Journal: Frontiers in immunology

Article Title: GSK3β Interacts With CRMP2 and Notch1 and Controls T-Cell Motility.

doi: 10.3389/fimmu.2021.680071

Figure Lengend Snippet: FIGURE 1 | Inhibition or depletion of GSK3b enhances T-cell motility. (A) Human primary T-cells were pre-treated with 5 µM CHIR-99021 for 2 h to inhibit GSK3b or DMSO (solvent control). Cells were then allowed to migrate on rICAM-1-coated plate and tracked in live cell microscopy. Spider plots showing the traced tracks of cells are presented. (B) Distance travelled by migrating T-cells over a 2-h period in mm. (C) Transwell chemotaxis of primary T-cells towards the chemokine SDF-1a, as determined using CIM-Plate 16 and real-time impedance-based measurements by the RTCA instrument. (D) HuT78 T-cells were nucleofected with 100 nM siRNA targeting GSK3b or non-specific (NS) siRNA. After 72 h, cells were lysed and the expression levels of GSK3b was determined by Western immunoblotting. Blots were re-probed for GAPDH as a loading control. The relative densitometry values for GSK3b were determined and plotted (mean ± SEM). (E) Transwell chemotaxis of control (NS siRNA) and GSK3b-depleted (GSK3b siRNA) HuT78 T-cells towards SDF-1a was determined using CIM-Plate 16 and real-time impedance-based measurements. Baseline was drawn automatically for wells without SDF-1a. Data represent at least three independent experiments. ***p < 0.001.

Article Snippet: The human T-cell line HuT78 was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in GibcoTM RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate and antibiotics (penicillin 100 units/ml, streptomycin 100 mg/ml) at 37°C and 5% CO2 as described (11).

Techniques: Inhibition, Solvent, Control, Microscopy, Chemotaxis Assay, Expressing, Western Blot

Journal: Frontiers in immunology

Article Title: GSK3β Interacts With CRMP2 and Notch1 and Controls T-Cell Motility.

doi: 10.3389/fimmu.2021.680071

Figure Lengend Snippet: FIGURE 2 | Identification of GSK3b interactome in migrating T-cells. (A) Schematic representation of workflow employed in mass spectrometry-based proteomics analysis of GSK3b interacting proteins in human HuT78 T-cells. (B) Image output from IPA Ingenuity Knowledge Base indicating statistically probable interactions of GSK3b in LFA-1-stimulated T-cells. Proteins depicted in red are high abundance in migrating T-cells and proteins shown in green are low in abundance, compared to resting T-cells. The protein symbols in purple highlight proteins curated from Ingenuity Knowledge Base with potential roles in cellular movement and modelled relationships among known functional networks in immune cells. The type of proteins, network, shapes, and relationships in IPA are provided in the legends.

Article Snippet: The human T-cell line HuT78 was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in GibcoTM RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate and antibiotics (penicillin 100 units/ml, streptomycin 100 mg/ml) at 37°C and 5% CO2 as described (11).

Techniques: Mass Spectrometry, Functional Assay

Journal: Frontiers in immunology

Article Title: GSK3β Interacts With CRMP2 and Notch1 and Controls T-Cell Motility.

