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: 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