Journal: Oncology Research

Article Title: uPAR Controls Vasculogenic Mimicry Ability Expressed by Drug-Resistant Melanoma Cells

doi: 10.3727/096504021X16273798026651

Figure Lengend Snippet: Extracellular acidosis induces VM in melanoma cells. (a) Representative pictures and relative quantification chart of capillary morphogenesis assay of A375-M6 wild type (WT) or chronically exposed to extracellular acidosis (chr.ac.). Two-way analysis of variance (ANOVA), GraphPad Prism. (b) Western blot (left) and flow cytometer analysis (right) with relative quantification charts of VM markers EphA2 and VE-cadherin of A375-M6 WT or chronically exposed to extracellular acidosis (chr.ac.). t -test, GraphPad Prism. (c) Representative pictures and relative quantification chart of capillary morphogenesis assay of A375-M6 WT or acid-adapted treated or not with 2 μM vemurafenib or 10 μM everolimus for 24 h. Two-way ANOVA, GraphPad Prism. Scale bar: 200 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Similar effects were observed in the A375 cell line, where uPAR KO was accompanied with a significantly decreased vascular-like channel formation in vitro and then restored in the uPAR rescue A375 cell line (Supplementary Fig. S4, available at https://www.sbsc.unifi.it/vp-351-supplementary-material-rev.html ).

Techniques: Quantitative Proteomics, Western Blot, Flow Cytometry

Journal: Oncology Research

Article Title: uPAR Controls Vasculogenic Mimicry Ability Expressed by Drug-Resistant Melanoma Cells

doi: 10.3727/096504021X16273798026651

Figure Lengend Snippet: Vemurafenib-resistant A375-M6 melanoma cells are capable of vasculogenic mimicry (VM). (a) Viability assay of A375-M6 WT and VEM-R cells treated with increasing doses of vemurafenib. One-way ANOVA, GraphPad Prism. (b) Cell cycle analysis of A375-M6 WT and VEM-R cells treated with 2 μM vemurafenib. One-way ANOVA, GraphPad Prism. (c) Representative pictures and relative quantification chart of capillary morphogenesis assay of A375-M6 WT and VEM-R cells in the presence or absence of 2 μM vemurafenib. Scale bar: 200 μm. Two-way ANOVA, GraphPad Prism. (d) Western blot of EphA2 and (e) flow cytometer analysis of VE-cadherin of A375-M6 WT and VEM-R cells treated or not with 2 μM vemurafenib. One-way ANOVA, GraphPad Prism. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Similar effects were observed in the A375 cell line, where uPAR KO was accompanied with a significantly decreased vascular-like channel formation in vitro and then restored in the uPAR rescue A375 cell line (Supplementary Fig. S4, available at https://www.sbsc.unifi.it/vp-351-supplementary-material-rev.html ).

Techniques: Viability Assay, Cell Cycle Assay, Quantitative Proteomics, Western Blot, Flow Cytometry

Journal: Oncology Research

Article Title: uPAR Controls Vasculogenic Mimicry Ability Expressed by Drug-Resistant Melanoma Cells

doi: 10.3727/096504021X16273798026651

Figure Lengend Snippet: Urokinase plasminogen activator receptor (uPAR) expression is required for VM in melanoma cells. (a) Real-time polymerase chain reaction (PCR) (left) and flow cytometer analysis (right) of uPAR expression in A375-M6 WT and acid-adapted cells. t -test, GraphPad Prism. (b) uPAR flow cytometer analysis (one-way ANOVA, GraphPad Prism) and (c) capillary morphogenesis assay (representative pictures on the top and quantification chart on the bottom; two-way ANOVA, GraphPad Prism) of WT, uPAR knockout (KO), and uPAR rescue A375-M6 cells maintained in standard condition. Scale bar: 200 μm. (d) Western blot of EphA2 (one-way ANOVA, GraphPad Prism) and (e) flow cytometer analysis of VE-cadherin (one-way ANOVA, GraphPad Prism) of WT, uPAR KO, and uPAR rescue A375-M6 cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Similar effects were observed in the A375 cell line, where uPAR KO was accompanied with a significantly decreased vascular-like channel formation in vitro and then restored in the uPAR rescue A375 cell line (Supplementary Fig. S4, available at https://www.sbsc.unifi.it/vp-351-supplementary-material-rev.html ).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Knock-Out, Western Blot

