Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 inhibits melanoma cell growth through induction of cell cycle arrest and apoptosis. ( A ) IC 50 values of SLMP53-2 in A375, G361, MEWO and SK-MEL-5 melanoma cells obtained by colony formation assay for 11 days; data were normalized to DMSO and correspond to mean ± SEM, n = 5 (two replicates each). ( B ) Colony formation assay for A375, G361, MEWO and SK-MEL-5 melanoma cells treated with SLMP53-2 for the indicated concentrations. Images are representative of five independent experiments. ( C ) Effect of SLMP53-2 on growth and morphology of A375 cells for the indicated time points; images are representative of five independent experiments (scale bar = 100 μm, magnification = ×100). ( D ) Apoptosis (Annexin V-positive cells) was evaluated in A375 cells after 24, 48 and 72 h of treatment with 12 μM SLMP53-2. ( E ) Cell cycle analysis in A375 cells was determined after 24, 48 and 72 h of treatment with 12 μM SLMP53-2. In ( D , E ), data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( F , G ) Effect of SLMP53-2 on three-day-old A375 spheroids, for up to 8 days of treatment. In G , data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( H , I ) Evaluation of spheroid formation after 10 days of treatment with SLMP53-2; treatment was performed at the seeding time of A375 cells. In I , data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. In ( F , H ), images are representative of five independent experiments; scale bar = 100 μm; magnification = 100×.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Colony Assay, Cell Cycle Assay

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 has p53-dependent growth inhibitory effect in melanoma cells with enhancement of p53 transcriptional activity. ( A – C ) Colony formation assay for silenced p53 (sip53) and control (CTRL) A375 cells treated with SLMP53-2, allowed to grow for 11 days. In ( A ), silencing efficacy of p53 by siRNA is shown; immunoblots are representative of five independent experiments and GAPDH was used as loading control; data plotted were normalized to CTRL and correspond to mean ± SEM, n = 5; values are significantly different from CTRL: * p < 0.05, unpaired Student’s t -test. In ( B ), images are representative of five independent experiments. In ( C ), data are normalized to DMSO and correspond to mean ± SEM, n = 5; values of sip53 cells significantly different from CTRL cells: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( D , E ) Protein levels of p53 transcriptional targets in A375 cells treated with SLMP53-2 for 24 h (p53, MDM2, PTEN, Cyclin D1, p21 and KILLER) or 48 h (GADD45, PUMA, BCL-2, BCL-xL and BAX). In ( D ), immunoblots are representative of five independent experiments; GAPDH was used as loading control. In ( E ), quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( F ) mRNA levels of p53 target genes were determined by RT-qPCR in A375 cells after 24 h treatment with SLMP53-2; fold change is relative to DMSO; data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, two-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Activity Assay, Colony Assay, Control, Western Blot, Expressing, Quantitative RT-PCR, Comparison

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 enhances p53 stabilization by disrupting the p53–MDM2 interaction and interferes with the miRNA network in melanoma cells. ( A ) p53 protein levels in A375 melanoma cells treated for 24 h with 12 µM SLMP53-2 or solvent followed by cycloheximide treatment from 0 to 2 h (CHX; 150 μg/mL). ( B ) Quantification of p53 protein expression levels; immunoblots are representative of five independent experiments; GAPDH was used as loading control. Values for cells nontreated with cycloheximide (0 h) were set as 1; data are mean ± SEM, n = 5. ( C , D ) Coimmunoprecipitation (Co-IP) was performed in A375 cells treated with SLMP53-2 for 4 h. In C, representative immunoblots of five independent experiments are shown—whole-cell lysate (Input). p53 from IP was used as loading control. In D, quantification of protein expression levels relative to DMSO is shown (set as 1). Data shown are mean ± SEM, n = 5. ( E ) Expression levels of miR-145 and miR-23a in A375 cells after 24 h of treatment with SLMP53-2 were determined by RT-qPCR; fold of change is relative to DMSO; data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( F , G ) Protein levels of miR-145 target genes, in A375 cells treated with SLMP53-2 for 24 h. In ( F ), immunoblots are representative of five independent experiments; GAPDH was used as loading control. In ( G ), quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Solvent, Expressing, Western Blot, Control, Co-Immunoprecipitation Assay, Quantitative RT-PCR

