Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation

doi: 10.1073/pnas.0805963106

Figure Lengend Snippet: LOK is abundant at the lymphocyte plasma membrane and colocalized with cpERM. (A) Tabulation of kinase peptides detected in mass spectrometric analysis of fractions from human lymphocytes. Fractions: MMV, membrane/microvillus; PNL, postnuclear lysate. (B) Colocalization of cpERM (green) and LOK (red) at human lymphocyte plasma membrane detected by immunofluorescence.

Article Snippet: The following antibodies were used: carboxy-terminal LOK rabbit pAb (Bethyl), β-actin mouse mAb (Sigma-Aldrich), cpERM mouse mAb from BD Biosciences. cpERM mAb was conjugated with AlexaFluor647 (Invitrogen).

Techniques: Immunofluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation

doi: 10.1073/pnas.0805963106

Figure Lengend Snippet: Evidence in Jurkat cells that LOK phosphorylates ERM and that ERM phosphorylation impedes migration. (A) Jurkat cells were transfected with LOK KD or GFP control and cpERM was detected by flow cytometry after 18 h. Cells from each preparation were separated into 4 categories by absolute GFP fluorescence i.e., nontransfected (0–10), low level expression (10–100), medium level expression (100–1000), and high level expression (>1000). (B) Same transfected cells as panel A analyzed for transmigration in response to SDF-1. (C) Jurkat cells were transfected with pseudophosphorylated (T558D), nonphosphorylated (T558A), or WT moesin GFP fusion protein and analyzed for transmigration in response to SDF-1.

Article Snippet: The following antibodies were used: carboxy-terminal LOK rabbit pAb (Bethyl), β-actin mouse mAb (Sigma-Aldrich), cpERM mouse mAb from BD Biosciences. cpERM mAb was conjugated with AlexaFluor647 (Invitrogen).

Techniques: Migration, Transfection, Flow Cytometry, Fluorescence, Expressing, Transmigration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation

doi: 10.1073/pnas.0805963106

Figure Lengend Snippet: Hematopoietic cells from LOK KO mice have decreased ERM phosphorylation without major change in lymphocyte microvilli. (A) Example of WB detection of cpERM, and actin in lysates from hematopoietic cells from different tissues, LN, lymph nodes; BM, bone marrow. (B) Comparison of cpERM level in spleen cells from 10 pairs of LOK KO mice and their heterozygote littermates. (C) SEM images of lymph node lymphocytes from a LOK KO mouse and its littermate.

Article Snippet: The following antibodies were used: carboxy-terminal LOK rabbit pAb (Bethyl), β-actin mouse mAb (Sigma-Aldrich), cpERM mouse mAb from BD Biosciences. cpERM mAb was conjugated with AlexaFluor647 (Invitrogen).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation

doi: 10.1073/pnas.0805963106

Figure Lengend Snippet: LOK depletion results in alteration of migration and polarization, but not in adhesion. (A) Splenic lymphocytes from LOK KO mice and littermate controls were analyzed for transmigration to SDF-1. (B) Splenic lymphocytes from LOK KO mice and littermate controls were analyzed for adhesion to VCAM-1 or ICAM-1. Assays were performed in the presence or absence of 1 mM MnCl2, which promotes conversion of integrins into active conformation. (C) Splenic lymphocytes from LOK KO mice and littermate controls were analyzed for cpERM phosphorylation under 4 standard conditions: R, resting, no treatment; SDF-1, 1 min after 100 ng/mL SDF-1 to assess physiological dephosphorylation; STA, 5 min after high concentration (500 nM) staurosporine to assess maximal dephosphorylation; CA, 5 min after 50 μM phosphatase inhibitor calyculin A to maximize phosphorylation; cpERM was assessed both by WB (Upper subpanel) and flow cytometry (Lower subpanel). (D) cpERM levels were measured for splenic lymphocytes from LOK KO mice and littermate controls before and after various times of stimulation with SDF-1. (E) Cells as in panel D, but stained with phalloidin to detect F-actin and scored blind for polarization. (F) Cells as in panel E, but analyzed for increase in F-actin by flow cytometry after 15 s of stimulation.

