Journal: Journal of Neuroscience

Article Title: CRMP2 Tethers Kainate Receptor Activity to Cytoskeleton Dynamics during Neuronal Maturation

doi: 10.1523/jneurosci.3136-13.2013

Figure Lengend Snippet: Figure 5. KARs regulate the phosphorylation state of CRMP2. A, In Western blots, KAR activation by 300 nM but not 3 M KA decreased T514 phosphorylation of CRMP2 (pCRMP2 T514) and increased the T555 phosphorylation of CRMP2 (pCRMP2 T555). CNQX (30 M) alone had no effect on the phosphorylation of CRMP2. **p 0.01 and ***p 0.001, one-sample t test (n 6). B, The increase in pCRMP2 T555 caused by 300 nM KA was blocked by the PKC inhibitor bisindolylmaleimide (Bisind; p 0.2683 vs Bisind alone, unpaired t test, n 6), whereas it was unaffected by the Rho-kinase inhibitor GSK429286 (5 M): ###p 0.001, unpaired t test (n 6); *p 0.05, **p 0.01, and ***p 0.001, one-sample t test (n 6). C, KA at300nMbutnot3MincreasedtheS9phosphorylationofGSK3(pGSK3 S9).NoneoftheKAconcentrationstestedmodifiedtheY216phosphorylationofGSK3(pGSK3 Y216).CNQX alone had no effect on any of the phosphorylation targets at GSK3. ***p 0.001, one-sample t test (n 6). D, Bisindolylmaleimide prevented the alterations to pGSK3 S9 and pCRMP2 T514 caused by 300 nM KA (p 0.2 vs Bisind alone in both cases; unpaired t test, n 6). *p 0.05, **p 0.01, and ***p 0.001, one-sample t test (n 6). The data are expressed as the fold change (mean SEM) in the ratio of phosphorylated to total protein compared with untreated or DMSO (0.1%)-treated cells. Representative Western blots are shown above the graphs.

Article Snippet: Phosphorylated forms of the CRMP2 protein were detected using rabbit antibodies directed against p-CRMP2 T555 (1:200; ECM Biosciences) and p-CRMP2 T514 (1:500; Cell Signaling Technology) diluted in TBS-T containing 5% BSA.

Techniques: Phospho-proteomics, Western Blot, Activation Assay

Journal: Communications Biology

Article Title: GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

doi: 10.1038/s42003-019-0524-1

Figure Lengend Snippet: PTEN −/− releases CRMP2 from GSK3 inhibition. a Schematic illustration of results shown in experiments of ( b – j ). b , e Western blots of T514-phosphorylated (pCRMP2) and total CRMP2 in optic nerve lysates from PTEN +/+ and PTEN −/− mice without and with Gsk3α S/A (α S/A ), or Gsk3β S/A (β S/A ) knockins. Animals were either left untreated (con, b ) or subjected to ONC ( e ) 5 days before tissue isolation. Optic nerve segments proximal to the lesion site were used for the analysis. βIII-tubulin (tubulin) was used as a loading control. c , d , f , g Densitometric quantification of total CRMP2 and pCRMP2 relative to tubulin and normalized to PTEN +/+ on Western blots as depicted in ( b ) and ( e ). Levels of pCRMP2 were considerably compromised upon PTEN −/− . Decreased levels were reversed in α S/A and β S/A genotypes, while total CRMP2 remained unaltered in all experimental groups. Values represent means ± SEM of three optic nerves ( n = 3) per group. h Retinal flat mounts isolated from animals treated as described in ( b ) and ( e ). Retinae were stained for T 514 -pCRMP2 (red) and βIII-tubulin (tubulin, green). ONC and to a stronger extent PTEN −/− reduced pCRMP2 levels in RGC somas and axons compared with the PTEN +/+ , while levels remained high in PTEN −/− /GSK3 S/A mice. Scale bar: 50 μm. i Retinal flat mounts from PTEN +/+ mice either untreated or 5 days upon ONC that were injected with AAV-HA-CRMP2 T514/A 3 weeks before the injury. The activity of mTOR was analyzed by pS6 levels (red), while HA-staining (HA-CRMP2 T/A , green) identified transduced βIII-tubulin-positive RGCs (tubulin, cyan). Scale bar: 25 µm. j Quantification of pS6-positive RGCs in retinal flat mounts as described in ( i ). Values represent means ± SEM of three retinae per group ( n = 3). Significances of intergroup differences were evaluated using one-way analysis of variance (ANOVA) with Holm-Sidak post hoc test ( c , d , f , g ), or student's t -test ( j ). Treatment effects were compared with PTEN +/+ con: ## p < 0.01, n.s. = non-significant; and PTEN +/+ ONC: *** p < 0.001, n.s. = non-significant. Treatment effect in ( j ): * p < 0.05

Article Snippet: Phospho-GSK3α and CRMP2 primary antibodies were detected using conformation-specific horseradish peroxidase conjugated anti-rabbit (IgG) secondary antibody (1:4000, Cell Signaling Technologies).

