Journal: Clinical Proteomics

Article Title: Current applications of antibody microarrays

doi: 10.1186/s12014-018-9184-2

Figure Lengend Snippet: Review of planar antibody microarray technologies and their applications in the field of proteomics. Images were adopted from Servier Medical Art by Servier ( http://www.servier.com/Powerpoint-image-bank ) and modified by the authors under the following terms: CREATIVE COMMONS Attribution 3.0 Unported (CC BY 3.0)

Article Snippet: Fullmoon Biosystems Inc , Prostate cancer , Phosphorylation-specific antibody microarray , 95 , 24009409.

Techniques: Microarray, Modification

Journal: Clinical Proteomics

Article Title: Current applications of antibody microarrays

doi: 10.1186/s12014-018-9184-2

Figure Lengend Snippet: Antibody microarray summary

Article Snippet: Fullmoon Biosystems Inc , Prostate cancer , Phosphorylation-specific antibody microarray , 95 , 24009409.

Techniques: Microarray, Phospho-proteomics, Infection, Biomarker Discovery, Virus

Journal: Blood

Article Title: Jun-regulated genes promote interaction of diffuse large B-cell lymphoma with the microenvironment

doi: 10.1182/blood-2014-04-568188

Figure Lengend Snippet: Immunohistochemical analysis of p-c-Jun (Ser73) expression in a subset of 25 DLBCL tumor tissues collected from patients with or without bone marrow involvement

Article Snippet: Antibody specific for p-c-Jun (Ser73) was purchased from Cell Signaling (#9164).

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: Blood

Article Title: Jun-regulated genes promote interaction of diffuse large B-cell lymphoma with the microenvironment

doi: 10.1182/blood-2014-04-568188

Figure Lengend Snippet: c-Jun is enriched in extranodal lymphoma. (A) Positron emission tomography/computed tomography (PET/CT) images from patients with different lymphoma localization. Patients #1 and #2 had only nodal sites of involvement; representative images are shown. Patient #3 had multifocal extranodal involvement in the bone marrow in the spine, as shown in sagittal images. Patient #4 had multiple extranodal sites of involvement, including bone marrow, subcutaneous tissues, and perineural spread. Expression of p-c-Jun (Ser73) in DLBCL tumor tissues from these patients was determined by immunohistochemistry; representative images are shown (×500). (B-D) Expression of c-Jun was stratified by the number of extranodal sites using microarray data available in a public repository (GSE10846). Log2 median-centered intensities of c-Jun (probe 201465_s) in 296 cases with a known number of extranodal sites were obtained by using Oncomine v4.5 (www.oncomine.org). Patients diagnosed with the ABC or GCB subtypes of DLBCL were analyzed in (C) and (D), respectively. Statistical significance was evaluated by using two-tailed Student t test. ****P < .0001; ***P < .001; **P < .01; *P < .05.

Article Snippet: Antibody specific for p-c-Jun (Ser73) was purchased from Cell Signaling (#9164).

Techniques: Positron Emission Tomography, Computed Tomography, Positron Emission Tomography-Computed Tomography, Expressing, Immunohistochemistry, Microarray, Two Tailed Test

Journal: PLoS ONE

Article Title: Dendritic Cell-Mediated Phagocytosis but Not Immune Activation Is Enhanced by Plasmin

doi: 10.1371/journal.pone.0131216

Figure Lengend Snippet: (A) MoDCs were incubated in the presence/absence of 1 nM t-PA and 100 nM plasminogen. 3 h later cultures were lysed and subject to kinomic microarray analysis yielding a short list of 31 signalling proteins that were differentially regulated upon plasmin-treatment (see for complete dataset). The short list was subjected to Ingenuity Pathway Analysis to yield three canonical signalling pathways (not shown) for plasmin-mediated immunomodulation, which were manually merged and refined to generate a single putative signalling pathway that underlies plasmin-mediated immunomodulation. Red symbols represent upregulated events. Blue symbols represent downregulated events. (B) The kinomic short list of differentially regulated signalling proteins was batch analysed using the Pathway Interaction Database (PID). Shown are the 5 most significantly perturbed pathways of interest (POI), the differentially regulated proteins within each POI and the degree of statistical significance (p-value computed using the default PID hypergeometric distribution test).

