Journal: Cancers

Article Title: SIRT2 Contributes to the Resistance of Melanoma Cells to the Multikinase Inhibitor Dasatinib

doi: 10.3390/cancers11050673

Figure Lengend Snippet: Cellular WM853 and MDA-MB-435S clone characteristics. ( a ) SIRT2 expression in SCW3 and SSW30 clones of WM853 cells and SCM1 and SSM15 clones of MDA-MB-435S as evidenced by Western blotting. ( b ) The cytotoxic effects of dasatinib treatment (48 h) on melanoma SCW3 and SSW30 clones of the WM853 cell line, as evidenced using the neutral red assay, mean ± SD, ( n = 3, independent experiments) ( c ) The cytotoxic effects of dasatinib treatment (48 h) on melanoma SCM1 and SSM15 clones of the MDA-MB-435S cell line, as evidenced using the neutral red assay, mean ± SD, ( n = 3, independent experiments).

Article Snippet: The human melanoma cell line WM853 (stage: primary/vertical growth) was purchased from Rockland (Rockland, Limerick, PA, USA) and was maintained in Tumor Specialized Medium containing 80% MCDB153 (Sigma-Aldrich, St. Louis, MO, USA), 20% Leibovitz’s (Sigma-Aldrich, St. Louis, MO, USA), 2% fetal bovine serum (Pan Biotech GmbH, Aidenbach, Germany), 1.68 mM calcium chloride, and 1.2 g (11.2 mM) sodium bicarbonate at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Expressing, Clone Assay, Western Blot, Neutral Red Assay

Journal: Cancers

Article Title: SIRT2 Contributes to the Resistance of Melanoma Cells to the Multikinase Inhibitor Dasatinib

doi: 10.3390/cancers11050673

Figure Lengend Snippet: Selected category of genes (based on a literature search) with changes in expression in WM853 cells with SIRT2 downregulation as determined using RNA-seq.

Article Snippet: The human melanoma cell line WM853 (stage: primary/vertical growth) was purchased from Rockland (Rockland, Limerick, PA, USA) and was maintained in Tumor Specialized Medium containing 80% MCDB153 (Sigma-Aldrich, St. Louis, MO, USA), 20% Leibovitz’s (Sigma-Aldrich, St. Louis, MO, USA), 2% fetal bovine serum (Pan Biotech GmbH, Aidenbach, Germany), 1.68 mM calcium chloride, and 1.2 g (11.2 mM) sodium bicarbonate at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Expressing

Journal: Cancers

Article Title: SIRT2 Contributes to the Resistance of Melanoma Cells to the Multikinase Inhibitor Dasatinib

doi: 10.3390/cancers11050673

Figure Lengend Snippet: Treatment with dasatinib impairs the migration of melanoma cells based on the scratch assay. ( a ) Image from a single representative experiment performed using WM853 SCW3 and SSW30 clones. ( b ) Image from a single representative experiment performed using MDA-MB-435S SCM1 and SSM15 clones. Quantification of above results is shown in the . 10 × 10 magnification.

Article Snippet: The human melanoma cell line WM853 (stage: primary/vertical growth) was purchased from Rockland (Rockland, Limerick, PA, USA) and was maintained in Tumor Specialized Medium containing 80% MCDB153 (Sigma-Aldrich, St. Louis, MO, USA), 20% Leibovitz’s (Sigma-Aldrich, St. Louis, MO, USA), 2% fetal bovine serum (Pan Biotech GmbH, Aidenbach, Germany), 1.68 mM calcium chloride, and 1.2 g (11.2 mM) sodium bicarbonate at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Migration, Wound Healing Assay, Clone Assay

Journal: Cancers

Article Title: SIRT2 Contributes to the Resistance of Melanoma Cells to the Multikinase Inhibitor Dasatinib

doi: 10.3390/cancers11050673

Figure Lengend Snippet: SIRT2 downregulation impairs the response of melanoma cells to EGFR and EPHA2 activators. The generated melanoma clones were treated with selected concentrations of EGF (EGFR activator) and ephrin A1 (EPHA2 activator) for 10 min and/or 30 min, respectively. Then, protein lysates were prepared and analyzed by Western blotting. ( a ) Experiments performed using WM853 SCW3 and SSW30 clones. ( b ) Experiments performed using MDA-MB-435S SCM1 and SSM15 clones.