Journal: Oncology Research

Article Title: uPAR Controls Vasculogenic Mimicry Ability Expressed by Drug-Resistant Melanoma Cells

doi: 10.3727/096504021X16273798026651

Figure Lengend Snippet: uPAR inhibition by M25 blocking peptide impairs VM and drug resistance in resistant melanoma cells. (a) Representative pictures of capillary morphogenesis assay with relative quantification chart of A375-M6 VEM-R treated with vemurafenib and M25 peptide alone or in combination. Scale bar: 200 μm. Two-way ANOVA, GraphPad Prism. (b) Representative pictures of capillary morphogenesis assay with relative quantification chart of A375-M6 WT or acid-adapted treated with scramble or uPAR-blocking peptide M25. Scale bar: 200 μm. Two-way ANOVA, GraphPad Prism. (c) Cell growth of A375-M6 VEM-R cells treated 24 h with vemurafenib and M25 peptide alone or in combination. Two-way ANOVA, GraphPad Prism. (d) Cell growth (upper) and representative pictures of colony formation assay (lower) of A375-M6 WT and acid-adapted cells treated for 24 h with vemurafenib and M25 peptide as a single or combined therapy. Two-way ANOVA, GraphPad Prism. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Similar effects were observed in the A375 cell line, where uPAR KO was accompanied with a significantly decreased vascular-like channel formation in vitro and then restored in the uPAR rescue A375 cell line (Supplementary Fig. S4, available at https://www.sbsc.unifi.it/vp-351-supplementary-material-rev.html ).

Techniques: Inhibition, Blocking Assay, Quantitative Proteomics, Colony Assay

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Effect of DOX on cell viability of A375 and MNT-1 cells. Cells were exposed to different concentrations of DOX for 24, 48, and 72 h, and cell viability was determined using MTT assay. Data shown are mean values ± standard deviation of three independent experiments with four technical replicates each. *—indicates statistical significance in comparison to the respective control ( p < 0.05).

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques: MTT Assay, Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Inhibitory concentrations (ICs) obtained for 24, 48, and 72 h DOX exposure. Values are expressed in μM.

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Effect of hyperthermia plus DOX on cell viability of A375 and MNT-1 cells. Cells were exposed to 43 °C for 30, 60, or 120 min, plus 0.012 μM or 0.043 μM and 0.68 μM or 1.38 μM during 24 h; 0.0056 μM or 0.0125 μM and 0.0066 μM or 0.0179 μM during 48 h; and 0.0012 μM or 0.0026 μM and 0.0042 μM or 0.0098 μM during 72 h; in cases of A375 or MNT-1, respectively. DMSO concentrations correspond to the equivalent percentage present in IC 20 of each cell line and time exposure. DOX concentrations correspond to the calculated IC 10 and IC 20 for each time exposure and for each cell line. Cell viability was determined using MTT assay. Data are shown as mean ± standard deviation of two independent experiments with four technical replicates each. *—indicates statistical significance in comparison to the control 37 °C; α indicates statistical significance in comparison to the respective control of each condition at 37 °C; and β indicates statistical significance of the combined treatment in comparison to hyperthermia alone ( p < 0.05).

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques: MTT Assay, Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Effect of hyperthermia plus DOX on morphology of A375 and MNT-1 cells. Cells were exposed to 43 °C for 30 min and 0.0125 μM or 0.0179 μM of DOX, in case of A375 or MNT-1 cells, respectively. ( A )—A375 cells; ( B )—MNT-1 cells.

