on aldehyde-coated microscope slides (Shanghai Baiao, China) by a GeSiM Nano-Plotter TM Micropipetting System (Radeberg, Germany): mouse monoclonal AFP antibody (1.25 mg/mL, Frpon Botech Inc, Shenzhen, China), mouse monoclonal GPC3 antibody (1 mg/mL, R&D Bio-techne, Minneapolis, USA), mouse monoclonal Golgi membrane protein 1 (GOLM1) antibody (0.5 mg/mL, Novus, Centennial, USA) and mouse monoclonal prothrombin factor II antibody (2.5mg/mL, Fujirebio Inc, Tokyo, Japan).

Techniques:

Journal: OncoTargets and therapy

Article Title: Dynamic Changes in Serum Markers and Their Utility in the Early Diagnosis of All Stages of Hepatitis B-Associated Hepatocellular Carcinoma

doi: 10.2147/OTT.S229835

Figure Lengend Snippet: Assessment of the diagnostic value of serum AFP, GPC3, GP73 and DCP in differentiating HBV-related HCC from controls. ( A ) All HCC vs LC, CHB, HC. ( B ) All HCC vs LC, CHB. ( C ) All HCC v s LC. ( D ) Very early and early stage HCC vs LC, CHB, HC. ( E ) Very early and early stage HCC vs LC, CHB. ( F ) Very early and early stage HCC vs LC. Abbreviations: HCC, hepatocellular carcinoma; LC, liver cirrhosis; CHB, chronic hepatitis B virus infection; HC, healthy controls; AFP, α-fetoprotein; GPC3, glypican 3; GP73, golgi protein 73; DCP, des-γ-carboxy prothrombin.

Article Snippet: The following antibodies were spotted on aldehyde-coated microscope slides (Shanghai Baiao, China) by a GeSiM Nano-Plotter TM Micropipetting System (Radeberg, Germany): mouse monoclonal AFP antibody (1.25 mg/mL, Frpon Botech Inc, Shenzhen, China), mouse monoclonal GPC3 antibody (1 mg/mL, R&D Bio-techne, Minneapolis, USA), mouse monoclonal Golgi membrane protein 1 (GOLM1) antibody (0.5 mg/mL, Novus, Centennial, USA) and mouse monoclonal prothrombin factor II antibody (2.5mg/mL, Fujirebio Inc, Tokyo, Japan).

Techniques: Diagnostic Assay, Virus, Infection

Journal: OncoTargets and therapy

Article Title: Dynamic Changes in Serum Markers and Their Utility in the Early Diagnosis of All Stages of Hepatitis B-Associated Hepatocellular Carcinoma

doi: 10.2147/OTT.S229835

Figure Lengend Snippet: The Value of Serum AFP, GPC3, GP73 and DCP in the Early Diagnosis of HCC (Including the Very Early HCC)

Article Snippet: The following antibodies were spotted on aldehyde-coated microscope slides (Shanghai Baiao, China) by a GeSiM Nano-Plotter TM Micropipetting System (Radeberg, Germany): mouse monoclonal AFP antibody (1.25 mg/mL, Frpon Botech Inc, Shenzhen, China), mouse monoclonal GPC3 antibody (1 mg/mL, R&D Bio-techne, Minneapolis, USA), mouse monoclonal Golgi membrane protein 1 (GOLM1) antibody (0.5 mg/mL, Novus, Centennial, USA) and mouse monoclonal prothrombin factor II antibody (2.5mg/mL, Fujirebio Inc, Tokyo, Japan).

Techniques:

Journal: OncoTargets and therapy

Article Title: Dynamic Changes in Serum Markers and Their Utility in the Early Diagnosis of All Stages of Hepatitis B-Associated Hepatocellular Carcinoma

doi: 10.2147/OTT.S229835

Figure Lengend Snippet: The Value of Serum AFP, GPC3, GP73 and DCP in the Very Early Diagnosis of HCC

Article Snippet: The following antibodies were spotted on aldehyde-coated microscope slides (Shanghai Baiao, China) by a GeSiM Nano-Plotter TM Micropipetting System (Radeberg, Germany): mouse monoclonal AFP antibody (1.25 mg/mL, Frpon Botech Inc, Shenzhen, China), mouse monoclonal GPC3 antibody (1 mg/mL, R&D Bio-techne, Minneapolis, USA), mouse monoclonal Golgi membrane protein 1 (GOLM1) antibody (0.5 mg/mL, Novus, Centennial, USA) and mouse monoclonal prothrombin factor II antibody (2.5mg/mL, Fujirebio Inc, Tokyo, Japan).

