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