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 inhibits melanoma cell migration and invasion. ( A ) A375 and SK-MEL-5 confluent cells were treated with 2 or 4 μM SLMP53-2, respectively; cells were observed at 24 and 32 h (A375) and 30 and 48 h (SK-MEL-5) in the wound-healing assay. Images are representative of five independent experiments; scale bar = 100 μM; magnification = 100×. ( B ) Quantification of wound closure using randomly selected microscopic fields (six fields per sample). Data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( C ) Effect of 2 μM SLMP53-2 on migration of A375 and SK-MEL-5 cells after 24 h of treatment. The relative number of migratory cells was determined by analysis of fluorescence signal intensity; values with DMSO were set as 1. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from DMSO: * p < 0.05, Student’s t -test. ( D ) Effect of 2 μM SLMP53-2 on the invasion of A375 and SK-MEL-5 cells after 24 h of treatment. Cells able to invade through an ECMatrix layer were quantified by fluorescence signal; values with DMSO were set as 1. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from DMSO: * p < 0.05, Student’s t -test. ( E ) Effect of SLMP53-2 on lactate secretion by A375 and SK-MEL-5 cells after 8 h of treatment. Cell density for each sample was used to normalize relative luminescence units (RLU) signal. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from DMSO: * p < 0.05; unpaired Student’s t -test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Migration, Wound Healing Assay, Fluorescence

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 interferes with key molecular players in epithelial-to-mesenchymal transition (EMT) and angiogenesis. ( A – D ) Protein expression levels of crucial regulators of EMT and angiogenesis in A375 ( A , B ) and SK-MEL-5 ( C , D ) melanoma cells after 48 h of treatment with SLMP53-2 (in A375 cells, β-catenin was detected for 8 h and E-cadherin and TWIST for 24 h of treatment). Immunoblots are representative of five independent experiments; GAPDH was used as a loading control. In ( B , D ), quantification of protein expression levels is shown; values with DMSO were set as 1; data are means ± SEM, n = 5.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Expressing, Western Blot, Control

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 sensitizes melanoma cells to clinically used chemotherapeutic agents. ( A – C ) Cells were treated with a concentration range of vemurafenib ( A ), dacarbazine ( B ) and cisplatin ( C ) alone and in combination with 2 μM SLMP53-2, for 48 h, and the growth was analysed by SBR assay. Growth with DMSO was set as 100%. For each combination, the combination index (C.I.) and dose reduction index (D.R.I.) values were obtained. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from chemotherapeutic drug alone: * p < 0.05; two-way ANOVA followed by Sidak’s test. ( D ) Apoptosis (Annexin V-positive cells) was evaluated in A375 cells after 48 h of treatment with 2 μM SLMP53-2 (SLMP) and 5 µM cisplatin and 0.03 µM vemurafenib. Data are mean ± SEM, n = 5; values are significantly different from drug alone: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( E , F ) Protein expression levels of BCL-2 after 48 h of treatment of SLMP53-2 with cisplatin (cisp) and with vemurafenib (vem). Immunoblots are representative of five independent experiments; GAPDH was used as a loading control. In F , quantification of protein expression levels; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( G ) Cell cycle analysis in A375 cells was determined after 48 h of treatment with 2 μM SLMP53-2 and 2 μM dacarbazine (Dac). Data are mean ± SEM, n = 5; values are significantly different from drug alone: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( H , I ) Protein expression levels of p21 after 48 h treatment of SLMP53-2 with dacarbazine (Dac). Immunoblots are representative of five independent experiments; GAPDH was used as a loading control. In I , quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( J , K ) Effect of 2 μM SLMP53-2 in combination with 0.027 μM Vemurafenib (Vem) on three-day-old A375 spheroids for up to 8 days of treatment. For the combination, the C.I. value was obtained. Images are representative of five independent experiments; scale bar = 100 μm; magnification = 100×. In ( K ), data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Concentration Assay, Expressing, Western Blot, Control, Cell Cycle Assay

