Journal: The American journal of pathology

Article Title: The Neuropeptide Alpha-Melanocyte-Stimulating Hormone Is Critical for Corneal Endothelial Cell Protection and Graft Survival after Transplantation.

doi: 10.1016/j.ajpath.2021.10.016

Figure Lengend Snippet: Figure 1 Corneal endothelial cells (CEnC) express high levels of the receptor for a-MSH. A: Immunostaining for a-MSH receptor, melanocortin receptor 1 (MC1R), on murine (mCEnC) and human (hCEnC) CEnC lines and primary CEnCs. Blue: DAPI, purple: MC1R. B and C: Representative Western blot bands of MC1R in murine and human CEnC lines (B) and in primary murine corneal endothelium lysates (C). SIT lysate: small intestine tissue lysate used as positive control for mouse MC1R (mMC1R). A-375: human skin epithelial cell lysate used as positive control for human MC1R (hMC1R). Scale bars Z 50 mm.

Article Snippet: The mouse small intestine tissue (SIT) lysate (No. PK-AB718-1408; PromoCell, Heidelberg, Germany) and A375 human skin epithelial cell lysate (No. sc-3811; Santa Cruz Biotechnology, Dallas, TX) were used as positive controls for mouse and human MC1R, respectively.

Techniques: Immunostaining, Western Blot, Positive Control

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: Scientific Reports

Article Title: Unraveling the role of HIF2α in melanoma progression and epithelial–mesenchymal transition

doi: 10.1038/s41598-025-02384-2

Figure Lengend Snippet: Effects of Hypoxia and HIF-2α Knockdown on HIF-2α, E-cadherin, N-cadherin, and Vimentin mRNA Expression in A375 Cells. ( A ) Hypoxia significantly increased the mRNA levels of HIF-2α in A375 cells (* P < 0.01); In the NC group, no significant changes in mRNA expression were observed under hypoxic conditions. HIF-2α knockdown under hypoxia led to a significant reduction in the mRNA levels of HIF-2αcompared to the NC group(# P < 0.01). ( B ) Hypoxia induced a modest yet statistically significant reduction in the mRNA levels of E-cadherin in A375 cells (* P < 0.01);In the NC group, no significant changes in mRNA expression were observed under hypoxic conditions.HIF-2α knockdown under hypoxia led to a modest but statistically significant upregulation of E-cadherin mRNA levels, as compared to the NC group(# P < 0.01). ( C , D ) Hypoxia induced a modest yet statistically significant upregulation in the mRNA levels of N-cadherin and Vimentin in A375 cells (* P < 0.01); In the NC group, no significant changes in mRNA expression were observed under hypoxic conditions.HIF-2α knockdown under hypoxia led to a modest but statistically significant reduction of N-cadherin and Vimentin mRNA levels, as compared to the NC group (# P < 0.01).

Article Snippet: Cell Grouping and Culture: The A375 cells were provided by Jiangsu KeyGEN BioTECH Co.,Ltd.

Techniques: Knockdown, Expressing

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: 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