Techniques:

Journal: OncoTargets and therapy

Article Title: Dynamic Changes in Serum Markers and Their Utility in the Early Diagnosis of All Stages of Hepatitis B-Associated Hepatocellular Carcinoma

doi: 10.2147/OTT.S229835

Figure Lengend Snippet: The value of serum AFP, GPC3, GP73 and DCP in the very early diagnosis of HBV-related HCC. ( A ) Very early stage HCC vs LC, CHB, HC. ( B ) Very early stage vs LC, CHB. ( C ) Very early stage vs LC. Abbreviations: HCC, hepatocellular carcinoma; LC, liver cirrhosis; CHB, chronic hepatitis B virus infection; HC, healthy controls; AFP, α-fetoprotein; GPC3, glypican 3; GP73, golgi protein 73; DCP, des-γ-carboxy prothrombin.

Article Snippet: The following antibodies were spotted on aldehyde-coated microscope slides (Shanghai Baiao, China) by a GeSiM Nano-Plotter TM Micropipetting System (Radeberg, Germany): mouse monoclonal AFP antibody (1.25 mg/mL, Frpon Botech Inc, Shenzhen, China), mouse monoclonal GPC3 antibody (1 mg/mL, R&D Bio-techne, Minneapolis, USA), mouse monoclonal Golgi membrane protein 1 (GOLM1) antibody (0.5 mg/mL, Novus, Centennial, USA) and mouse monoclonal prothrombin factor II antibody (2.5mg/mL, Fujirebio Inc, Tokyo, Japan).

Techniques: Virus, Infection

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) promotes the aggressiveness of human uveal melanoma through dephosphorylation of CRMP2

doi: 10.1038/s41598-019-39643-y

Figure Lengend Snippet: PTP4A3 expression reduces CRMP2 phosphorylation. ( A ) The phosphorylation state of CRMP2 was compared between OCM-1-EGFP-PTP4A3 and OCM1-EGFP-C104S cells by 2D gel electrophoresis. After Western blotting, detection was performed using an anti-CRMP2 antibody that recognizes total CRMP2 or phospho-specific anti-CRMP2 (S522) and anti-CRMP2 (T514) antibodies. ( B ) Spot densitometric profiles of phosphorylated CRMP2 were generated using ImageJ64. The grey peaks indicate those present for OCM1-EGFP-C104S cells which are absent from OCM1-EGFP-PTP4A3 cells. ( C ) Western blot analysis of total lysates (input) and lysates after phosphoprotein purification as loading controls.

Article Snippet: CRMP2 shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (sc-44485-V).

Techniques: Expressing, Phospho-proteomics, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Generated, Purification

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) promotes the aggressiveness of human uveal melanoma through dephosphorylation of CRMP2

doi: 10.1038/s41598-019-39643-y

Figure Lengend Snippet: PTP4A3 and CRMP2 interact. ( A ) Coimmunoprecipitation of PTP4A3 and CRMP2 using a GFP-Trap_A kit. This kit allows the immunoprecipitation of GFP recombinant proteins from OCM-1-EGFP, OCM-1-EGFP-PTP4A3, and OCM-1-EGFP-C104S cells. GFP recombinant protein and binding partners were revealed by Western blotting with anti-CRMP2 and anti-GFP antibodies. Input: 10% of the loading. ( B ) Phosphatase activity test of GST fusion proteins. The dephosphorylation capacity of GST-PTP4A3 or GST-C104S was assessed by incubation with OMPF substrate and measuring the absorbance at 450 nm. ( C ) Immunoprecipitation of CRMP2 from OCM-1 cells. After immunoprecipitation, CRMP2 was eluted in three successive fractions. The presence of CRMP2 and its phosphorylation state (T514) was verified by Western blotting. ( D ) GST pull-down experiment. Purified phosphorylated CRMP2 was incubated with GST-PTP4A3 and GST-C104S. After the pull-down, the ability of CRMP2 to bind GST fusion proteins was assessed by Western blotting with an anti-CRMP2 antibody and the phosphorylation state of CRMP2 with an anti-CRMP2 (T514). ( E ) Quantification of CRMP2 T514 phosphorylation by analysis of the densitometric profiles of the bands using ImageJ64. Band intensity is represented by the peak area. The ratio of peak area of CRMP2 to that of CRMP2 (T514) was determined.