Article Snippet: The human melanoma cell line WM853 (stage: primary/vertical growth) was purchased from Rockland (Rockland, Limerick, PA, USA) and was maintained in Tumor Specialized Medium containing 80% MCDB153 (Sigma-Aldrich, St. Louis, MO, USA), 20% Leibovitz’s (Sigma-Aldrich, St. Louis, MO, USA), 2% fetal bovine serum (Pan Biotech GmbH, Aidenbach, Germany), 1.68 mM calcium chloride, and 1.2 g (11.2 mM) sodium bicarbonate at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Generated, Clone Assay, Western Blot

Journal: Cancers

Article Title: SIRT2 Contributes to the Resistance of Melanoma Cells to the Multikinase Inhibitor Dasatinib

doi: 10.3390/cancers11050673

Figure Lengend Snippet: SIRT2 downregulation impairs the expression and phosphorylation of tyrosine kinase receptor-associated pathways. The generated melanoma clones were treated with selected concentrations of dasatinib for 1 h. Then, protein lysates were prepared and analyzed by Western blotting. ( b ) Experiments performed using WM853 SCW3 and SSW30 clones. ( a ) Experiments performed using MDA-MB-435S SCM1 and SSM15 clones.

Article Snippet: The human melanoma cell line WM853 (stage: primary/vertical growth) was purchased from Rockland (Rockland, Limerick, PA, USA) and was maintained in Tumor Specialized Medium containing 80% MCDB153 (Sigma-Aldrich, St. Louis, MO, USA), 20% Leibovitz’s (Sigma-Aldrich, St. Louis, MO, USA), 2% fetal bovine serum (Pan Biotech GmbH, Aidenbach, Germany), 1.68 mM calcium chloride, and 1.2 g (11.2 mM) sodium bicarbonate at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Expressing, Phospho-proteomics, Generated, Clone Assay, Western Blot

Journal: Oncogene

Article Title: Pyruvate dehydrogenase inactivation causes glycolytic phenotype in BAP1 mutant uveal melanoma

doi: 10.1038/s41388-021-02154-0

Figure Lengend Snippet: a. Average expression of PDH (E1), DLAT (E2) and PDHK1 between BAP1 wild type (92.1, OMM1.3, UM001, UM004, UM002B and MM66) and mutant (MP46, MP65, MP38, MM28, PDX4 and WM3618F) UM cells were analyzed by RPPA analysis. RPPA data were used to determine antibodies that are significantly different between mutant and wild-type cell lines. Comparison of average normalized log2 values were performed by the two-sample t-test method and assumed unequal variance. Data points are shown as individual lysate collections **p<0.01. b. Validation of protein expression by Western blot.

Article Snippet: WM3618F was purchased from Rockland Immunochemicals (Limerick, PA) and cultured routinely in 1× MCDB media containing 10% heat-inactivated FBS, CaCl 2 , 50 IU penicillin and 50 μg/mL streptomycin.

Techniques: Expressing, Mutagenesis, Western Blot

Journal: Biomolecules

Article Title: Targeting Mitochondria in Melanoma

doi: 10.3390/biom10101395

Figure Lengend Snippet: Melanoma cell lines show higher levels of OXPHOS compared to HDFs. ( a ) The left and right panels represent two separate western blots because of the equal size of the MTCO2 (CIV) and the NDUFS4 (CI) protein. ( b – g ) Western blot analysis for subunits of OXPHOS complexes (CI-CV), VDAC1 and vinculin in HDF1, WM3311, A375, WM47 and WM3000 cells. ( h – m ) Absolute activities of citrate synthase and OXPHOS complexes in HDF1, WM3311, A375, WM47 and WM3000 cells. Values are given as mean ± SD. One-way ANOVA followed by Dunnett’s Multiple Comparison Test; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. For western blot and enzymatic activity: post-nuclear supernatant containing the mitochondrial fraction was used. Two and three independent experiments were performed for western blot (n = 2) and enzymatic activity (n = 3), respectively. The levels of OXPHOS complexes and VDAC1 were normalized to vinculin. The activities of the OXPHOS complexes were normalized to protein concentration in cell lysates. Citrate synthase (CS); Complex I (CI); Complex II (CII); Complex III (CIII); Complex IV (CIV); Complex V (CV).