Article Snippet: Accordingly, to assist the future identification of putative signalling events that may underlie plasmin-mediated immunomodulation, we performed a kinomic screen whereby MoDCs were treated with/without plasmin for 3 h, after which cell lysates were harvested and subjected to Kinexus antibody microarray (which utilises ~500 pan- and ~340 phospho-specific antibodies).

Techniques: Incubation, Microarray

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN-gamma-primed macrophages exhibit increased CCR2-dependent migration and altered IFN-gamma responses mediated by Stat1.

doi: 10.4049/jimmunol.175.6.3637

Figure Lengend Snippet: FIGURE 2. IFN- priming selectively suppresses the induction of a subset of IFN--activated genes. A, Control or primed human macrophages were activated with 100 U/ml IFN- for 3 h (for analysis of IL-1, IL-6, and Cox-2) or 24 h (for analysis of VCAM-1). Steady-state mRNA levels were determined by real-time PCR, and fold induction by IFN- relative to basal expression in control and IFN--primed cells is shown. Each line rep- resents an individual experiment performed with an independent blood donor. B, Stat1-independent induction of IFN--responsive genes. Murine macro- phages from Stat1-deficient mice were cultured with 10 ng/ml IFN- for the indicated time periods, and mRNA levels of IL-1, IL-6, and Bcl2A1 were analyzed using real-time PCR and normalized relative to expression of GAPDH. Results are expressed as mean SD of triplicate determinants.

Article Snippet: Phosphorylation-specific (tyrosine 701) Stat1 Ab and phosphorylation-specific (tyrosine 705) Stat3 Ab were obtained from Cell Signaling Technology.

Techniques: Control, Real-time Polymerase Chain Reaction, Expressing, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN-gamma-primed macrophages exhibit increased CCR2-dependent migration and altered IFN-gamma responses mediated by Stat1.

doi: 10.4049/jimmunol.175.6.3637

Figure Lengend Snippet: FIGURE 3. Stat1 overexpression suppresses IFN- activation of genes induced by Stat1-independent pathways. A, Upper and middle panels, Human primary macrophages were treated with 3 U/ml IFN- as indicated. Stat1 and SOCS1 mRNA levels were quantitated using real-time PCR. Lower panel shows the average signal intensities of SOCS1 derived from microarray analysis. B, THP-1 monocytic cells transduced with eGFP, Stat1, or Stat1- encoding lentiviral particles were activated with 100 U/ml IFN- for 15 min. Whole-cell lysates were subjected to immunoblotting with anti-tyrosine phosphorylated Stat1 (pY-Stat1), anti-Stat1, and anti-Stat3 Abs. C, Transduced THP-1 cells were stimulated with 100 U/ml IFN- for 3 h and mRNA levels of Mig were quantitated using real-time PCR. D, THP-1 cells transduced with eGFP or Stat1-encoding lentiviral particles were stimulated with 100 U/ml IFN- for the indicated time periods, and mRNA expression of CD63, EGR2, Fas, and VCAM-1 was measured by real-time PCR. E, THP-1 cells were stimulated with 100 U/ml IFN- for 24 h, and mRNA levels were determined using real-time PCR.

Article Snippet: Phosphorylation-specific (tyrosine 701) Stat1 Ab and phosphorylation-specific (tyrosine 705) Stat3 Ab were obtained from Cell Signaling Technology.