Article Snippet: CRMP2 shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (sc-44485-V).

Techniques: Immunoprecipitation, Recombinant, Binding Assay, Western Blot, Activity Assay, De-Phosphorylation Assay, Incubation, Phospho-proteomics, Purification

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) promotes the aggressiveness of human uveal melanoma through dephosphorylation of CRMP2

doi: 10.1038/s41598-019-39643-y

Figure Lengend Snippet: CRMP2 expression downstream of PTP4A3 reduces cell migration and invasiveness. ( A ) Western blot showing the knockdown of CRMP2 in OCM-1 cells expressing EGFP, EGFP-PTP4A3, or EGFP-C104S. Twenty micrograms of protein extract was loaded. The detection of α-tubulin was performed as a loading control. ( B ) The mean overall speed of OCM-1 cells was assessed by a 2D random cell migration assay by time-lapse video microscopy. Data are shown as the mean values +/− SD. ~100 cells were tracked per condition. ***p < 0.001, Student’s t-test. ( C ) Western blot showing the knockdown of CRMP2 in human PDX-MP41 cells and endogenous expression of PTP4A3. Twenty micrograms of protein extract was loaded. The detection of β-actin was performed as a loading control. ( D ) The mean overall speed of MP41 cells was assessed by a 2D random cell migration assay by time-lapse video microscopy. Data are shown as the mean values +/− SD. ~100 cells were tracked per condition. ***p < 0.001, Student’s t-test. ( E ) Quantitative analysis of the relative invasiveness of EGFP-PTP4A3shCtrl and EGFP-PTP4A3shCRMP2 cells. Cells (0.25 × 10 6 ) were inoculated into the CAM of chick embryos and the presence of human cells assessed by real-time PCR analysis of chick GAPDH and human alu sequences in the chick femur DNA. Values for the calibrator were arbitrarily defined as 1. The graphs show the means +/− SD. *P < 0.05 (embryos n = 14/cell line), Student test. ( F ) MMP14 localization on the cell surface assessed by flow cytometry of nonpermeabilized OCM-1 cells. n > 8000 cells analyzed, (****p < 0.0001), Student test.

Article Snippet: CRMP2 shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (sc-44485-V).

Techniques: Expressing, Migration, Western Blot, Knockdown, Control, Cell Migration Assay, Microscopy, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) promotes the aggressiveness of human uveal melanoma through dephosphorylation of CRMP2

doi: 10.1038/s41598-019-39643-y

Figure Lengend Snippet: CRMP2 affects the actin network. ( A ) Visualization of the actin cytoskeleton network in OCM-1 cells as assessed by Phalloïdin staining. After deconvolution, images were processed using ImageJ64. Scale bar = 20 μm. ( B ) Quantification of the number of fragments of < 40 adjoining pixels normalized to the cell area in OCM-1 cells. Data are shown as the mean values +/− SEM. ~20 cells were analyzed. ***p < 0.001, **p < 0.01, Student’s t-test. ( C ) Visualization of the actin cytoskeleton network in OCM-1 cells as assessed by Phalloïdin staining of cells treated with Y27632 for 24 h. After deconvolution, images were processed using ImageJ64. Scale bar = 20 μm. ( D ) Quantification of the number of fragments of < 40 adjoining pixels normalized to the cell area in OCM-1 cells treated or not with Y27632. Data are shown as the mean values +/− SEM. ~60 cells were analyzed. ***p < 0.001,*p < 0.01, *p < 0.05, Student’s t-test. ( E ) The mean overall speed of OCM-1 cells treated with Y27632 as assessed by a 2D random cell migration assayed by time-lapse video microscopy. Data are shown as the mean values +/− SD. ~100 cells were tracked per condition. ***p < 0.001, **p < 0.01, Student’s t- test.

Article Snippet: CRMP2 shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (sc-44485-V).

