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a375m human melanoma cell line  (ATCC)


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    ATCC a375m human melanoma cell line
    A375m Human Melanoma Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 1151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/a375m+human+melanoma+cell+line/A375%3B+Melanoma%3B+Human/pmc12828906-90-0-9
    Average 97 stars, based on 1151 article reviews
    a375m human melanoma cell line - by Bioz Stars, 2026-09
    97/100 stars

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    Related Articles

    Isolation:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Virus:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Subcloning:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Bacteria:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Recombinant:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Multiplex sample analysis:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Labeling:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Reverse Transcription:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    RNA Extraction:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Reporter Assay:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Bicinchoninic Acid Protein Assay:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Flow Cytometry:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Enzyme-linked Immunosorbent Assay:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Quantitative RT-PCR:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Control:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Plasmid Preparation:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Expressing:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Construct:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Luciferase:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Software:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Protease Inhibitor:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Purification:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Mutagenesis:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Fluorescence:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .

    Sequencing:

    Article Title: Lineage-restricted regulation of SCD and fatty acid saturation by MITF controls melanoma phenotypic plasticity
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , ( Fogh et al., 1977 ; Giard et al., 1973 )( Fogh et al., 1977 ; Giard et al., 1973 ).

    Article Title: MITF, TFEB, and TFE3 drive distinct adaptive gene expression programs and immune infiltration in melanoma
    Article Snippet: A375M Human melanoma cell line (male) , Obtained from ATCC , Giard et al. .



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    PGE2 increases migration and invasion of <t>A375M</t> (CAV1/E-cad) cells. A375 (Mock) and A375 (CAV1) cell lines cultured in the presence of IPTG were transiently transfected with pBATEM for 48 h to express E-cad. A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (CAV1/E-cad) cells were treated with 50 μM of PGE2 for 5 h. Then, the extracts were either prepared for immunoprecipitation experiments or cell migration and invasion measurements in transwell and matrigel assays, respectively. ( A ) CAV1 was immunoprecipitated from protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via Western blotting. ( B ) Representative Western blot of immunoprecipitates obtained in three independent experiments. E-cad and CAV1 protein levels were quantified in several experiments via densitometry and normalized to actin (mean ± SD, n = 3; ** p < 0.01). A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (E-cad/CAV1) cells were serum-deprived for 5 h and treated with PGE2 (50 μM) for 2 h. Then, CAV1 was immunoprecipitated from the protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via Western blotting. ( B ) Graph showing the CAV1 as normalized pixels (mean ± SEM) for each condition; n = 3; and ** p < 0.01 with respect to A375 (mock). ( C ) Graph showing E-cad as normalized pixels (mean ± SEM) for each condition; n = 3; ** p < 0.01 with respect to A375 (mock). ( D ) CAV1/E-cad as normalized pixels (mean ± SEM) for each condition; n = 3; and * p < 0.05 or ** p < 0.01 with respect to A375 (mock). ( E ) A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (E-cad/CAV1) cells were treated with 50 μM of PGE2 for 5 h. Then, 150.000 cells were seeded into Boyden chambers (transwells) in the presence of 50 μM PGE2. Migration was measured after 2 h. Significant differences are shown (* p < 0.05; n = 3). ( F ) Panels showing representative images of results obtained for cells migrating either without treatment (basal condition, upper panel ) or when treated with 50 μM of PGE2 for 5 h ( lower panel ), in both cases stained with crystal violet. ( H ) Cells cultured in the presence or absence of 50 µM of PGE2 for 5 h were used for the invasion assays. Cells (150.000) were seeded into matrigel Boyden chambers in the presence of 50 μM of PGE2. Invasion was measured after 48 h. Graphs show averages (mean ± SEM; n = 3). Significant differences are indicated (* p < 0.05). ( G ) Panel showing representative images obtained in invasion assays for cells cultured in the absence (basal condition, upper panel ) or presence ( lower panel ) of 50 μM of PGE2, in both cases stained with DAPI Bar = 50 μm. (* p < 0.05; ** p < 0.01; *** p < 0.001; n = 3).
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    Figure 2. In vitro antitumor activity of EV20‑based ADCs. (A) The cytotoxic response of LC cells to EV20‑based ADCs treatment was evaluated by MTT after 120 h of treatment with increasing doses ranging between 0.006 nM and 100 nM. The IC50 values were calculated with GraphPad Prism 5.0 software and reported. (B) <t>A375m</t> and HepG2 cells were maintained for 30 min on ice in the presence of 10 µg/ml EV20 and placed again in the incubator at 37˚C for 1 h. The internalization rate of the antibody was evaluated by flow cytometry and by confocal microscopy imaging. The histogram represents the percentage of MFI referred to control (cells maintained on ice). Plotted results are an average ± SD of three independent experiments. For confocal microscopy imaging, EV20 and nuclei were visualized on green and blue channels, respectively. (C) A375m and HepG2 cells were incubated for 72 h with eight increasing concentrations of free MMAF, diluted from 10 µM to 10 pM, in 1:10 dilution increments. Proliferation was evaluated by MTT assay (left panel). HepG2 cells were incubated for 72 h with the same increasing doses of free MMAF used in (C) in absence or presence of MK571 (25 µM), PSC833 (3 µM) and Reversan (15 µM) and proliferation was evaluated by MTT assay (right panel). (D) EV20‑sss‑vc/MMAF characterization. HIC was used for DAR calculation; melanoma A375m HER3+ cells were used for cell binding by flow cytometry. ELISA was performed for in vitro binding with naked EV20‑sss mAb used as control. (E) PLC/PRF/5 LC cells were incubated for 2 h or not with naked or conjugated EV20 mAb, at a dose of 10 µg/ml, before NRG‑1β stimulation (10 min, 10 ng/ml). Total and phosphorylated HER‑3 receptor was analysed by western blotting and bands were quantified using actin as loading control. Histograms represent densitometric analysis of a single experiment, expressed as arbitrary units. MFI, mean fluorescence intensity; ADCs, antibody‑drug conjugates; LC, liver cancer; MTT, 3‑(4,5‑dimethyldiazol‑2‑yl)‑2,5‑diphenyl tetrazolium bromide; MMAF, monomethyl auristatin F; vc, valine‑citrulline; HIC, hydrophobic chromatography; DAR, drug antibody ratio.
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    Image Search Results