Techniques: Over Expression, Activation Assay, Real-time Polymerase Chain Reaction, Derivative Assay, Microarray, Transduction, Western Blot, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN-gamma-primed macrophages exhibit increased CCR2-dependent migration and altered IFN-gamma responses mediated by Stat1.

doi: 10.4049/jimmunol.175.6.3637

Figure Lengend Snippet: FIGURE 4. Stat3 mediates IFN- activation of certain Stat1-indepen- dent genes. A, THP-1 cells were transduced with lentiviral particles en- coding DSred2 small interfering RNA (siRNA) or Stat3 siRNA. Cell ly- sates from lentiviral-transduced THP-1 cells were subjected to immunoblotting. B, Lentiviral-transduced THP-1 cells were stimulated with 100 U/ml IFN- for 3 h, and mRNA levels of SOCS3 were quantitated using real-time PCR. C, Transduced THP-1 cells were stimulated with 100 U/ml IFN- for the indicated time periods, and mRNA expression of CD63, EGR2, Fas, and VCAM-1 was measured by real-time PCR.

Article Snippet: Phosphorylation-specific (tyrosine 701) Stat1 Ab and phosphorylation-specific (tyrosine 705) Stat3 Ab were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Transduction, Small Interfering RNA, Western Blot, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN-gamma-primed macrophages exhibit increased CCR2-dependent migration and altered IFN-gamma responses mediated by Stat1.

doi: 10.4049/jimmunol.175.6.3637

Figure Lengend Snippet: FIGURE 5. Regulation of Stat3 activation in primed macrophages ex- pressing high Stat1 levels. A, Primary human macrophages were primed with 3 U/ml IFN- for 2 days and restimulated with 10 U/ml IFN- for the indicated time periods. Whole-cell extracts were subjected to immunoblot- ting. B, Control or IFN--primed primary macrophages were stimulated with 10 U/ml IFN- for 15 min. Nuclear extracts were subjected to EMSA with high-affinity SIS-inducible element oligonucleotide probe. Stat3 was specifically supershifted using anti-Stat3 Abs as previously described (44). C, THP-1 cells transduced with eGFP, Stat1, or Stat1-encoding lentiviral particles were stimulated with 100 U/ml IFN- for the indicated time pe- riods, and mRNA expression of SOCS3 was measured by real-time PCR.

Article Snippet: Phosphorylation-specific (tyrosine 701) Stat1 Ab and phosphorylation-specific (tyrosine 705) Stat3 Ab were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot, Control, Transduction, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN-gamma-primed macrophages exhibit increased CCR2-dependent migration and altered IFN-gamma responses mediated by Stat1.

doi: 10.4049/jimmunol.175.6.3637

Figure Lengend Snippet: FIGURE 6. Hypothetical model for regulation of IFN- transcriptional responses by priming. The thickness of arrow lines and the font size of signaling molecules represent the intensity of signal transduction. In con- trol human macrophages, IFN- induces the expression of both Stat1- and Stat3-dependent genes, with minimal repression of Stat3 function by basal Stat1 levels. In IFN--primed cells, high Stat1 levels suppress Stat3-me- diated gene activation without altering Stat3 tyrosine phosphorylation.

Article Snippet: Phosphorylation-specific (tyrosine 701) Stat1 Ab and phosphorylation-specific (tyrosine 705) Stat3 Ab were obtained from Cell Signaling Technology.

Techniques: Transduction, Expressing, Activation Assay, Phospho-proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN-gamma-primed macrophages exhibit increased CCR2-dependent migration and altered IFN-gamma responses mediated by Stat1.