Article Snippet: The following antibodies were used: carboxy-terminal LOK rabbit pAb (Bethyl), β-actin mouse mAb (Sigma-Aldrich), cpERM mouse mAb from BD Biosciences. cpERM mAb was conjugated with AlexaFluor647 (Invitrogen).

Techniques: Migration, Transmigration Assay, De-Phosphorylation Assay, Concentration Assay, Flow Cytometry, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: The Transcription Factor ETV5 Mediates BRAFV600E-Induced Proliferation and TWIST1 Expression in Papillary Thyroid Cancer Cells 1

doi: 10.1016/j.neo.2018.09.003

Figure Lengend Snippet: Meta-analysis of ETV5 expression in cohorts of PTC patients showing its upregulation in comparison to normal tissues samples. (A-D) Analysis of ETV5 expression in the thyroid of patients harboring a BRAF (V600E) mutation in comparison to normal individuals using the NCBI GEO database (29-32). (E) Analysis of ETV5 expression in thyroid lesions harboring a BRAF (V600E) or a RAS mutation in comparison to normal samples using the NCBI GEO database (29, 32). (F) Analysis of ETV5 expression in BRAF (V600E) or RAS-mutated thyroid cancer samples in comparison to normal contralateral tissue using TCGA (33).

Article Snippet: To transiently overexpress ETV5, KTC1 and Nthy-ori 3-1 cells were transfected with pCMV6 expressing both ETV5 and GFP (#RG200366, Origene, Rockville, MD), or the empty expression vector pCMV6-GFP (#PS100010) as control.

Techniques: Expressing, Comparison, Mutagenesis

Journal: Neoplasia (New York, N.Y.)

Article Title: The Transcription Factor ETV5 Mediates BRAFV600E-Induced Proliferation and TWIST1 Expression in Papillary Thyroid Cancer Cells 1

doi: 10.1016/j.neo.2018.09.003

Figure Lengend Snippet: Immunohistochemistry of ETV5 expression in normal and PTC samples. (A) ETV5 expression in a normal thyroid sample. Most follicular cells are negative or weakly positive for ETV5 expression, which is confined to the nucleus. (B) Magnification of A. Parafollicular cells (C cells) appear positive (arrows). (C and D) ETV5 is strongly upregulated in two different PTC samples, with expression in both nucleus and cytoplasm.

Article Snippet: To transiently overexpress ETV5, KTC1 and Nthy-ori 3-1 cells were transfected with pCMV6 expressing both ETV5 and GFP (#RG200366, Origene, Rockville, MD), or the empty expression vector pCMV6-GFP (#PS100010) as control.

Techniques: Immunohistochemistry, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: The Transcription Factor ETV5 Mediates BRAFV600E-Induced Proliferation and TWIST1 Expression in Papillary Thyroid Cancer Cells 1

doi: 10.1016/j.neo.2018.09.003

Figure Lengend Snippet: Rates of proliferation of KTC1 cells after different treatments. (A) Inhibition of the MAPK pathway using the pan-ERK inhibitor GDC0994 (10 μM) will decrease the rate of proliferation of KTC1 cells in comparison to cells treated with DMSO alone, while the TGFbeta receptor inhibitor LY210976 (10 μM) has less effect. (B) Inhibition of the PI3K-AKT pathway will also decrease the rate of proliferation of KTC1 cells in comparison to DMSO alone. MK2206-2HCL, a pan-AKT inhibitor, was used at final concentrations of 10 μM. LY294002, a pan-PI3K inhibitor, was used at a final concentration of 20 μM; mTOR inhibition using rapamycin (20 nM) confirms that the PI3K-AKT pathway is used for KTC1 cell proliferation. (C) Downregulation of ETV5 expression by RNAi causes significant reduction of the rate of proliferation of KTC1 cells in comparison to control cultured (mock and scrambled conditions). (D) Downregulation of ETV5 expression with siRNA causes simultaneous downregulation of CCND1 and CCND2 expression in KTC1 cells. ** P < .005, *** P < .001.