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: Melanoma cells do not develop resistance to SLMP53-2: vemurafenib-resistant melanoma cells show no cross-resistance to SLMP53-2 and are resensitized to vemurafenib by SLMP53-2. ( A ) A375 cells were exposed to six rounds of treatment with 6, 9, 12, 18, 24 and 30 μM of SLMP53-2. IC 50 values were determined at the end of each round by SRB assay after 48 h of treatment. Data were normalized to DMSO and correspond to mean ± SEM, n = 5 (two replicates each); values not significantly different from parental cells: p > 0.05, two-way ANOVA followed by Sidak’s test. ( B ) Representative images of parental, vemurafenib-resistant (Vem-res) A375 cells; scale bar = 100 μm; magnification = 100×. ( C ) Concentration–response curves for vemurafenib in parental and Vem-res A375 cells after 48 h of treatment. Data were normalized to DMSO and correspond to mean ± SEM, n = 6 (two replicates each); values of Vem-res cells significantly different from parental cells: * p < 0.05; two-way ANOVA followed by Sidak’s test. ( D , E ) Protein levels of p-AKT/AKT, p-ERK/ERK, PTEN and MDR-1 in untreated parental and Vem-res A375 cells. In D, immunoblots are representative of five independent experiments; GAPDH was used as loading control. In E, quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( F ) Concentration–response curves for SLMP53-2 in parental and Vem-res A375 cells after 48 h of treatment. Data were normalized to DMSO and correspond to mean ± SEM, n = 6 (two replicates each); values of Vem-res cells are not significantly different from parental cells: two-way ANOVA followed by Sidak’s test. ( G ) Vem-res A375 cells were treated with a concentration range of vemurafenib alone and in combination with 2 μM of SLMP53-2. Cell growth was evaluated for 48 h of treatment; growth obtained with DMSO was set as 100%. For each combination, the C.I. and D.R.I. values were obtained. Data are mean ± SEM, n = 6 (two replicates each); values are significantly different from vemurafenib alone: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( H ) Representative images of Vem-res A375 cells treated with DMSO, 2 μM SLMP53-2, 1.3 μM vemurafenib (Vem) and the combination (SLMP53-2 + Vem) for 48 h; images are representative of five treatments; scale bar = 100 μm; magnificatio n = 100×. ( I , J ) Protein levels of PTEN, BCL-2, MDR-1 and p-AKT/AKT, in Vem-res cells after 48 h of treatment with 2 µM SLMP53-2. In I, immunoblots are representative of five independent experiments; GAPDH was used as loading control. In J, quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Sulforhodamine B Assay, Concentration Assay, Western Blot, Control, Expressing

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: In vivo melanoma antitumour activity of SLMP53-2. C57BL/6-Rag2 −/− IL2rg −/− mice carrying A375 xenografts were treated with 50 mg∙kg −1 SLMP53-2 or vehicle by intraperitoneal injection twice a week for a total of six administrations. ( A ) Tumour volume curves of mice carrying A375 xenografts treated with SLMP53-2 or vehicle. Fold change is relative to the start of treatments; data are mean ± SEM, n = 7; values are significantly different from vehicle: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( B ) Tumour weights measured at the end of the in vivo experiment; data are mean ± SEM, n = 7; values are significantly different from vehicle: * p < 0.05, unpaired Student’s t -test. Representative images of the tumours treated with SLMP53-2 or vehicle at the end of the experiment. ( C ) Body weight of the mice registered during the course of the experiment. Data are mean ± SEM, n = 7; values are not significantly different from vehicle: p > 0.05, two-way ANOVA followed by Sidak’s test. ( D ) Weight of heart, spleen, kidney and livers from animals treated with SLMP53-2 or vehicle. Data are mean ± SEM, n = 7; values are not significantly different from vehicle: p > 0.05, two-way ANOVA followed by Sidak’s test. ( E ) Representative images of p53, Ki-67, BAX, BCL-2, TUNEL, β-catenin, Vimentin, and Slug detection in tumour tissues of A375 xenografts treated with SLMP53-2 or vehicle, collected at the end of treatment (scale bar = 5 μm; magnificatio n = 200×); haematoxylin and eosin (H&E). ( F – H ) Quantification of immunohistochemistry of A375 xenograft tumour tissues treated with SLMP53-2 or vehicle. In F, quantification of the number of Ki-67-positive and -negative cells; values are significantly different from vehicle: * p < 0.05, two-way ANOVA followed by Sidak’s test. In G, quantification of the percentage of positive-staining cells with TUNEL, n = 5; values are significantly different from vehicle: * p < 0.05, unpaired Student’s t -test. In H, quantification of the p53, Vimentin, BAX, BCL-2, β-catenin and Slug staining by evaluation of 3,3′-diaminobenzidine (DAB) intensity is shown, n = 5; values are significantly different from vehicle: * p < 0.05, unpaired Student’s t -test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: In Vivo, Activity Assay, Injection, TUNEL Assay, Immunohistochemistry, Staining