doi: 10.4049/jimmunol.175.6.3637

Figure Lengend Snippet: FIGURE 3. Stat1 overexpression suppresses IFN- activation of genes induced by Stat1-independent pathways. A, Upper and middle panels, Human primary macrophages were treated with 3 U/ml IFN- as indicated. Stat1 and SOCS1 mRNA levels were quantitated using real-time PCR. Lower panel shows the average signal intensities of SOCS1 derived from microarray analysis. B, THP-1 monocytic cells transduced with eGFP, Stat1, or Stat1- encoding lentiviral particles were activated with 100 U/ml IFN- for 15 min. Whole-cell lysates were subjected to immunoblotting with anti-tyrosine phosphorylated Stat1 (pY-Stat1), anti-Stat1, and anti-Stat3 Abs. C, Transduced THP-1 cells were stimulated with 100 U/ml IFN- for 3 h and mRNA levels of Mig were quantitated using real-time PCR. D, THP-1 cells transduced with eGFP or Stat1-encoding lentiviral particles were stimulated with 100 U/ml IFN- for the indicated time periods, and mRNA expression of CD63, EGR2, Fas, and VCAM-1 was measured by real-time PCR. E, THP-1 cells were stimulated with 100 U/ml IFN- for 24 h, and mRNA levels were determined using real-time PCR.

Article Snippet: Phosphorylation-specific (tyrosine 701) Stat1 Ab and phosphorylation-specific (tyrosine 705) Stat3 Ab were obtained from Cell Signaling Technology.

Techniques: Over Expression, Activation Assay, Real-time Polymerase Chain Reaction, Derivative Assay, Microarray, Transduction, Western Blot, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN-gamma-primed macrophages exhibit increased CCR2-dependent migration and altered IFN-gamma responses mediated by Stat1.

doi: 10.4049/jimmunol.175.6.3637

Figure Lengend Snippet: FIGURE 4. Stat3 mediates IFN- activation of certain Stat1-indepen- dent genes. A, THP-1 cells were transduced with lentiviral particles en- coding DSred2 small interfering RNA (siRNA) or Stat3 siRNA. Cell ly- sates from lentiviral-transduced THP-1 cells were subjected to immunoblotting. B, Lentiviral-transduced THP-1 cells were stimulated with 100 U/ml IFN- for 3 h, and mRNA levels of SOCS3 were quantitated using real-time PCR. C, Transduced THP-1 cells were stimulated with 100 U/ml IFN- for the indicated time periods, and mRNA expression of CD63, EGR2, Fas, and VCAM-1 was measured by real-time PCR.

Article Snippet: Phosphorylation-specific (tyrosine 701) Stat1 Ab and phosphorylation-specific (tyrosine 705) Stat3 Ab were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Transduction, Small Interfering RNA, Western Blot, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN-gamma-primed macrophages exhibit increased CCR2-dependent migration and altered IFN-gamma responses mediated by Stat1.

doi: 10.4049/jimmunol.175.6.3637

Figure Lengend Snippet: FIGURE 5. Regulation of Stat3 activation in primed macrophages ex- pressing high Stat1 levels. A, Primary human macrophages were primed with 3 U/ml IFN- for 2 days and restimulated with 10 U/ml IFN- for the indicated time periods. Whole-cell extracts were subjected to immunoblot- ting. B, Control or IFN--primed primary macrophages were stimulated with 10 U/ml IFN- for 15 min. Nuclear extracts were subjected to EMSA with high-affinity SIS-inducible element oligonucleotide probe. Stat3 was specifically supershifted using anti-Stat3 Abs as previously described (44). C, THP-1 cells transduced with eGFP, Stat1, or Stat1-encoding lentiviral particles were stimulated with 100 U/ml IFN- for the indicated time pe- riods, and mRNA expression of SOCS3 was measured by real-time PCR.

Article Snippet: Phosphorylation-specific (tyrosine 701) Stat1 Ab and phosphorylation-specific (tyrosine 705) Stat3 Ab were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot, Control, Transduction, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN-gamma-primed macrophages exhibit increased CCR2-dependent migration and altered IFN-gamma responses mediated by Stat1.

doi: 10.4049/jimmunol.175.6.3637

Figure Lengend Snippet: FIGURE 6. Hypothetical model for regulation of IFN- transcriptional responses by priming. The thickness of arrow lines and the font size of signaling molecules represent the intensity of signal transduction. In con- trol human macrophages, IFN- induces the expression of both Stat1- and Stat3-dependent genes, with minimal repression of Stat3 function by basal Stat1 levels. In IFN--primed cells, high Stat1 levels suppress Stat3-me- diated gene activation without altering Stat3 tyrosine phosphorylation.

