Journal: Current Issues in Molecular Biology

Article Title: Hyperthermia Enhances Adeno-Associated Virus Vector Transduction Efficiency in Melanoma Cells

doi: 10.3390/cimb45100537

Figure Lengend Snippet: Expression of AAVR, HSPG1, and HSPG2 receptors for AAV in melanoma cells (A375, G-361, and SK-MEL-1) and normal fibroblasts (CCD-1064Sk). The results are presented as a mean of three independent repeats ± SD. ( A ) Expression levels (2 −dCt ) of receptors for AAV under standard conditions—constitutive levels. Statistically significant differences between CCD-1064Sk and cancer cells are marked with asterisks (**** p < 0.0001). ( B ) Averaged constitutive expressions (2 −dCt ) of receptors for AAV in the examined cells. The expression levels (2 −dCt ) of the three receptors were averaged for each cell line. ( C ) Changes in the expression (2 −ddCt ) of receptors after exposure to hyperthermia. For each receptor, lighter colors indicate expression at 37 °C and darker at 43 °C. Statistically significant differences between 37 °C and 43 °C are marked with asterisks (** p < 0.01; *** p < 0.001; **** p < 0.0001).

Article Snippet: Cell lines of melanoma A375 (CRL-1619), G-361 (CRL-1424), and SK-MEL-1 (HTB-67) from the Melanoma Cancer Cell Panel (TCP-1013) and the cell line of normal fibroblasts CCD-1064Sk were obtained from the ATCC (American Type Culture Collection, Manassas, VA, USA).

Techniques: Expressing

Journal: Cell adhesion & migration

Article Title: Expression of kindlin-3 in melanoma cells impedes cell migration and metastasis.

doi: 10.1080/19336918.2016.1243645

Figure Lengend Snippet: Figure 6. Analyses of kindlin-3 expression in M10 human melanoma cell line. (A) qRT-PCR analyses of kindlin-1, ¡2 and ¡3 mRNA expression levels in M10 cells and the human chronic myeloid leukemia cell line K562. Values are mean § SD of technical triplicates. A representative experiment from 3 independent experiments is shown. (B) qRT-PCR analyses of kindlin-3 expression in M10, K562 and the human fibroblast cell line 293T. In (A) and (B), GAPDH and actin were used as internal controls. (C) Immunoblot analyses of kindlin-3 protein expression in M10 cells, K562, 293T and the human breast cancer cell line MDA-MB-231 using the anti-kindlin-3 mAb clone 9. Actin serves as loading control. (D) Immunoblot analyses using the anti-kindlin-3 mAb 3D6.

Article Snippet: Human melanoma cell line M10 was purchased from American Type Culture Collection (Manassas, VA) and cultured in RPMI1640 fullmedium containing supplements aforementioned.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Cell adhesion & migration

Article Title: Expression of kindlin-3 in melanoma cells impedes cell migration and metastasis.

doi: 10.1080/19336918.2016.1243645

Figure Lengend Snippet: Figure 7. Analyses of the migratory properties of M10 cells transfected with GFP-kindlin-3. (A) Flow cytometry analyses and sorting of cells transiently transfected with GFP or GFP-kindlin-3 expression plasmid. (B) Sorted cells were subjected to 2D-random migration assays on fibronectin-coated (2 mg/cm2) coverslip-bottom culture dishes. Twenty cells were tracked and migratory profiles were plotted. Representative data from at least 2 independent experiments are shown. X and Y axes are relabeled with larger fonts for clarity.

Article Snippet: Human melanoma cell line M10 was purchased from American Type Culture Collection (Manassas, VA) and cultured in RPMI1640 fullmedium containing supplements aforementioned.

Techniques: Transfection, Flow Cytometry, Expressing, Plasmid Preparation, Migration

Journal: Frontiers in Veterinary Science

Article Title: COX-2 Silencing in Canine Malignant Melanoma Inhibits Malignant Behaviour

doi: 10.3389/fvets.2021.633170

Figure Lengend Snippet: Construction of an inducible COX-2 miRNA expression vector in canine melanoma cell lines. (A) Canine double-stranded COX-2 miRNAs were generated and cloned into (i) pcDNA™6.2-GW/EmGFP-miR vector (Invitrogen) and transferred to (ii) pTRIPZdest destination vector. (B) Basal expression level of COX-2 in CMGD2, CMGD5, TLM1, and CML10 canine melanoma cell lines. (C) Validation of inducible COX-2 mi-RNA expression: (i) CMGD2 cells and (ii) TLM1 cells were transfected with 2.5 μg of the vector or negative control after 24 h cells were treated with 1 μg/ml DOX (+) or vehicle control (–) and COX-2 expression was determined by qRT-PCR. Data shown as fold change relative to miRneg (+) expression ( p = 0.03*, p = 0.002**).

Article Snippet: The panel of canine melanoma cell lines used in this study included CMGD2, CMGD-5, CML-10 and TLM1 (Kerafast, Boston, MA, USA), cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum, 100 μg/ml streptomycin (ThermoFisher Scientific) and 5% HEPES (ThermoFisher Scientific).

Techniques: Expressing, Plasmid Preparation, Generated, Clone Assay, RNA Expression, Transfection, Negative Control, Quantitative RT-PCR

Journal: Frontiers in Veterinary Science

Article Title: COX-2 Silencing in Canine Malignant Melanoma Inhibits Malignant Behaviour

doi: 10.3389/fvets.2021.633170

Figure Lengend Snippet: Melanoma cell viability and colony-forming ability is COX-2 dependent. (A) Cell viability of CMGD2 (i) and TLM1 (ii) was determined after transfection with either miRNACOX2-346 or the miRneg negative control with (+) or without (–) DOX. Cell viability was assayed 48 h after DOX treatment ( p = 0.001*, p = 0.003**). (B) Colony forming ability of CMGD2 (i) and TLM1 (ii) was determined after transfection with either miRNACOX2-346 or the negative control with (+) or without (–) DOX ( p = 0.0001*, p = 0.01**).