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Effects of hyperthermia combined with DOX on cell cycle distribution. Cells were exposed to 43 °C for 30 min and 0.0125 μM or 0.0179 μM of DOX, in case of A375 or MNT-1 cells, respectively. ( A ) Cell cycle distribution (%) in A375 and MNT-1 cells; ( B ) histograms representative of cell distribution of A375 and MNT-1 cells. Data shown are mean values ± standard deviation of two independent experiments with two technical replicates each and each replicate with at least 5000 events. *—indicates statistical significance in comparison to the control 37 °C; α indicates statistical significance in comparison to the respective control of each condition at 37 °C; and β indicates statistical significance of the combined treatment in comparison to hyperthermia alone ( p < 0.05).

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques: Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Effects of hyperthermia combined with DOX on production of intracellular ROS. Cells were exposed to 43 °C for 30 min and 0.0125 μM or 0.0179 μM of DOX for 48 h, in case of A375 or MNT-1 cells, respectively. ( A ) Relative abundance of intracellular ROS of A375 and MNT-1 cells; ( B ) histograms representative of abundance of intracellular ROS of A375 and MNT-1 cells. Data shown are mean values ± standard deviation of two independent experiments with two technical replicates each and each replicate with at least 5000 events. *—indicates statistical significance in comparison to the control 37 °C; α indicates statistical significance in comparison to the respective control of each condition at 37 °C; and β indicates statistical significance of the combined treatment in comparison to hyperthermia alone ( p < 0.05).

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques: Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

doi: 10.3390/ijms23010035

Figure Lengend Snippet: Effects of hyperthermia in combination with DOX on apoptotic profile. Both cell lines were exposed to 43 °C for 30 min and A375 cells were treated with 0.0125 μM and MNT-1 cells with 0.0179 μM of DOX for 48 h. ( A ) Percentage of apoptotic cells after treatment in populations corresponding to viable and non-apoptotic, early and late apoptotic A375 and MNT-1 cells; ( B ) histograms representative of Annexin V-FITC. Data shown are mean values ± standard deviation of two independent experiments with two technical replicates each and each replicate with at least 5000 events. *— indicates statistical significance in comparison to the control 37 °C; α indicates statistical significance in comparison to the respective control of each condition at 37 °C; and β indicates statistical significance of the combined treatment in comparison to hyperthermia alone ( p < 0.05).

Article Snippet: Human melanoma cell line A375 was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and supplied by Sigma-Aldrich (Madrid, Spain) and the MNT-1 melanoma cell line was kindly provided by Dr. Manuela Gaspar (iMed.ULisboa, Lisbon, Portugal).

Techniques: Standard Deviation, Comparison, Control

Journal: Cell Research

Article Title: Cortical branched actin determines cell cycle progression

doi: 10.1038/s41422-019-0160-9

Figure Lengend Snippet: Cell transformation is generally associated with insensitivity to Arp2/3 inhibition. a Various immortalised cell lines from human breast were treated with the Arp2/3 inhibitor, CK-666, or with the inactive control, CK-869, at the indicated concentrations. These immortalised human breast cell lines stopped cycling in response to Arp2/3 inhibition, in a dose-dependent manner. Mouse Embryonic Fibroblasts (MEF) behaved the same. b Various mammary carcinoma cell lines and the melanoma cell line A375M were insensitive to Arp2/3 inhibition. c siRNA mediated inactivation of the tumour suppressor genes RB1 and CDKN1A , which encodes the CDK inhibitory protein p21 WAF1/CIP1 , prevents the cell cycle arrest due to Arp2/3 inhibition. A similar effect is seen in MCF10A cells knock-out for CDKN1A , and mouse embryonic fibroblasts knock-out for all three genes encoding retinoblastoma proteins. Data are mean ± s.e.m of five technical repeats ( a – c ). One experiment representative of 2 biological repeats is displayed, Mann–Whitney test at 200 µM of drug for a – c (middle and right), twoway ANOVA followed by Sidak’s test for c (left). For b and c , no significant difference between groups was found