Article Snippet: Phosphorylation-specific (tyrosine 701) Stat1 Ab and phosphorylation-specific (tyrosine 705) Stat3 Ab were obtained from Cell Signaling Technology.

Techniques: Transduction, Expressing, Activation Assay, Phospho-proteomics

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: Rabbit polyclonal anti-CRMP2 (CP2161) and rabbit polyclonal anti-CRMP2 (S522) (CP2191) antibodies were purchased from ECM Biosciences.

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: Rabbit polyclonal anti-CRMP2 (CP2161) and rabbit polyclonal anti-CRMP2 (S522) (CP2191) antibodies were purchased from ECM Biosciences.

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: Rabbit polyclonal anti-CRMP2 (CP2161) and rabbit polyclonal anti-CRMP2 (S522) (CP2191) antibodies were purchased from ECM Biosciences.

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: Rabbit polyclonal anti-CRMP2 (CP2161) and rabbit polyclonal anti-CRMP2 (S522) (CP2191) antibodies were purchased from ECM Biosciences.

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: Rabbit polyclonal anti-CRMP2 (CP2161) and rabbit polyclonal anti-CRMP2 (S522) (CP2191) antibodies were purchased from ECM Biosciences.

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: Rabbit polyclonal anti-CRMP2 (CP2161) and rabbit polyclonal anti-CRMP2 (S522) (CP2191) antibodies were purchased from ECM Biosciences.

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: Rabbit polyclonal anti-CRMP2 (CP2161) and rabbit polyclonal anti-CRMP2 (S522) (CP2191) antibodies were purchased from ECM Biosciences.

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

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR21 modulates the Hippo signaling pathway via interference with PP2A Bβ to inhibit trophoblast invasion and cause preeclampsia

doi: 10.1016/j.omtn.2022.09.006

Figure Lengend Snippet: PE-complicated trophoblasts are associated with upregulated expression of miR21, which impairs invasion and migration (A) Heatmap of gene expression from a microarray of normal and preeclamptic term placentae; n = 5, two-tailed t test, ∗p < 0.05. (B) ddPCR analysis of miR21 expression in human term placentae from normal and PE-complicated pregnancies; n = 19 in the control and n = 20 in the PE group, two-tailed t test, ∗p < 0.05. (C) RT-qPCR analysis of miR21 expression in primary human trophoblasts (PHTs) isolated from normal and preeclamptic term placentae; n = 3, two-tailed t test, ∗p < 0.05. FISH of miR21 and IF staining of CK7 and HLA-G in term normal and PE-complicated placentae (D) and decidua (E); scale bar, 100 μm. HTR8/SVneo cells transfected with mimic NC (NC), inhibitor NC (in NC), miR21 mimic (mimic), or miR21 inhibitor (inhibitor) for 6 h and then cultured in fresh medium for 48 h and blank control cells were subjected to (F) Matrigel Transwell assays (scale bar, 400 μm), (G) wound-healing assays (scale bar, 100 μm), (H) CCK-8 staining assays, (I) EdU assay, and (J) flow cytometry assay for measuring apoptosis by staining with Annexin V-FITC and PI (n = 3 in each group, one-way ANOVA and Tukey’s multiple comparison test; ns, nonsignificant; ∗p < 0.05 versus mimic NC; #p < 0.05 versus inhibitor NC). The data are presented as the means ± SEMs.