doi: 10.3389/fimmu.2021.680071

Figure Lengend Snippet: FIGURE 3 | GSK3b-Notch1 interactions in motile T-cells. (A, B) Human primary T-cells, untreated or pre-treated with 10 mM DAPT, were stimulated to migrate on rICAM-1-coated plates for 30 min and lysed. Cellular lysates were Western immunoblotted for pGSK3b-S9, GSK3b, NICD and total Notch1. The expression levels of proteins were quantified by determining relative densitometry (mean ± SEM) of pGSK3b-S9/GSK3b and NICD/Notch1. (C) Resting and LFA-1-stimulated HuT78 T- cells were immunostained with anti-pGSK3b-S9/Alexa Fluor® 488 (green), anti-NICD/Alexa Fluor® 568 (red), Phalloidin-Alexa Fluor® 647 (actin, cyan) and Hoechst (nucleus, blue) and then imaged by confocal laser scanning microscopy, 63X oil objective. Overlay images with intensity profiles (replotted using the GraphPad Prism software) of nucleus, pGSK3b-S9, NICD and actin are shown. Scale bar = 5 mm. (D) T-cells (2×104 cells/per well) were allowed to migrate on rICAM-1-coated 96- well plate for multiple time-points up to 2 h and fixed. Cells were stained for pGSK3b-S9 or GSK3b and imaged by an automated IN Cell Analyzer 2200 microscope using 20X objective (6 fields/well). Cellular images containing >500 cells/well were subsequently analysed cell-by-cell by the IN Cell Investigator software. (E) HuT78 T-cells were pre-treated with 10 µM DAPT, nocodazole (positive control), or DMSO (solvent control) and then allowed to migrate on rICAM-1-coated E-Plate 16. Cell migration was recorded in real-time using impedance-based measurements by the RTCA instrument. Wells without cells were used to automatically draw the baseline. Data represent at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared to corresponding resting T-cells (0 min) control; RFU, relative fluorescent intensity.

Article Snippet: The human T-cell line HuT78 was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in GibcoTM RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate and antibiotics (penicillin 100 units/ml, streptomycin 100 mg/ml) at 37°C and 5% CO2 as described (11).

Techniques: Western Blot, Expressing, Confocal Laser Scanning Microscopy, Software, Staining, Microscopy, Positive Control, Solvent, Control, Migration

Journal: Frontiers in immunology

Article Title: GSK3β Interacts With CRMP2 and Notch1 and Controls T-Cell Motility.

doi: 10.3389/fimmu.2021.680071

Figure Lengend Snippet: FIGURE 4 | GSK3b-CRMP2 interactions in motile T-cells. (A) Human primary T-cells were stimulated to migrate on rICAM-1-coated plates for 30 min and lysed. Cellular lysates from unstimulated or LFA-1/ICAM-1-stimulated T-cells were immunoprecipitated (IP) with anti-CRMP2 and Western immunoblotted for GSK3b and CRMP2. The amounts of GSK3b co-precipitating with CRMP2 were quantified by densitometry analysis (mean ± SEM). (B) Human primary T-cells, untreated or pre- treated with 10 mM DAPT, were stimulated to migrate on rICAM-1-coated plates for 30 min and lysed. (C) Human primary T-cells were treated with 5 mM CHIR- 99021 or an equivalent amount of DMSO (solvent control) for 2 h and lysed. Cellular lysates in “B” and “C” were Western immunoblotted for pCRMP2-T514 and total CRMP2. The relative expression levels of pCRMP2-T514 were quantified by densitometry analysis (mean ± SEM). (D) Resting and LFA-1-stimulated migrating HuT78 T-cells were immunostained with anti-pGSK3b-S9/Alexa Fluor® 488 (green), anti-CRMP2/Alexa Fluor® 568 (red), Phalloidin-Alexa Fluor® 647 (actin, cyan) and Hoechst (nucleus, blue), and then imaged by confocal laser scanning microscopy, 63X oil objective. Overlay images with intensity profiles (replotted using the GraphPad Prism software) of nucleus, pGSK3b-S9, CRMP2 and actin are shown. Scale bar = 5 mm. (E) T-cells (2×104 cells/per well) were allowed to migrate on rICAM-1-coated 96-well plate for multiple time-points up to 2 h and fixed. Cells were stained for CRMP2 or pGSK3b-S9 and imaged by an automated IN Cell Analyzer 2200 microscope using 20X objective (6 fields/well). Cytoplasmic and nuclear intensities of CRMP2 and pGSK3b-S9, respectively, from >500 cells/well were subsequently analysed cell-by-cell by the IN Cell Investigator software. Each dot represents mean intensity values from >500 cells. Data represent at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared to resting T-cells (0 min) control; RFU, relative fluorescent intensity.

Article Snippet: The human T-cell line HuT78 was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in GibcoTM RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate and antibiotics (penicillin 100 units/ml, streptomycin 100 mg/ml) at 37°C and 5% CO2 as described (11).