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Synthesis, in silico modelling, and in vitro biological evaluation of substituted pyrazole derivatives as potential anti-skin cancer, anti-tyrosinase, and antioxidant agents

doi: 10.1080/14756366.2023.2205042

Figure Lengend Snippet: Cytotoxicity of pyrazole (celecoxib analogs), isoxazole, pyrazolone, and positive control compounds P1 – P25 (structures, Figure 3 ) against cells of human cutaneous melanoma and non-melanoma skin cancer lines relative to standard control noncancerous immortalised HaCaT cells.

Article Snippet: Human-derived GFP-expressing melanoma A375 and epidermoid carcinoma A431 cell lines were purchased from Angio-Proteomie (Boston, MA).

Techniques: Positive Control, Control

Journal: PLoS ONE

Article Title: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

doi: 10.1371/journal.pone.0086338

Figure Lengend Snippet: The invasive capacity of BRAF mutated (A375, SK-MEL-28 and RPMI-7951), NRAS mutated (SK-MEL-119) and BRAF-NRAS wild type (Hs294T) melanoma cells was determined in vitro using Boyden chamber assay. Melanoma cells (3×10 4 cells/200 µl serum-reduced medium) were placed in the upper chamber of Boyden chamber containing 0, 5, 10 and 20 µM of fisetin. The lower chamber contained 110 µl of medium supplemented with 10% FBS. After 24 hours of incubation, the invaded cells on the lower surface of the membranes were fixed with chilled methanol and stained with crystal violet. A representative picture from three independent experiments is shown. The invaded cells were counted in at least four to five randomly selected microscopic fields on the membrane and the results are summarized and expressed as the mean number of invaded cells ± SEM per microscopic field. Significant difference versus control group, * P <0.05, ** P <0.01. Bar = 100 µm.

Article Snippet: Three-dimensional skin equivalents of A375 melanoma cells (MLNM-FT-A375) were obtained from MatTek Corporation (Ashland, MA).

Techniques: In Vitro, Boyden Chamber Assay, Incubation, Staining, Membrane, Control

Journal: PLoS ONE

Article Title: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

doi: 10.1371/journal.pone.0086338

Figure Lengend Snippet: [ A ] The three-dimensional skin equivalents containing A375 cells were treated with fisetin (5–20 µM) for 7 days. After treatment with fisetin, skin samples were collected and H&E was performed. A representative picture from three independent experiments is shown. Arrows indicate invading A375 cells. Bar = 25 µm. [ B ] The three-dimensional skin equivalents containing A375 cells were treated with 20 µM of fisetin, PD98059 or CAPE for 12 days. After treatment samples were collected and H&E was performed. A representative picture from three independent experiments is shown. Arrows indicate invading A375 cells. Bar = 25 µm. [ C ] A375 cells (3×10 4 cells/200 µl serum-reduced medium) were placed in the upper chamber of Boyden chamber containing 10 and 20 µM of fisetin, PD98059 or CAPE. The lower chamber contained 110 µl of medium supplemented with 10% FBS. After 24 hours of incubation, the invaded cells on the lower surface of the membranes were fixed with chilled methanol and stained with crystal violet. The invaded cells were counted on the membrane in at least four to five randomly selected microscopic fields. The results are summarized and expressed as the mean number of invaded cells ± SEM per microscopic field. Significant difference versus control group, * P <0.05, ** P <0.01. [D] A375 cells were transfected with MEK1-GFP or GFP-N2 control vector using Xfect Transfection Reagent as per the manufacturer’s protocol. Forty-eight hours after transfection, the cells were harvested and invasion assay was performed as described in “Materials and Methods” section. Significant difference versus control group, ** P <0.01.