Article Snippet: Primary antibodies against PP2A Bβ (1:1,000, 13123-1-AP), CK7 (1:1,000, 17513-1-AP), human leukocyte antigen G (HLA-G) (1:1,000, 66447-1-Ig), anti-ɑ-tubulin (1:1,000, 11224-1-AP), and anti-β-actin (1:1,000, 66009-1-Ig) were purchased from Proteintech (China).

Techniques: Expressing, Migration, Gene Expression, Microarray, Two Tailed Test, Control, Quantitative RT-PCR, Isolation, Staining, Transfection, Cell Culture, CCK-8 Assay, EdU Assay, Flow Cytometry, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR21 modulates the Hippo signaling pathway via interference with PP2A Bβ to inhibit trophoblast invasion and cause preeclampsia

doi: 10.1016/j.omtn.2022.09.006

Figure Lengend Snippet: miR21 regulates the subcellular distribution of YAP in trophoblasts by modulating phosphorylation (A) Venn analysis and (B) GO enrichment analysis of biological process of mRNAs decreased by miR21 mimic and increased by miR21 inhibitor in HTR8/SVneo cells as determined by whole-genome RNA sequencing. HTR8/SVneo cells transfected with miR21 mimic or miR21 inhibitor and blank and scramble controls were subjected to (C) western blotting of p-MST1/2, MST1, MST2, p-LATS1 Thr1079 , p-LATS1 Ser909 , LATS1, p-YAP Ser127 , p-YAP Ser397 , and YAP; (D) western blotting of YAP in cytoplasm and nuclei (n = 3 in each group, one-way ANOVA and Tukey’s multiple comparison test; ns, nonsignificant; ∗p < 0.05 versus mimic NC; #p < 0.05 versus inhibitor NC); (E) IF staining of YAP (green), CK7 (red), and DAPI (blue) (scale bar, 50 μm); and (F) RT-qPCR of CTGF , AMOTL2 , and CTNNB1 (n = 3 in each group, one-way ANOVA and Tukey’s multiple comparison test; ns, nonsignificant; ∗p < 0.05 versus mimic NC; #p < 0.05 versus inhibitor NC). (G) Western blotting of p-MST1/2, MST1, MST2, p-LATS1 Thr1079 , LATS1, p-YAP Ser127 , and YAP in normal and preeclamptic term placentae; n = 6, two-tailed t test; ns, nonsignificant; ∗p < 0.05. (H) Western blotting of YAP in cytoplasm and nuclei of normal and PE term placentae; n = 6, two-tailed t test, ∗p < 0.05. (I) FISH of miR21 (red) and IF costaining of YAP (green) in normal (upper) and preeclamptic (lower) term placentae. Trophoblasts were stained for CK7 (green) on serial sections, and nuclei were counterstained by DAPI (blue); scale bar, 100 μm. (J) RT-qPCR of CTGF , AMTOL2 , and CTNNB1 in normal and preeclamptic term placentae; n = 6, two-tailed t test, ∗p < 0.05. (K) Western blotting of p-MST1/2, MST1, p-LATS1 Thr1079 , LATS1, p-YAP Ser127 , and YAP in PHTs isolated from normal and preeclamptic term placentae; n = 3, two-tailed t test; ns, nonsignificant; ∗p < 0.05. The data are presented as the means ± SEMs.

Article Snippet: Primary antibodies against PP2A Bβ (1:1,000, 13123-1-AP), CK7 (1:1,000, 17513-1-AP), human leukocyte antigen G (HLA-G) (1:1,000, 66447-1-Ig), anti-ɑ-tubulin (1:1,000, 11224-1-AP), and anti-β-actin (1:1,000, 66009-1-Ig) were purchased from Proteintech (China).