Article Snippet: The panel of canine melanoma cell lines used in this study included CMGD2, CMGD-5, CML-10 and TLM1 (Kerafast, Boston, MA, USA), cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum, 100 μg/ml streptomycin (ThermoFisher Scientific) and 5% HEPES (ThermoFisher Scientific).

Techniques: Transfection, Negative Control

Journal: Frontiers in Veterinary Science

Article Title: COX-2 Silencing in Canine Malignant Melanoma Inhibits Malignant Behaviour

doi: 10.3389/fvets.2021.633170

Figure Lengend Snippet: COX-2 regulates cell invasion. (A) CMGD2 and (B) TLM1 cells were transfected with either miRNACOX2-346 or the miRneg negative control with (+) or without (–) DOX. Invasion potential was determined 48 h after seeding into a transwell chamber assay (QCM™ collagen-based cell invasion assay kit) ( p = 0.0004*, p = 0.0002**).

Article Snippet: The panel of canine melanoma cell lines used in this study included CMGD2, CMGD-5, CML-10 and TLM1 (Kerafast, Boston, MA, USA), cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum, 100 μg/ml streptomycin (ThermoFisher Scientific) and 5% HEPES (ThermoFisher Scientific).

Techniques: Transfection, Negative Control, Transwell Chamber Assay, Invasion Assay

Journal: Cell Death & Disease

Article Title: Role of non-genomic androgen signalling in suppressing proliferation of fibroblasts and fibrosarcoma cells

doi: 10.1038/cddis.2014.497

Figure Lengend Snippet: Effect of different concentrations of androgens on DNA synthesis of mesenchymal cells. Quiescent NIH3T3 cells were used and left untreated or treated with the indicated compounds. R1881 (Perkin-Elmer) or DHT (Sigma) were used at 1 pM or 10 nM; bicalutamide (Sigma-Aldrich; Bic) was added at 1000-fold excess. In ( a and c ), cells on coverslips were pulsed with 100 μ M BrdU and 18 h later BrdU incorporation was analyzed by IF. Data were expressed as % of cells. In ( b and d ), cell growth was measured 24 h later by MTT assay and data were expressed as relative increase. Quiescent mouse embryo fibroblasts (MEFs in e ) or HT1080 cells ( f ) or primary mouse fibroblasts (MFs in g and h ) on coverslips were left untreated or treated for 18 h with the indicated concentration of R1881 or DHT. Bicalutamide was added at 1000-fold excess. BrdU incorporation was analyzed as above and expressed as % of cells. In ( a–h ), means and S.E.M. are shown. n represents the number of experiments throughout the figures

Article Snippet: Karyotypically heterogeneous human fibrosarcoma HT1080 cells were a gift from P. Friedl (Department of Cell Biology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands).

Techniques: DNA Synthesis, BrdU Incorporation Assay, MTT Assay, Concentration Assay

Journal: Cell Death & Disease

Article Title: Role of non-genomic androgen signalling in suppressing proliferation of fibroblasts and fibrosarcoma cells

doi: 10.1038/cddis.2014.497

Figure Lengend Snippet: Rac inhibition triggers DNA synthesis and suppresses p27 Ser10 phosphorylation in NIH3T3 fibroblasts and human fibrosarcoma HT1080 cells treated with 10 nM R1881. In ( a – c ), NIH3T3 cells were used. In ( a ), quiescent cells on coverslips were left untreated or treated for 18 h with 10 nM R1881 in the absence or presence of EHT1864 (10 μ M). Control cells were treated with EHT1864 alone. After in vivo pulse, BrdU incorporation was analyzed by IF and expressed as % of cells. In ( b ), quiescent cells were left untreated or treated for 30 min with 10 nM R1881 in the absence or presence of EHT1864 (10 μ M). Control cells were treated with EHT1864 alone. Lysate proteins were analyzed by western blot, using antibodies against the indicated proteins. In ( c ), growing cells were transfected with non-targeting siRNA (ctrl siRNA) or Rac1 siRNA (Rac1 siRNA). Cells were co-transfected with negative control siRNA Alexa-Fluor 488 to help identification of transfected cells. After transfection, the cells were made quiescent and then left unstimulated or stimulated for 18 h with 10 nM R1881. After in vivo pulse, BrdU incorporation was analyzed by IF and expressed as % of transfected cells. Data are derived from at least 500 scored cells for each experiment. Inset in ( c ) shows the western blot with anti-Rac antibody of lysate proteins from cells transfected with Rac1 or non-targeting (ctrl) siRNA. The filter was re-probed with anti-tubulin antibody as a loading control (tub). In ( d ), quiescent HT1080 cells on coverslips were left untreated or treated for 18 h with 10 nM R1881 in the absence or presence of EHT1864 (10 μ M). Control cells were treated with EHT1864 alone. BrdU incorporation was analyzed as in ( a ). Means and S.E.M. are shown

Article Snippet: Karyotypically heterogeneous human fibrosarcoma HT1080 cells were a gift from P. Friedl (Department of Cell Biology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands).

Techniques: Inhibition, DNA Synthesis, Phospho-proteomics, Control, In Vivo, BrdU Incorporation Assay, Western Blot, Transfection, Negative Control, Derivative Assay