Article Snippet: To transiently overexpress ETV5, KTC1 and Nthy-ori 3-1 cells were transfected with pCMV6 expressing both ETV5 and GFP (#RG200366, Origene, Rockville, MD), or the empty expression vector pCMV6-GFP (#PS100010) as control.

Techniques: Inhibition, Comparison, Concentration Assay, Expressing, Control, Cell Culture

Journal: Neoplasia (New York, N.Y.)

Article Title: The Transcription Factor ETV5 Mediates BRAFV600E-Induced Proliferation and TWIST1 Expression in Papillary Thyroid Cancer Cells 1

doi: 10.1016/j.neo.2018.09.003

Figure Lengend Snippet: ETV5 is expressed downstream of the MAPK pathway. (A) ETV5 mRNA expression is strongly downregulated by MAPK pathway inhibition but not by PI3K/AKT pathway inhibition. (B) Western blot analysis confirming that ETV5 protein expression parallels expression/phosphorylation of ERK1/2. The chosen antibody (Thermo Fisher) detects the two predicted alternative spliced isoforms of human ETV5 (Uniprot kDa 57.8 and kDa 62.4). ERKi = ERK1/2 inhibitor (GDC0994); PI3Ki = PI3K inhibitor (LY294002). (C) Significant downregulation of ETV5 mRNA expression in KTC1 cells after 48-hour exposure to 1 μM vemurafenib, a specific BRAF (V600E) inhibitor. (D) Significant downregulation of ETV5 protein expression in KTC1 cells after 48-hour exposure to 1 μM vemurafenib. (a) Quantitation of ETV5 protein expression after standardization with ACTB, showing a 55% reduction after vemurafenib treatment. (b) Representative Western blot showing ETV5 protein expression without (A) or with 1 μM vemurafenib (B). The antibody used (Genentech) distinguishes only one isoform. **** P < .0001, ** P < .005, n ≥ 3.

Article Snippet: To transiently overexpress ETV5, KTC1 and Nthy-ori 3-1 cells were transfected with pCMV6 expressing both ETV5 and GFP (#RG200366, Origene, Rockville, MD), or the empty expression vector pCMV6-GFP (#PS100010) as control.

Techniques: Expressing, Inhibition, Western Blot, Phospho-proteomics, Quantitation Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: The Transcription Factor ETV5 Mediates BRAFV600E-Induced Proliferation and TWIST1 Expression in Papillary Thyroid Cancer Cells 1

doi: 10.1016/j.neo.2018.09.003

Figure Lengend Snippet: Expression of some EMT markers correlates with ETV5. (A) SNAI1 expression is downregulated in KTC1 cells following knockdown of ETV5 by RNAi. (B) SNAI1 expression in immortalized thyrocytes is downregulated after knockdown of ETV5 by RNAi. (C) Downregulation of the EMT markers TWIST1, SNAI2, CDH2, and TGFBR1 is observed after ETV5 knockdown with ETV5 shRNA.

Article Snippet: To transiently overexpress ETV5, KTC1 and Nthy-ori 3-1 cells were transfected with pCMV6 expressing both ETV5 and GFP (#RG200366, Origene, Rockville, MD), or the empty expression vector pCMV6-GFP (#PS100010) as control.

Techniques: Expressing, Knockdown, shRNA

Journal: Neoplasia (New York, N.Y.)

Article Title: The Transcription Factor ETV5 Mediates BRAFV600E-Induced Proliferation and TWIST1 Expression in Papillary Thyroid Cancer Cells 1

doi: 10.1016/j.neo.2018.09.003

Figure Lengend Snippet: ETV5 downregulation impairs gap closure in a scratch/wound healing assay. (A + B) Gap closure of wound healing by control nontreated KTC1 cells (representative sample). (C + D) Gap closure of control KTC1 cells after transduction of an shRNA empty vector showing no significant difference with nontreated cells. Gap nears closure at approximately 36 hours (dotted line) after scratches were made. (E + F) Delay in gap closure of KTC1 cells after downregulation of ETV5 expression through shRNA vector transduction. (G) Quantitation of gap closure over time. The rate of closure is significantly lower when ETV5 expression is knocked down.