Article Snippet: The melanoma cell lines WM3311 (BRAF/NRAS wild-type; Wistar Institute, Philadelphia, PA, USA), WM3000 (BRAFwt/NRASQ61R; Rockland Inc., Philadelphia, PA, USA), WM47 (BRAFV600E/NRASwt; Wistar Institute, Philadelphia, PA, USA) were cultured in MCDB153 Medium (Sigma Aldrich, Darmstadt, Germany) supplemented with 20% Leibovitz’s L-15 Medium (Sigma Aldrich, Darmstadt, Germany), 10% fetal bovine serum (FBS) (Gibco, Darmstadt, Germany), 1% Penicillin-Streptomycin-Amphotericin mixture (Lonza, Basel, Switzerland), 1.68 mM CaCl 2 (Sigma Aldrich, Darmstadt, Germany) and 5 μg/mL insulin (Sigma Aldrich, Darmstadt, Germany).

Techniques: Western Blot, Comparison, Activity Assay, Protein Concentration

Journal: Biomolecules

Article Title: Targeting Mitochondria in Melanoma

doi: 10.3390/biom10101395

Figure Lengend Snippet: Results of the Mito Stress ( a ) and Glycolysis Stress ( b ) tests in HDF1, WM3311, A375, WM47 and WM3000 cells are presented as real-time measurements of OCR. ( c ) Basal respiration, ( d ) maximal respiration, ( e ) ATP-linked respiration, ( f ) proton leak, ( g ) coupling efficiency %, ( h ) basal glycolysis, ( i ) glycolytic capacity, ( j ) glycolytic reverse, ( k ) ratio of respiration to glycolysis. Values are given as mean ± SD. One-way ANOVA followed by Dunnett’s Multiple Comparison Test; * p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001. For every cell line, two independent measurements with 6 to 8 replicates were performed. 10 mM Glucose (Glu); 5 µM Oligomycin (O); 2 µM FCCP (F); 0.5 µM Rotenone and Antimycin A (R/A); 50 mM 2-Deoxy-d-glucose (2DG).

Article Snippet: The melanoma cell lines WM3311 (BRAF/NRAS wild-type; Wistar Institute, Philadelphia, PA, USA), WM3000 (BRAFwt/NRASQ61R; Rockland Inc., Philadelphia, PA, USA), WM47 (BRAFV600E/NRASwt; Wistar Institute, Philadelphia, PA, USA) were cultured in MCDB153 Medium (Sigma Aldrich, Darmstadt, Germany) supplemented with 20% Leibovitz’s L-15 Medium (Sigma Aldrich, Darmstadt, Germany), 10% fetal bovine serum (FBS) (Gibco, Darmstadt, Germany), 1% Penicillin-Streptomycin-Amphotericin mixture (Lonza, Basel, Switzerland), 1.68 mM CaCl 2 (Sigma Aldrich, Darmstadt, Germany) and 5 μg/mL insulin (Sigma Aldrich, Darmstadt, Germany).

Techniques: Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thymosin β4 Regulates Focal Adhesion Formation in Human Melanoma Cells and Affects Their Migration and Invasion

doi: 10.3389/fcell.2019.00304

Figure Lengend Snippet: Thymosin β4 is a constituent of focal adhesions (FAs) and forms complexes with FAs constituents in WM1341D and A375 cell lines. (A) Schematic representation of DNA constructs used in this study. (B) Immunocytochemical stainings visualizing FAs components: vinculin and αVβ3 integrin and co-localizing with them Tβs. (C) Visualization of vinculin and exogenous HA-Tβ4 in transfected cells. (D) PLA of HA-Tβ4 vs. vinculin, ILK, FAK, Pinch 2, α parvin, and αVβ3 integrin in transfected cells. Cell nuclei are shown in dark gray. Arrows point at FAs, while red arrowheads indicate PLA complexes.