Article Snippet: Three-dimensional skin equivalents of A375 melanoma cells (MLNM-FT-A375) were obtained from MatTek Corporation (Ashland, MA).

Techniques: Incubation, Staining, Membrane, Control, Transfection, Plasmid Preparation, Invasion Assay

Journal: PLoS ONE

Article Title: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

doi: 10.1371/journal.pone.0086338

Figure Lengend Snippet: The melanoma cells (A375 and RPMI-7951) were treated with fisetin (5–20 µM; 24 hours) and then the cells were harvested. Total cell lysates were prepared and [ A ] Western blot analysis for protein expression and [ B ] relative density was performed, as described in the ‘Materials and Methods’ section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblots shown here are representative of three independent experiments with similar results, shown in relative units ± SEM. Significant difference versus control group, * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Three-dimensional skin equivalents of A375 melanoma cells (MLNM-FT-A375) were obtained from MatTek Corporation (Ashland, MA).

Techniques: Western Blot, Expressing, Stripping Membranes, Control

Journal: PLoS ONE

Article Title: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

doi: 10.1371/journal.pone.0086338

Figure Lengend Snippet: The melanoma cells (A375 and RPMI-7951) were treated with fisetin (5–20 µM; 24 hours) and then the cells were harvested. Nuclear and cytosolic lysates were prepared and [A&C] Western blot analysis for protein expression and [B&D] relative density was performed, as described in the ‘Materials and Methods’ section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for Lamin (nuclear fraction) and β-actin (cytosolic fraction). The immunoblots shown here are representative of three independent experiments with similar results, shown in relative units ± SEM. Significant difference versus control group, * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Three-dimensional skin equivalents of A375 melanoma cells (MLNM-FT-A375) were obtained from MatTek Corporation (Ashland, MA).

Techniques: Western Blot, Expressing, Stripping Membranes, Control

Journal: PLoS ONE

Article Title: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

doi: 10.1371/journal.pone.0086338

Figure Lengend Snippet: The melanoma cells (A375 and RPMI-7951) were treated with fisetin (5–20 µM; 24hours) and then cells were harvested. Total cell lysates were prepared and [ A ] Western blot analysis for protein expression and [ B ] relative density was performed, as described in the ‘Materials and Methods’ section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblots shown here are representative of three independent experiments with similar results, shown in relative units ± SEM. Significant difference versus control group, * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Three-dimensional skin equivalents of A375 melanoma cells (MLNM-FT-A375) were obtained from MatTek Corporation (Ashland, MA).

Techniques: Western Blot, Expressing, Stripping Membranes, Control

Journal: PLoS ONE

Article Title: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

doi: 10.1371/journal.pone.0086338

Figure Lengend Snippet: The tissue-engineered three-dimensional skin equivalents consisting A375 cells were treated with (10 and 20 µM fisetin for 7 days. After treatment, samples were collected, fixed in formalin and paraffin blocks were prepared. Five micormeter sections were cut, deparaffinized, rehydrated and were heated at 95°C for 20 min in citrate buffer (pH 6.0) for antigen retrieval. Sections were incubated with primary antibody against E-cadherin or vimentin overnight at 4°C followed by incubation with specific Alexa Flour 488 or 594 labeled secondary antibody for 1 hour at room temperature in the dark. After washing, the sections were incubated with vectashield mounting media containing DAPI for 10 min in the dark and analyzed under microscope immediately. A representative picture from three independent experiments is shown. E-cadherin is shown in red, vimentin in green and DAPI in blue. Bar = 25 µm.

Article Snippet: Three-dimensional skin equivalents of A375 melanoma cells (MLNM-FT-A375) were obtained from MatTek Corporation (Ashland, MA).

Techniques: Incubation, Labeling, Microscopy

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: 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