Article Snippet: To transiently overexpress ETV5, KTC1 and Nthy-ori 3-1 cells were transfected with pCMV6 expressing both ETV5 and GFP (#RG200366, Origene, Rockville, MD), or the empty expression vector pCMV6-GFP (#PS100010) as control.

Techniques: Wound Healing Assay, Control, Transduction, shRNA, Plasmid Preparation, Expressing, Quantitation Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: The Transcription Factor ETV5 Mediates BRAFV600E-Induced Proliferation and TWIST1 Expression in Papillary Thyroid Cancer Cells 1

doi: 10.1016/j.neo.2018.09.003

Figure Lengend Snippet: ETV5 levels of expression influence cell invasion and cell morphology. (A + B) KTC1 and KTC1-VA7 cell migration versus invasion. ETV5 downregulation does not significantly impair cell migration in a Boyden chamber assay (A). Cell invasion through a collagen layer is significantly reduced when ETV5 expression is downregulated (B). (C-E) Levels of ETV5 expression significantly alter KTC1 cell morphology in culture. Control cells (C) and cells with a downregulated ETV5 expression (D) retain a cuboidal or fusiform shape, while cells that overexpress ETV5 (E) increase in size and exhibit prominent actin stress fibers (stars). Cells were stained with DAPI (nuclear staining) and DyLight 488 Phalloidin (ACTB, β-actin).

Article Snippet: To transiently overexpress ETV5, KTC1 and Nthy-ori 3-1 cells were transfected with pCMV6 expressing both ETV5 and GFP (#RG200366, Origene, Rockville, MD), or the empty expression vector pCMV6-GFP (#PS100010) as control.

Techniques: Expressing, Migration, Boyden Chamber Assay, Control, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: The Transcription Factor ETV5 Mediates BRAFV600E-Induced Proliferation and TWIST1 Expression in Papillary Thyroid Cancer Cells 1

doi: 10.1016/j.neo.2018.09.003

Figure Lengend Snippet: ETV5 binds to the TWIST1 promoter. (A) Downregulation of TWIST1 expression after ETV5 shRNA vector transduction. (B) Analysis of the human TWIST1 promoter with respect to ETV5 putative binding sites. The human TWIST1 proximal promoter contains six ETV5 consensus sequences for possible binding of ETV5 to DNA. Regions amplified with three different primer pairs (PP1, PP2, PP3) are represented, which were used after ChIP (Supplemental Table 3). (C) ChIP-qPCR analysis of the human TWIST1 promoter showing binding of ETV5 in the region bp −179 to −87 probed with primer pair 3 (PP3). Primer pair 1 (PP1) and primer pair 2 (PP2) did not amplify, indicating no chromatin immunoprecipitation. Similarly, no amplification was obtained with PPC = control primer pair (~−6000 bp from TSS). Results show fold enrichment in comparison to immunoprecipitation using IgG instead of ETV5 antibody and are given as means ± standard error of the mean. ** P < .01 , *** P < .001.

Article Snippet: To transiently overexpress ETV5, KTC1 and Nthy-ori 3-1 cells were transfected with pCMV6 expressing both ETV5 and GFP (#RG200366, Origene, Rockville, MD), or the empty expression vector pCMV6-GFP (#PS100010) as control.