Article Snippet: WM1341D and WM9 cell lines were bought from Rockland Immunochemicals.

Techniques: Construct, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thymosin β4 Regulates Focal Adhesion Formation in Human Melanoma Cells and Affects Their Migration and Invasion

doi: 10.3389/fcell.2019.00304

Figure Lengend Snippet: Focal adhesion formation in WM1341D and A375 cells and effect of TMSB4X expression silencing on formation of FAs in melanoma cells. (A,F) Immunocytochemical analysis of cells in order to visualize vinculin. Cells’ borders were marked for better orientation with white lines. Quantitation of FAs’ number (B,G) , size of FA’s (C,H), and cell’s area (D,I) ( n = 20). (E,J) Adhesion assays of investigated cell lines and clones ( n = 3). Arrows point at FAs. The significance level was set at ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 ( www.oncomine.org , February 2018, Thermo Fisher Scientific, Ann Arbor, MI, United States).

Article Snippet: WM1341D and WM9 cell lines were bought from Rockland Immunochemicals.

Techniques: Expressing, Quantitation Assay, Clone Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thymosin β4 Regulates Focal Adhesion Formation in Human Melanoma Cells and Affects Their Migration and Invasion

doi: 10.3389/fcell.2019.00304

Figure Lengend Snippet: Characterization of 2D motility on non-coated and Matrigel TM -coated surface of WM1341D and A375 cells. The cells were seeded either on non-coated surface (A–E) or on Matrigel TM -coated surface (F–J) . (A–C,F–H) Spontaneous 2D-migration of tested cells. The cells were recorded over 72 h ( n = 30): (A,F) trajectories of single cells migration, (B,G) calculated migrated distances of the cells, (C,H) estimation of directionality of cells’ migration. Wound healing assay performed on tested cells ( n = 3): (D,I) representative pictures of cells migrating collectively and (E,J) graphs representing scratch closure expressed in % of closed scratch. The significance level was set at ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 ( www.oncomine.org , February 2018, Thermo Fisher Scientific, Ann Arbor, MI, United States).

Article Snippet: WM1341D and WM9 cell lines were bought from Rockland Immunochemicals.

Techniques: Migration, Wound Healing Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thymosin β4 Regulates Focal Adhesion Formation in Human Melanoma Cells and Affects Their Migration and Invasion

doi: 10.3389/fcell.2019.00304

Figure Lengend Snippet: A375 cells are more progressed in epithelial-mesenchymal transition (EMT) than WM1341D cells. (A) Analysis of 3D motility type exhibited by studied cell lines by inhibitors application: 25 μM GM6001, 10 μM NSC23766, 10 μM Y-27632. The cells were seeded onto Matrigel TM gel placed in the upper compartment of a Transwell TM in medium with added inhibitors. 24 h later the assay was terminated ( n = 3). (B) Immunostainings performed on cells growing under 2D or 3D (the cells were embedded in Matrigel TM gel) conditions. F-actin and cortactin were visualized by application of fluorescently labeled phalloidin and antibodies recognizing cortactin. Arrowheads point at invadopodia. (C) Visualization of N-cadherin and F-actin in studied cells by immucytochemical staining. (D) Representative immunoblots of EMT markers levels in tested cell lines. 30 μg of protein was loaded on every lane. Membranes prior to incubation with antibodies were stained with Ponceau S to show equal protein loading on lanes. The significance level was set at ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 ( www.oncomine.org , February 2018, Thermo Fisher Scientific, Ann Arbor, MI, United States).

Article Snippet: WM1341D and WM9 cell lines were bought from Rockland Immunochemicals.

Techniques: Labeling, Staining, Western Blot, Incubation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Thymosin β4 Regulates Focal Adhesion Formation in Human Melanoma Cells and Affects Their Migration and Invasion

doi: 10.3389/fcell.2019.00304

Figure Lengend Snippet: Comparison of WM1341D and A375 melanoma cell lines studied features.

Article Snippet: WM1341D and WM9 cell lines were bought from Rockland Immunochemicals.