Article Snippet: All cell lines derived from human breast were from the collection of breast cell lines organised by Thierry Dubois (Institut Curie, Paris), A375M, WM1960, IGR1 were from Lionel Larue (Institut Curie, Orsay), 83 T, 104 T were from Yardena Samuels (The Weizmann Institute of Science, Rehovot), MEF triple knockout for Rb were from Julien Sage (Stanford University of Medicine), MCF10A p21 −/− were from Ben Ho Park (Johns Hopkins School of Medicine, Baltimore).

Techniques: Transformation Assay, Inhibition, Control, Knock-Out, MANN-WHITNEY

Journal: Analytical Chemistry

Article Title: Mass Spectrometry Analysis of Glycopeptides Enriched by Anion Exchange-Mediated Methods Reveals PolyLacNAc-Extended N -Glycans in Integrins and Tetraspanins in Melanoma Cells

doi: 10.1021/acs.analchem.3c04045

Figure Lengend Snippet: Specificity of Enrichment of PolyLacNAc-Containing Glycopeptide of Anion Exchange Columns from A375 Cell Lysates a

Article Snippet: The A375 malignant melanoma cell line was purchased from Ubigene (Brooklyn, NY).

Techniques:

Journal:

Article Title: Changes in the gene expression profile of A375 human melanoma cells induced by over-expression of multifunctional pigment epithelium-derived factor

doi: 10.1097/CMR.0b013e32834495c3

Figure Lengend Snippet: Characterization of PEDF-over-expressing A375 cell lines. (a) Western blot analysis of secreted extracellular PEDF (PEDFe) protein in 48 h conditioned medium (CM) from control (A375-pCEP4 Pool) and PEDF-over-expressing (A375-pCEP4-PEDF Pool) cells. Numbers below blot show densitometry values normalized to A375-pCEP4 Pool expression. (b) Quantitative RT-PCR analysis of PEDF mRNA levels in A375-pCEP4 Pool and A375-pCEP4-PEDF Pool cells. PEDF mRNA levels are shown relative to A375-pCEP4 Pool after normalization to GAPDH. Bars represent average ± standard deviation (SD). (c) Invasion assay of A375-pCEP4 Pool and A375-pCEP4-PEDF Pool cells toward 10% FBS for 24 h. Statistical significance was determined by Student’s t-test (****, p<0.0001). Results are representative of two independent experiments.

Article Snippet: A375 human melanoma cell line, originally established in culture from a lymph node metastasis of a melanoma patient [ 28 , 29 ], was kindly provided by Dr Vidal-Vanaclocha (Universidad del Pais Vasco, Bilbao, Spain).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Standard Deviation, Invasion Assay

Journal:

Article Title: Changes in the gene expression profile of A375 human melanoma cells induced by over-expression of multifunctional pigment epithelium-derived factor

doi: 10.1097/CMR.0b013e32834495c3

Figure Lengend Snippet: Distribution in functional categories of genes regulated by PEDF in A375 melanoma cells. (a) Heat map of expression of genes regulated by PEDF in two independent hybridizations (Hybr. 1 and Hybr. 2) of A375-pCEP4-PEDF Pool cells compared to control A375-pCEP4 Pool cells. Coloring represents normalized signal intensity of the ratio log2 (A375-pCEP4-PEDF Pool/A375-pCEP4 Pool): red, upregulation; black, no change; green, downregulation. (b) Diagram showing genes regulated by PEDF grouped into biological processes and/or functional categories as described in the main text. Percentage of regulated genes in each category relative to total number of regulated genes is shown. For some categories the most relevant genes are listed in the lower boxes, being shown in red when upregulated and in green when downregulated in A375-pCEP4-PEDF Pool compared to A375-pCEP4 Pool.