Techniques: Inhibition, Western Blot, Isolation, Control, Staining, Injection, Activity Assay

Journal: Communications Biology

Article Title: GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

doi: 10.1038/s42003-019-0524-1

Figure Lengend Snippet: PTEN −/− stimulated neurite growth is CRMP2 dependent. a Representative pictures of retinal cell cultures plated on PDL. PTEN +/+ and PTEN −/− mice were subjected to ONC to transform RGCs into a regenerative state. Cells were cultured 5 days after that. Cultures were either untreated (−) or incubated with rapamycin (rap; 10 nM), lacosamide (LCM, 5 µM), or a combination of both. After 2 days in culture, RGCs were stained for phosphorylated S6 (pS6, red) and βIII-tubulin (tubulin, green). b Quantification of neurite growth of RGCs as described in ( a ) in the absence (PDL) or presence of CNS myelin (myelin). Some of the cultures were treated with the ROCK inhibitor Y27632 (Y27, 10 µM). Values represent means ± SEM of three ( n = 3) independent experiments with four wells as technical replicate per group and were normalized to the PTEN −/− , PDL group with an average neurite length of 6.12 µm per RGC of in total ~500 RGCs with ~30 RGCs showing neurite growth. c Quantification of the percentage of pS6-positive RGCs in cultures as described in ( a ). Values represent means ± SEM of 4 independent experiments ( n = 4). d ) Quantification of neurite length of βIII-tubulin-positive RGCs from PTEN +/+ , PTEN −/− , or PTEN −/− mice with a Gsk3α S/A (PTEN −/− /α S/A ) or Gsk3β S/A (PTEN −/− /β S/A ) knockin. Animals were subjected to ONC 5 days before culture preparation. Cultures were grown in the in the absence or presence of CNS myelin and either left untreated (−) or incubated with lacosamide (LCM, 5 µM) or Y27632 (Y27, 10 µM) and fixed after 48 h. Values represent means ± SEM of n = 4 independent experiments with four wells as technical replicate per group. Significances of intergroup differences were evaluated using two-way ( b , c ) or three-way ( d ) analysis of variance (ANOVA) with Holm-Sidak post hoc test. Treatment effects: compared with PTEN −/− , PDL: +++ p < 0.001; PTEN −/− , myelin: ### p < 0.001; or as indicated: * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. = non-significant

Article Snippet: Phospho-GSK3α and CRMP2 primary antibodies were detected using conformation-specific horseradish peroxidase conjugated anti-rabbit (IgG) secondary antibody (1:4000, Cell Signaling Technologies).

Techniques: Cell Culture, Incubation, Staining, Knock-In

Journal: Communications Biology

Article Title: GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

doi: 10.1038/s42003-019-0524-1

Figure Lengend Snippet: CRMP2 T/A overcomes GSK3β S/A -mediated inhibition on nerve regeneration. a Retinal flat-mounts from PTEN −/− mouse 3 weeks after intravitreal injection of AAV-Cre + AAV-HA-CRMP2 T/A , immunohistochemically stained for Cre (red), the HA-tag of CRMP2 T/A (HA-CRMP2 T/A , green), and βIII-tubulin (tubulin, blue). Scale bar: 50 μm. b Representative longitudinal optic nerve sections with regenerating, CTB-labeled axons 3 weeks after ONC. PTEN −/− or PTEN −/− /GSK3β S/A mice had received treatment with either AAV-GFP (GFP) or AAV-HA-CRMP2 T/A (CRMP2 T/A ) 3 weeks before ONC. Asterisks indicate the lesion site. Scale bar: 200 µm. c – f Magnifications of optic nerve sections as indicated in ( b ) at 2.5 mm beyond the lesion site. Scale bar: 100 µm. g Quantification of regenerating axons at indicated distances beyond the crush site in optic nerves as described in ( b ). CRMP2 T/A rescued adverse effect of Gsk3β S/A knockin on PTEN −/− -induced optic nerve regeneration. Values represent means ± SEM of at least seven animals per group (PTEN −/− /GFP, n = 13; PTEN −/− /β S/A /GFP, n = 7; PTEN −/− /CRMP2 T/A , n = 12; PTEN −/− /β S/A /CRMP2 T/A , n = 8). h βIII-tubulin-stained whole-mount retinae from AAV-HA-CRMP2 T/A treated PTEN −/− and PTEN −/− /GSK3β S/A mice as described in ( b ). Scale bar: 50 µm. i Quantification of RGCs per mm 2 in retinal whole-mounts as described in ( h ). The number of surviving RGCs 21 days after ONC were similar for the respective genotypes. The dashed bar represents values from PTEN −/− mice as shown in Fig. for comparison. All values represent means ± SEM of 4–10 retinae per group (PTEN −/− , n = 10; PTEN −/− /CRMP2 T/A , n = 6; PTEN −/− /β S/A /CRMP2 T/A , n = 4). Significances of intergroup differences were evaluated using two-way ( g ) or one-way ( i ) analysis of variance (ANOVA) followed by Holm-Sidak post hoc test. Treatment effects: ** p < 0.01, *** p < 0.001, n.s. = non-significant