Techniques: Comparison, Expressing, Migration

Journal: PLoS ONE

Article Title: CCT196969 effectively inhibits growth and survival of melanoma brain metastasis cells

doi: 10.1371/journal.pone.0273711

Figure Lengend Snippet: Cell viability in the vemurafenib-naïve and resistant cell lines after exposure to increasing concentrations of vemurafenib (A, C) and CCT196969 (B, D). (A) Dose response curves after exposure to vemurafenib for H1 and H1-R using the following concentrations: untreated, 1, 2, 4 and 6 μM. (B) Dose response curves for H1 and H1-R after exposure to CCT196969 using the following concentrations: untreated, 0.1, 0.5, 1 and 2 μM. (C) Dose response curves after exposure to vemurafenib for Wm3248 and Wm3248-DR using the following concentrations: untreated, 1, 2, 4 and 6 μM. (D) Dose response curves for Wm3248 and Wm3248-DR after exposure to CCT196969 using the following concentrations: untreated, 0.1, 0.5, 1 and 2 μM. Abbreviations: ns: not significant, **: p < 0.01, ****: p < 0.0001.

Article Snippet: The BRAF V600E -mutated melanoma cell line Wm3248 was purchased from Rockland (Limerick, PA, USA).

Techniques:

Journal: mAbs

Article Title: Generation and in vivo characterization of a novel high-affinity human antibody targeting carcinoembryonic antigen

doi: 10.1080/19420862.2023.2217964

Figure Lengend Snippet: In vitro characterization of new anti-CEA antibodies. (a) SPR sensorgram of G9, F7, and F4 in a scFv format. K D values were calculated to be 640 nM, 50 nM, and 7.7 nM, respectively. (b) Flow cytometry analysis with the new antibodies in IgG format on CEA-expressing CT26 cells and on CEA-negative CT26 wild-type cells. (c) Immunofluorescence staining with IgG formats on the human colon adenocarcinoma xenograft LS174T. Anti-CEA antibodies were detected in green. Blood vessels were detected by CD31 staining (red). 20× magnification, scale bars = 100 μm.

Article Snippet: Immunofluorescence analysis was performed on LS174T and HT-29 xenografts harvested from mice, on patient-derived colon cancer samples, and on a human tissue microarray (Amsbio, T6235700–5).

Techniques: In Vitro, Flow Cytometry, Expressing, Immunofluorescence, Staining

Journal: mAbs

Article Title: Generation and in vivo characterization of a novel high-affinity human antibody targeting carcinoembryonic antigen

doi: 10.1080/19420862.2023.2217964

Figure Lengend Snippet: Ex vivo immunofluorescence-based biodistribution analysis. Immunofluorescence analysis assessed tumor targeting of new anti-CEA antibodies in IgG format. Two hundred micrograms of IgG-FITC were injected intravenously into LS174T-bearing mice. Tumors were excised 24 hours after injection. IgG-FITC was detected in green; blood vessels were detected through CD31 staining (red). 20× magnification, scale bars = 100 μm.

Article Snippet: Immunofluorescence analysis was performed on LS174T and HT-29 xenografts harvested from mice, on patient-derived colon cancer samples, and on a human tissue microarray (Amsbio, T6235700–5).

Techniques: Ex Vivo, Immunofluorescence, Injection, Staining

Journal: mAbs

Article Title: Generation and in vivo characterization of a novel high-affinity human antibody targeting carcinoembryonic antigen

doi: 10.1080/19420862.2023.2217964

Figure Lengend Snippet: Quantitative biodistribution with radiolabeled anti-CEA antibodies in diabody format. Quantitative biodistribution analysis of radio iodinated anti-CEA diabodies in BALB/c nude mice bearing subcutaneous LS174T colon adenocarcinomas. Organs were harvested 24 hours after intravenous injection, and radioactivity was quantified. Results are shown as the percentage of injected dose per gram (ID/g (%)). Error bars = SEM; n = 4.

Article Snippet: Immunofluorescence analysis was performed on LS174T and HT-29 xenografts harvested from mice, on patient-derived colon cancer samples, and on a human tissue microarray (Amsbio, T6235700–5).

Techniques: Injection, Radioactivity