Article Snippet: A375 human melanoma cell line, originally established in culture from a lymph node metastasis of a melanoma patient [ 28 , 29 ], was kindly provided by Dr Vidal-Vanaclocha (Universidad del Pais Vasco, Bilbao, Spain).

Techniques: Functional Assay, Expressing, Control

Journal:

Article Title: Changes in the gene expression profile of A375 human melanoma cells induced by over-expression of multifunctional pigment epithelium-derived factor

doi: 10.1097/CMR.0b013e32834495c3

Figure Lengend Snippet: Distribution in functional categories of genes regulated by PEDF in A375 human melanoma cell line Selected relevant genes regulated by PEDFe are shown classified into functional categories described in the main text. Upregulated genes are included at the top of the list followed by downregulated genes. Shown are gene symbol, description and the linear ratio of GCRMA-normalized expression PEDF/control in the two hybridizations from independently isolated RNAs from A375-pCEP4-PEDF Pool y A375-pCEP4 Pool cells (Hybr. 1 and Hybr. 2).

Article Snippet: A375 human melanoma cell line, originally established in culture from a lymph node metastasis of a melanoma patient [ 28 , 29 ], was kindly provided by Dr Vidal-Vanaclocha (Universidad del Pais Vasco, Bilbao, Spain).

Techniques: Functional Assay, Expressing, Isolation, Permeability

Journal:

Article Title: Changes in the gene expression profile of A375 human melanoma cells induced by over-expression of multifunctional pigment epithelium-derived factor

doi: 10.1097/CMR.0b013e32834495c3

Figure Lengend Snippet: Validation of candidate target genes regulated by PEDF in A375 melanoma cells. (a) Quantitative RT-PCR analysis of mRNA levels of genes related to: 1) angiogenesis/adhesion/migration/invasion: COL4A2, FN1, IL8 and TGFA; 2) melanoma progression: FGF13, IGFBP3, JAG1 and LGALS3; 3) melanoma markers: MIA and S100B; 4) melanin secretion: MLPH and RAB27A. Empty bars correspond to A375-pCEP4 Pool cells and filled bars to A375-pCEP4-PEDF Pool cells. For each gene mRNA levels are shown relative to A375-pCEP4 Pool after normalization to GAPDH. Bars represent average ± SD. Statistical significance was determined by Student’s t-test or Welch-corrected t-test (the latter for COL4A2, TGFA, LGALS3, MLPH and S100B) (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001). (b) ELISA analysis of secreted IL8 protein levels in 48 h CM from A375-pCEP4 Pool and A375-pCEP4-PEDF Pool cells. Bars represent average ± SD. Statistical significance was determined by Student’s t-test (****, p<0.0001). Results are representative of two independent experiments.

Article Snippet: A375 human melanoma cell line, originally established in culture from a lymph node metastasis of a melanoma patient [ 28 , 29 ], was kindly provided by Dr Vidal-Vanaclocha (Universidad del Pais Vasco, Bilbao, Spain).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Migration, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Changes in the gene expression profile of A375 human melanoma cells induced by over-expression of multifunctional pigment epithelium-derived factor

doi: 10.1097/CMR.0b013e32834495c3

Figure Lengend Snippet: Confirmation of regulation of several target genes in other PEDF-over-expressing melanoma cell lines. (a) Quantitative RT-PCR analysis of PEDF mRNA levels in A375-pCEP4 Pool, A375-pCEP4-PEDF Clone 1 and A375-pCEP4-PEDF Clone 4 cells. PEDF mRNA levels are shown relative to A375-pCEP4 Pool after GADPH normalization. Bars represent average ± SD. (b) Quantitative RT-PCR analysis of IL8 (left panel), JAG1 (middle panel) and MLPH (right panel) mRNA levels in A375-pCEP4 Pool, A375-pCEP4-PEDF Clone 1 and A375-pCEP4-PEDF Clone 4 cells. For each gene mRNA levels are shown relative to A375-pCEP4 Pool after GAPDH normalization. Bars represent average ± SD. Statistical significance was determined by one-way ANOVA test using Tukey-Kramer post-test (*, p<0.05; ***, p<0.001). Results are representative of two independent experiments. (c) Western blot analysis of PEDFe protein in 48 h CM from control (UCD-GFP or C8161-GFP) and PEDF-over-expressing (UCD-PEDF or C8161-PEDF) cells. Numbers below blots show densitometry values normalized to each control cells expression. (d) Quantitative RT-PCR analysis of IGFBP3, MIA and IL8 mRNA levels in UCD-GFP, UCD-PEDF, C8161-GFP and C8161-PEDF cells. For each gene mRNA levels are shown relative to control cells after GAPDH normalization. Bars represent average ± SD. Statistical significance was determined by Student’s t-test (***, p<0.001; ****, p<0.0001). Results are representative of two independent experiments.