Techniques: Immunoprecipitation, Western Blot, Solvent, Control, Expressing, Confocal Laser Scanning Microscopy, Software, Staining, Microscopy

Journal: Frontiers in immunology

Article Title: GSK3β Interacts With CRMP2 and Notch1 and Controls T-Cell Motility.

doi: 10.3389/fimmu.2021.680071

Figure Lengend Snippet: FIGURE 5 | CRMP2 colocalization to the MTOC and the effect of CRMP2 depletion on T-cell motility. (A) Resting and LFA-1-stimulated HuT78 T-cells were immunostained with anti-CRMP2/Alexa Fluor® 568 (red), anti-pericentrin/Alexa Fluor® 488 (green), phalloidin-Alexa Fluor® 647 (actin, pink) and Hoechst (nucleus, blue). Cells were then imaged by confocal laser scanning microscopy, array scan 63X objective. (B) 3D and 2.5D projections, zoomed overlay and intensity profiles (replotted using the GraphPad Prism software) of CRMP2 and pericentrin are shown. (C) Pearson Correlation Coefficient between CRMP2 and pericentrin was assessed using Carl Zeiss ZEN Black software. Each dot represents a single T-cell, and the images were taken from at least three independent experiments; n=20 for resting T-cells and n=15 for migrating T-cells; error bar, mean ± SEM. (D) PBL T-cells were nucleofected with 100 nM siRNA targeting CRMP2 or non-specific (NS) siRNA. After 72 h, cells were lysed, Western immunoblotted and probed for CRMP2. Blots were re-probed for GAPDH as a loading control. Relative densitometry values for CRMP2/GAPDH were determined and plotted (mean ± SEM). (E) The control (NS siRNA) and CRMP2-depleted (CRMP2 siRNA) PBL T-cells were treated with 5 µM CHIR-99021 for 2 h and then allowed to migrate on rICAM-1-coated E-Plate 16 for 4 h. Cell migration was recorded in real-time using impedance-based measurements by the RTCA instrument. Wells without cells were used to automatically draw the baseline. Data represent at least three independent experiments. ***p < 0.001.

Article Snippet: The human T-cell line HuT78 was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in GibcoTM RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate and antibiotics (penicillin 100 units/ml, streptomycin 100 mg/ml) at 37°C and 5% CO2 as described (11).

Techniques: Confocal Laser Scanning Microscopy, Software, Western Blot, Control, Migration

Journal: Experimental dermatology

Article Title: RARα/RXR Synergism Potentiates Retinoid Responsiveness in Cutaneous T Cell Lymphoma Cell Lines

doi: 10.1111/exd.13348

Figure Lengend Snippet: RARα activation prompts β7 expression and function in CTCL. (a) MJ or HuT78 cells were treated with DMSO or 2×EC50 of well-established RAR isotype agonists for 24, 48 or 72 hrs. Surface β7 integrin expression was determined through flow cytometry and displayed as mean fluorescence intensity (MFI). (b) MJ or HuT78 cells were cultured in the presence of DMSO, 100 nM ATRA, 2×EC50 RARα, β, or γ agonists for the indicated time. Static cell adhesion assays were assessed on 0.75 μg/ml of the β7 specific ligand MAdCAM-1. (c) MJ cells were cultured with the indicated RAR isotype antagonists at 500 nM for 24 hrs. Cells were further subcultured for an additional 24 hrs in the presence of 200 nM ATRA. Static cell adhesion assays were conducted as described before. Ordinate represents data that have been normalized to adhesion levels obtained with ATRA in the absence of antagonist. (d) Adhesion assays were repeated as previously described in (B) with the non-CTCL cell line, Jurkat. (e) Whole cell lysates (30 μg/lane) of CTCL or non-CTCL (Jurkat) cell lines were examined for the presence and relative abundance of the various RAR receptor isotypes.

Article Snippet: Cell culture The human T leukemia/lymphoma cell lines Jurkat and CCRFCEM and human cutaneous T cell lymphoma lines HuT78, MJ and HuT102 were purchased from ATCC (Manassas, VA).