Techniques: Staining, Migration, Microscopy

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) promotes the aggressiveness of human uveal melanoma through dephosphorylation of CRMP2

doi: 10.1038/s41598-019-39643-y

Figure Lengend Snippet: CRMP2 affects the microrheological properties of the cells. ( A ) Sketch of the microrheology experiments. A 2-µm-diameter bead internalized in the cell is trapped with an optical tweezer. At time t = 0 s, the microscope stage is moved in a X s = 0.5-µm step displacement. After the initial rapid displacement of the bead from the trap center, the bead position x b ( t ) relaxes towards the center of the optical trap, which acts as a spring. Single particle tracking of the bead allows determination of the viscoelastic relaxation curves shown in B. ( B ) Average bead displacement curves showing viscoelastic relaxation of the bead towards the trap center following a 0.5-µm step displacement of the microscope stage for OCM-1-EGFP-PTP4A3 and OCM1-EGFP-C104S cells treated with shCtrl or shCRMP2. ( C ) Quantification of the relaxation curves using a phenomenological model-independent approach (upper graphs) yielding the rigidity index and the bead-step amplitude, and the Standard Linear Liquid (SLL) viscoelastic model (lower graphs) yielding the elasticity and viscosity of the cytoplasm. Data were obtained from N = 16 and 14 beads for the OCM-1-EGFP-PTP4A3 cells treated with shCtrl or shCRMP2, respectively, and from N = 13 and 18 beads for OCM1-EGFP-C104S cells treated with shCtrl or shCRMP2, respectively. Error bars represent the standard error. p-values were determined using Student’s t-test for unpaired samples (***p < 0.001, *p < 0.05).

Article Snippet: CRMP2 shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (sc-44485-V).

Techniques: Microscopy, Single-particle Tracking, Viscosity

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) promotes the aggressiveness of human uveal melanoma through dephosphorylation of CRMP2

doi: 10.1038/s41598-019-39643-y

Figure Lengend Snippet: Expression level of PTP4A3 and CRMP2 in normal melanocytes, uveal melanoma cell lines, tumors and survival probability in patients. ( A ) A Transcriptome analysis was conducted on replicates of normal melanocytes (NM), and PDX of primary tumors such as MP41, MP46, MP158, and a PDX of a metastasis MM224. Normal melanocytes, and uveal melanoma tumor cells from cell sorting of dissociated PDX were analyzed on Affymetrix Human Transcriptome Array v2.0 and on Illumina to performed gene expression analysis . Signal from microarray data were normalized according Genosplice’s pipeline based on a RMA normalization step as described previously . RNAseq signals are Deseq. 2-Normalized counts processed by Genosplice’s pipeline too . A paired t-test was applied between UM and normal melanocytes. In our comparisons, CRMP2 is down regulated in UM vs NM, as observed in RNASeq and in microarray datasets. PTP4A3 is upregulated in UM vs normal melanocytes. No p-value are calculated for MP158 and MM224 in RNAseq dataset because a unique sample was sequenced contrary to first experiments done on microarrays. MP41 and MP46 were described previously , . MP158 and MM224 are GNAQ mutated and BAP-1 mutated. ( B ) PTP4A3 and CRMP2 expression were determined by microarrays analysis. Specimens were analyzed on GeneChip Human Genome U133 Plus 2.0 microarrays (Affymetrix) as described previously . PTP4A3 and CRMP2 expression are inversely correlated in tumors (Spearman coefficient of −0,4). Spearman instead of Pearson correlation, because the Spearman correlation is less sensitive than the Pearson correlation to strong outliers. 63 tumors were analysed, each one divided in two groups: the red dots correspond to meta0 tumors (low metastatic risk) and blue dots to meta1 tumors (high metastatic risk) . ( C ) Effect of CRMP2 expression on UM survival. Kaplan-Meier analysis of CRMP2 expression in 80 uveal melanoma tumors from the TCGA database ( http://ualcan.path.uab.edu/index.html ).

Article Snippet: CRMP2 shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (sc-44485-V).

Techniques: Expressing, FACS, Gene Expression, Microarray

Journal: Scientific Reports

Article Title: Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) promotes the aggressiveness of human uveal melanoma through dephosphorylation of CRMP2

doi: 10.1038/s41598-019-39643-y

Figure Lengend Snippet: Model for the regulation of PTP4A3 and CRMP2 expression in uveal melanoma cells. The phosphatase PTP4A3 dephosphorylates CRMP2 on the T514 affecting cytoskeleton (microtubule function and actin fibers formation) which affect cells micro-rheoligical properties and MMP14 membrane accumulation leading to an increase of both migration and invasion. Arrow: activation, blind-ended arrow: inhibition.

Article Snippet: CRMP2 shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (sc-44485-V).

Techniques: Expressing, Membrane, Migration, Activation Assay, Inhibition