Article Snippet: A375 human melanoma cell line, originally established in culture from a lymph node metastasis of a melanoma patient [ 28 , 29 ], was kindly provided by Dr Vidal-Vanaclocha (Universidad del Pais Vasco, Bilbao, Spain).

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

Journal:

Article Title: Changes in the gene expression profile of A375 human melanoma cells induced by over-expression of multifunctional pigment epithelium-derived factor

doi: 10.1097/CMR.0b013e32834495c3

Figure Lengend Snippet: Validation of regulation of IL8 by transient transduction of A375 melanoma cells using lentivirus-PEDF. (a) Transduction efficiency of A375 melanoma cell line after infection with control (A375-lenti-GFP) or PEDF (A375-lenti-PEDF) lentivirus. Fluorescence images show more than 95% GFP-positive cells after 72 h of infection. Corresponding phase-contrast images are shown. (b) Western blot analysis of secreted extracellular PEDF (PEDFe) protein in 48 h CM from A375 melanoma cell line after 72 h of transduction with control lentivirus-GFP (A375-lenti-GFP) or lentivirus-PEDF (A375-lenti-PEDF). Numbers below blot show densitometry values relative to A375-lenti-GFP. (c) Quantitative RT-PCR analysis of PEDF mRNA levels in A375 melanoma cell line after 72 h of transduction with control lentivirus-GFP (A375-lenti-GFP) or lentivirus-PEDF (A375-lenti-PEDF). PEDF mRNA levels are shown relative to A375-lenti-GFP after normalization to GAPDH. Bars represent average ± SD. (d) Quantitative RT-PCR analysis of IL8 mRNA levels in A375 melanoma cell line after 72 h of transduction with control lentivirus-GFP (A375-lenti-GFP) or lentivirus-PEDF (A375-lenti-PEDF). IL8 mRNA levels are shown relative to A375-lenti-GFP after normalization to GAPDH. Bars represent average ± SD. Statistical significance was determined by Student’s t-test (*, p<0.01). (e) ELISA analysis of secreted IL8 protein levels in 48 h CM from A375 melanoma cell line after 72 h of transduction with control lentivirus-GFP (A375-lenti-GFP) or lentivirus-PEDF (A375-lenti- PEDF). Bars represent average ± SD. Statistical significance was determined by Student’s t-test (**, p<0.01). (f) IL8 transcriptional activity reporter assay in the A375-lenti-GFP and A375-lenti-PEDF melanoma cells mentioned above after 24 h treatment with 10% FBS or 10 ng/ml TNFα. Luciferase levels relative to A375-lenti-GFP in 10% FBS after normalization to renilla levels are shown. Bars represent average ± SD. Results are representative of two independent experiments.

Article Snippet: A375 human melanoma cell line, originally established in culture from a lymph node metastasis of a melanoma patient [ 28 , 29 ], was kindly provided by Dr Vidal-Vanaclocha (Universidad del Pais Vasco, Bilbao, Spain).

Techniques: Biomarker Discovery, Transduction, Infection, Control, Fluorescence, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Activity Assay, Reporter Assay, Luciferase