Techniques: Activation Assay, Expressing, Flow Cytometry, Fluorescence, Cell Culture

Journal: Experimental dermatology

Article Title: RARα/RXR Synergism Potentiates Retinoid Responsiveness in Cutaneous T Cell Lymphoma Cell Lines

doi: 10.1111/exd.13348

Figure Lengend Snippet: RARα/RXR coactivation induces CTCL cell adhesion in a synergistic manner. (a) MJ or HuT78 cells were cultured in the presence of RAR isotype-specific agonists (1×EC50) alone or in combination with 5 nM Bexarotene for 24 hrs. Cells were then assessed for adhesion to 0.75 μg/ml of MAdCAM-1. (b) CTCL cell line SeAx or MyLa cells were cultured with DMSO, 2×EC50 RARα agonist, 100 nM Bexarotene, or a combination of agonists for 72 hrs. The extent of adhesion was determined on the integrin β7-ligand MAdCAM-1. (c) HuT78 cells were cultured in the presence of an RAR isotype-specific agonist (1×EC50) alone or in combination with 100 nM of the pan-RXR activator SR11237 for 48 hrs. Cell adhesion was then determined as previously described. (d) Real-time quantitative PCR was conducted with primers that amplify regions encoding the human integrin α4 or β7 subunits. Templates were derived from MJ cells cultured with vehicle or RARα/RXR agonists. The ordinate reflects normalized data obtained by dividing values for RARα/RXR agonists with those from vehicle treated samples.

Article Snippet: Cell culture The human T leukemia/lymphoma cell lines Jurkat and CCRFCEM and human cutaneous T cell lymphoma lines HuT78, MJ and HuT102 were purchased from ATCC (Manassas, VA).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: Experimental dermatology

Article Title: RARα/RXR Synergism Potentiates Retinoid Responsiveness in Cutaneous T Cell Lymphoma Cell Lines

doi: 10.1111/exd.13348

Figure Lengend Snippet: CCR9-mediated chemotaxis to CCL25 is enhanced by RARα/RXR activity. (a) CTCL cell lines MJ and HuT78 or the non-CTCL T cell lines CCRFCEM and Jurkat were treated with 1 μM ATRA or 1 μM Bexarotene for 72 hrs. Percentage of CCR9+ cells was detected through flow cytometry. (b) MJ and HuT78 cells were cultured with DMSO, 1 μM ATRA or 10 μM Bexarotene for 72 hrs. Directed migration towards 100 ng/ml of CCL25 was determined and displayed on the ordinate in arbitrary units (AU). (c) HuT78 or HuT102 cells were incubated with the vehicle DMSO, 1×EC50 RARα agonist, 5 nM Bexarotene or combination of the two agonists. As described in (b), the relative extent of chemotaxis towards 100 ng/ml of CCL25 was measured.

Article Snippet: Cell culture The human T leukemia/lymphoma cell lines Jurkat and CCRFCEM and human cutaneous T cell lymphoma lines HuT78, MJ and HuT102 were purchased from ATCC (Manassas, VA).

Techniques: Chemotaxis Assay, Activity Assay, Flow Cytometry, Cell Culture, Migration, Incubation

Journal: Experimental dermatology

Article Title: RARα/RXR Synergism Potentiates Retinoid Responsiveness in Cutaneous T Cell Lymphoma Cell Lines

doi: 10.1111/exd.13348

Figure Lengend Snippet: RARα/RXR cooperatively induce apoptosis and inhibit CTCL cell proliferation. (a) HuT78, MJ cells, MyLa cells or (b) the non-CTCL cell line Jurkat were treated with DMSO, 1×EC50 RARα agonist, 5 nM Bexarotene or combination thereof for 48 hrs. Data shown are the percentage of Annexin V positive cells. (c) Total cell lysates (40 μg/lane) from CTCL or non-CTCL cell lines were assessed for the indicated apoptotic protein marker. Lysates were derived from untreated cells or cells treated with RARα/RXR agonists for 72 hrs. (d) HuT78 or SeAx cells were incubated with DMSO or retinoids as described above for 48 hrs or 72 hrs, respectively. BrdU cell proliferation assays were conducted and the relative extent of proliferation is shown as arbitrary units (AU).

Article Snippet: Cell culture The human T leukemia/lymphoma cell lines Jurkat and CCRFCEM and human cutaneous T cell lymphoma lines HuT78, MJ and HuT102 were purchased from ATCC (Manassas, VA).

Techniques: Marker, Derivative Assay, Incubation