Article Snippet: Phospho-GSK3α and CRMP2 primary antibodies were detected using conformation-specific horseradish peroxidase conjugated anti-rabbit (IgG) secondary antibody (1:4000, Cell Signaling Technologies).

Techniques: Inhibition, Injection, Staining, Labeling, Knock-In, Comparison

Journal: Journal of Neuroscience

Article Title: CRMP2 Tethers Kainate Receptor Activity to Cytoskeleton Dynamics during Neuronal Maturation

doi: 10.1523/jneurosci.3136-13.2013

Figure Lengend Snippet: Figure 1. Identification of KAR interacting proteins. A, Proteins interacting with the GluK5 KAR subunit were purified by immunoprecipitation of detergent-solubilized membrane frac- tions from mouse brain homogenate using rabbit anti-GluK5 antibody and separated by 2D electrophoresis. MW, molecular weight; IEF, isoelectric focusing. The specific protein spots re- cruited by rabbit anti-GluK5, identified by MALDI-TOF mass spectrometry, corresponded to DPYL2 or CRMP2 (spot 1), DPYL3 or CRMP4 (spot 2), and MARE-3 (spot 3). B, GluK5 antibody immunoprecipitates CRMP2 and CRMP4 in mouse brain extracts. C, Exogenously expressed CRMP2 and GluK5 colocalize in the plasma membrane of COS-7 cells transfected with N-terminal Myc-tagged GluK5 (blue), untagged GluK1-a, and a CRMP2 construct (yellow). Co- localization is shown in white. D, GST-pull-down analysis of CRMP2 from mouse brain extracts targeting the C-terminal domain of GluK5.

Article Snippet: Phosphorylated forms of the CRMP2 protein were detected using rabbit antibodies directed against p-CRMP2 T555 (1:200; ECM Biosciences) and p-CRMP2 T514 (1:500; Cell Signaling Technology) diluted in TBS-T containing 5% BSA.

Techniques: Purification, Immunoprecipitation, Membrane, Two-Dimensional Gel Electrophoresis, Molecular Weight, Mass Spectrometry, Clinical Proteomics, Transfection, Construct

Journal: Journal of Neuroscience

Article Title: CRMP2 Tethers Kainate Receptor Activity to Cytoskeleton Dynamics during Neuronal Maturation

doi: 10.1523/jneurosci.3136-13.2013

Figure Lengend Snippet: Figure 5. KARs regulate the phosphorylation state of CRMP2. A, In Western blots, KAR activation by 300 nM but not 3 M KA decreased T514 phosphorylation of CRMP2 (pCRMP2 T514) and increased the T555 phosphorylation of CRMP2 (pCRMP2 T555). CNQX (30 M) alone had no effect on the phosphorylation of CRMP2. **p 0.01 and ***p 0.001, one-sample t test (n 6). B, The increase in pCRMP2 T555 caused by 300 nM KA was blocked by the PKC inhibitor bisindolylmaleimide (Bisind; p 0.2683 vs Bisind alone, unpaired t test, n 6), whereas it was unaffected by the Rho-kinase inhibitor GSK429286 (5 M): ###p 0.001, unpaired t test (n 6); *p 0.05, **p 0.01, and ***p 0.001, one-sample t test (n 6). C, KA at300nMbutnot3MincreasedtheS9phosphorylationofGSK3(pGSK3 S9).NoneoftheKAconcentrationstestedmodifiedtheY216phosphorylationofGSK3(pGSK3 Y216).CNQX alone had no effect on any of the phosphorylation targets at GSK3. ***p 0.001, one-sample t test (n 6). D, Bisindolylmaleimide prevented the alterations to pGSK3 S9 and pCRMP2 T514 caused by 300 nM KA (p 0.2 vs Bisind alone in both cases; unpaired t test, n 6). *p 0.05, **p 0.01, and ***p 0.001, one-sample t test (n 6). The data are expressed as the fold change (mean SEM) in the ratio of phosphorylated to total protein compared with untreated or DMSO (0.1%)-treated cells. Representative Western blots are shown above the graphs.