Techniques: Phospho-proteomics, RNA Sequencing, Transfection, Western Blot, Comparison, Staining, Quantitative RT-PCR, Two Tailed Test, Isolation

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR21 modulates the Hippo signaling pathway via interference with PP2A Bβ to inhibit trophoblast invasion and cause preeclampsia

doi: 10.1016/j.omtn.2022.09.006

Figure Lengend Snippet: The upregulation of miR21 reduced the dephosphorylation of LATS1 Thr1079 and YAP Ser127 by suppressing PP2A Bβ in vivo Western blotting of (A) PP2A Bβ, p-LATS1 (Thr1079), LATS1, p-YAP (Ser127), and YAP in placentae at E18.5 and (B) cytoplasmic and nuclear YAP in placentae at E18.5 (n = 4 placentae from four dams in the agomir-NC, agomir-miR21, and control groups; one-way ANOVA and Tukey’s multiple comparison test; ns, nonsignificant). (C) IF staining of CK7(red) and YAP (green) in placentae of the agomir-NC (upper) or agomir-miR21 group (lower) at E18.5. Nuclei were counterstained by DAPI (blue); scale bar, 25 μm; two-way ANOVA and Tukey’s multiple comparison test, ∗p < 0.05. (D) RT-qPCR of ctgf , amotl2 , and ctnnb1 in mouse placentae at E18.5, n = 6 placentae from six dams in each group, two-way ANOVA and Tukey’s multiple comparison test; ns, nonsignificant; ∗p < 0.05 versus the control group; #p < 0.05 versus the agomir-NC group. (E) Schematic representation of the mechanism underlying the miR21/PP2A Bβ/Hippo axis in extravillous trophoblast (EVT) invasion and placental development. The data are presented as the means ± SEMs.

Article Snippet: Primary antibodies against PP2A Bβ (1:1,000, 13123-1-AP), CK7 (1:1,000, 17513-1-AP), human leukocyte antigen G (HLA-G) (1:1,000, 66447-1-Ig), anti-ɑ-tubulin (1:1,000, 11224-1-AP), and anti-β-actin (1:1,000, 66009-1-Ig) were purchased from Proteintech (China).

Techniques: De-Phosphorylation Assay, In Vivo, Western Blot, Control, Comparison, Staining, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Altered splicing leads to reduced activation of CPEB3 in high-grade gliomas

doi: 10.18632/oncotarget.9735

Figure Lengend Snippet: ( A ) Evaluation of CPEB3 and phospho-CPEB3 protein expression in gliomas. Light grey graph sections correspond to the lack of CPEB expression, while grey and black sections to CPEB expression. Immunoreactivity was divided into intensity groups (1: weak, 2: intermediate, 3: strong). Upregulation of CPEB3 expression and reduction of phospho-CPEB3 activity was observed in parallel to the growing grade of glioma malignancy. ( B ) Expression of CaMKII and PKA kinases induce the activation of CPEB3 protein in gliomas. Representative staining of pGBM: a, c, e, g, i, k and AII: b, d, f, h, j, l tissue samples. Pictures a, b present cell nuclei stained with violet hematoxylin, and cell cytoplasm stained with pink eosin. On the remaining pictures (c-l), dark blue areas represent cell nuclei stained by hematoxylin, while brown areas correspond to respective antibody staining: GFAP: c, d; CPEB3: e, f; phospho-CPEB3: g, h; phospho-CaMKII: i, j; PKA: k, l. ( C ) Correlation between phospho-CPEB3 expression and overall patient survival observed in all investigated glioma samples ( p < 0.001, upper panel ). The positive link between phospho-CPEB3 expression and prolonged mean patient survival was particularly evident for AII ( n = 8), AAIII ( n = 20, p < 0.01) and sGBM ( n = 8) (Figure 6C- lower panel ). Error bars indicate standard error of the mean.

Article Snippet: In addition we have incubated the tissue microarrays with a phosphorylation specific antibody detecting the active forms of CPEB3, CaMKII and PKA proteins: phospho-CPEB3 (1:50, custom-made [ ]), phospho- CaMKII (1:1000, Cell Signaling, 3361) and PKA (1:500, Abcam, ab59218).

Techniques: Expressing, Activity Assay, Activation Assay, Staining