Techniques: Expressing, shRNA, Plasmid Preparation, Transduction, Binding Assay, Amplification, ChIP-qPCR, Chromatin Immunoprecipitation, Control, Comparison, Immunoprecipitation

Journal: Breast Cancer Research : BCR

Article Title: LIN7A is a major determinant of cell-polarity defects in breast carcinomas

doi: 10.1186/s13058-016-0680-x

Figure Lengend Snippet: Polarity abnormalities in invasive micropapillary carcinoma (IMPC). a Representative views of cell-division control protein 42 (CDC42), cis-Golgi marker (GM130) and occludin (OCLN) immunostainings in three normal ducts ( left panel ) and in three IMPC ( right panel ). Scale bars 10 μm, L lumen. G × 400 magnification. b Analysis of polarity protein expression and subcellular localization in 24 IMPC. Expression and cellular localization (sub-apical, apical, cytoplasmic, membranous, cell/cell: lateral membrane staining between two cells and basolateral) of polarity proteins was compared to that in normal cells. p-ERM phospho-ezrin-radixin-moesin, p-aPKCζ phospho-atypical PKC, PALS1 protein associated with Lin seven 1, SCRIB Scribble, ZO-1 zonula occludens 1

Article Snippet: Antibodies used were directed against phospho-ezrin-radixin-moesin (p-ERM,1:200 dilution, pH6.1, BD Biosciences), GM130 (clone 35, 1:100 dilution, pH9, BD Biosciences), cell division control protein 42 (CDC42, 1:300 dilution, pH6.1, Lifespan Biosciences, Seattle, WA, USA), phospho-atypical PKCζ (p-aPKCζ, clone190D10, 1:250 dilution, pH6.1, Cell Signaling), protein associated with Lin seven 1 (PALS1, 1:50 dilution, pH9, Lifespan Biosciences), occludin (OCLN, 1:200 dilution, pH6.1, Lifespan Biosciences), Scribble (SCRIB, 1:50 dilution, pH6.1, Santa Cruz Biotechnology, Dallas, TX, USA), zonula occludens 1 (ZO-1, 1:500 dilution, pH6.1, BD Biosciences), β-catenin (clone 14/Beta-catenin, 1:200 dilution, pH6.1, BD Biosciences), E-cadherin (clone 4A2C7, 1:100 dilution, pH9, Invitrogen, Camarillo, CA, USA), KI-67 (clone MIB1, 1:100 dilution, pH6.1, Dako A/S) or active-caspase-3 (1:250 dilution, pH6.1, Cell Signaling).

Techniques: Marker, Expressing, Staining

Journal: Molecular neurobiology

Article Title: A2A adenosine receptor regulates the human blood brain barrier permeability

doi: 10.1007/s12035-014-8879-2

Figure Lengend Snippet: (A) Western blot on phosphorylated ERM (p-ERM) and phosphorylated focal adhesion kinase (p-FAK) were performed in Lexiscan treated primary human brain endothelial cells (HBMVEC) upto 30 minutes. (B) Intensity of the band of phosphorylated form was divided by that of total protein to obtain the ratio. Ratio from treated group was normalized by GAPDH and was divided by the value of control group at each time point (from A) and plotted as graph. (C) Western blot analysis of p-ERM, p-FAK were performed in NECA treated HBMVEC upto 120 minutes. (D) Intensity of the band of phosphorylated form was divided by that of total protein to obtain the ratio. Ratio from treated group was normalized by GAPDH and was divided by the value of control group at each time point (from C) and plotted as graph. (E) Western blot analysis of p-ERM in Lexiscan treated HCMEC-D3 cells up to 60 minutes. (F) Intensity of the band of phosphorylated form was divided by that of total protein to obtain the ratio. Normalized ratio by GAPDH from treated group was divided by that of control group at each time point (from E) and plotted as graph. (G) Western blot analysis of p-ERM in NECA treated HCMEC D3 cells up to 120 minutes. (H) Intensity of the band of phosphorylated form was divided by that of total protein to obtain the ratio. Normalized ratio by GAPDH from treated group was divided by that of control group at each time point (from G) and plotted as graph. In all images M indicates media only control.

Article Snippet: Similar kinetics was observed in HCMEC-D3 cells, which showed that phosphorylated ERM decreased very rapidly after Lexiscan treatment (within 5 minutes), and this decrease was maintained up to 60 minutes ( ).

Techniques: Western Blot