Article Snippet: Phosphorylated forms of the CRMP2 protein were detected using rabbit antibodies directed against p-CRMP2 T555 (1:200; ECM Biosciences) and p-CRMP2 T514 (1:500; Cell Signaling Technology) diluted in TBS-T containing 5% BSA.

Techniques: Phospho-proteomics, Western Blot, Activation Assay

Journal: Journal of Neuroscience

Article Title: CRMP2 Tethers Kainate Receptor Activity to Cytoskeleton Dynamics during Neuronal Maturation

doi: 10.1523/jneurosci.3136-13.2013

Figure Lengend Snippet: Figure 7. KAR-induced delayed neuronal maturation is mediated by increased T555 phosphorylation of CRMP2, whereas the KAR-induced increase in neurite outgrowth is mediated by decreased phosphorylation of T514 of CRMP2. DRG neurons were infected with Sindbis virus encoding GFP-CRMP2 and phospho-null or phospho-mimetic mutants of CRMP2 T514 and CRMP2 T555

Article Snippet: Phosphorylated forms of the CRMP2 protein were detected using rabbit antibodies directed against p-CRMP2 T555 (1:200; ECM Biosciences) and p-CRMP2 T514 (1:500; Cell Signaling Technology) diluted in TBS-T containing 5% BSA.

Techniques: Phospho-proteomics, Infection, Virus

Journal: Journal of Neuroscience

Article Title: CRMP2 Tethers Kainate Receptor Activity to Cytoskeleton Dynamics during Neuronal Maturation

doi: 10.1523/jneurosci.3136-13.2013

Figure Lengend Snippet: Figure8. KAR-induceddelayofDRGneuronalmaturationinvolvesthedownregulationofCav2.2channeldensity.A,TheCav2.2 inhibitor -conotoxin (50 nM) occluded the inhibitory effect of 300 nM but not the facilitatory effect of 3 M KA on neuronal maturation. Data are expressed as the percentage of neurons (mean SEM) at each stage of maturation quantified from five different cultures with a minimum of 100 neurons analyzed per condition and culture. ***p 0.001, two-way ANOVA with Bonferroni’s post hoc test. B, KA exposure (300 nM, 20–36 h) reduced N-type Ca 2 currents elicited by the application of 50 ms voltage steps (10 mV increments) from a holding potential of 80 mV in patch-clamped GFP -DRG neurons. C, DRG neurons overexpressingGFP-CRMP2presentedasignificanthigherpeakCa 2currentdensity(pulsesfrom80to0mV)comparedwith GFP-expressing cells. Ca 2 current density was similar in cells overexpressing GFP-CRMP2 and the phospho-null mutant GFP- CRMP2 T555A, but significantly lower in cells expressing the phospho-mimic mutant GFP-CRMP2 T514D. The data are mean SEM ofpeakCa 2currentdensity(pA/pF)of30–37cellsfromfivetosixdifferentcultures.**p0.01,one-wayANOVAwithTukey’s multiple-comparisonstest.Dotsrepresentsinglecurrentdensityvaluesfromeachindividualcell.D,Summaryofthealterationsin Ca 2 current density induced by exposure to 300 nM KA in cells expressing GFP, GFP-CRMP2, GFP-CRMP2 T555A, and GFP- CRMP2 T555D.Thedataareexpressedasthepercentagechangerelativetotheuntreatedneurons(fivetosixdifferentcultures,six neurons minimum per condition per culture). **p 0.01, one-sample t test.

Article Snippet: Phosphorylated forms of the CRMP2 protein were detected using rabbit antibodies directed against p-CRMP2 T555 (1:200; ECM Biosciences) and p-CRMP2 T514 (1:500; Cell Signaling Technology) diluted in TBS-T containing 5% BSA.

Techniques: Expressing, Mutagenesis

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