    PGE2 increases migration and invasion of A375M (CAV1/E-cad) cells. A375 (Mock) and A375 (CAV1) cell lines cultured in the presence of IPTG were transiently transfected with pBATEM for 48 h to express E-cad. A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (CAV1/E-cad) cells were treated with 50 μM of PGE2 for 5 h. Then, the extracts were either prepared for immunoprecipitation experiments or cell migration and invasion measurements in transwell and matrigel assays, respectively. ( A ) CAV1 was immunoprecipitated from protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via Western blotting. ( B ) Representative Western blot of immunoprecipitates obtained in three independent experiments. E-cad and CAV1 protein levels were quantified in several experiments via densitometry and normalized to actin (mean ± SD, n = 3; ** p < 0.01). A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (E-cad/CAV1) cells were serum-deprived for 5 h and treated with PGE2 (50 μM) for 2 h. Then, CAV1 was immunoprecipitated from the protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via Western blotting. ( B ) Graph showing the CAV1 as normalized pixels (mean ± SEM) for each condition; n = 3; and ** p < 0.01 with respect to A375 (mock). ( C ) Graph showing E-cad as normalized pixels (mean ± SEM) for each condition; n = 3; ** p < 0.01 with respect to A375 (mock). ( D ) CAV1/E-cad as normalized pixels (mean ± SEM) for each condition; n = 3; and * p < 0.05 or ** p < 0.01 with respect to A375 (mock). ( E ) A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (E-cad/CAV1) cells were treated with 50 μM of PGE2 for 5 h. Then, 150.000 cells were seeded into Boyden chambers (transwells) in the presence of 50 μM PGE2. Migration was measured after 2 h. Significant differences are shown (* p < 0.05; n = 3). ( F ) Panels showing representative images of results obtained for cells migrating either without treatment (basal condition, upper panel ) or when treated with 50 μM of PGE2 for 5 h ( lower panel ), in both cases stained with crystal violet. ( H ) Cells cultured in the presence or absence of 50 µM of PGE2 for 5 h were used for the invasion assays. Cells (150.000) were seeded into matrigel Boyden chambers in the presence of 50 μM of PGE2. Invasion was measured after 48 h. Graphs show averages (mean ± SEM; n = 3). Significant differences are indicated (* p < 0.05). ( G ) Panel showing representative images obtained in invasion assays for cells cultured in the absence (basal condition, upper panel ) or presence ( lower panel ) of 50 μM of PGE2, in both cases stained with DAPI Bar = 50 μm. (* p < 0.05; ** p < 0.01; *** p < 0.001; n = 3).

    Journal: International Journal of Molecular Sciences

    Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

    doi: 10.3390/ijms242316947

    Figure Lengend Snippet: PGE2 increases migration and invasion of A375M (CAV1/E-cad) cells. A375 (Mock) and A375 (CAV1) cell lines cultured in the presence of IPTG were transiently transfected with pBATEM for 48 h to express E-cad. A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (CAV1/E-cad) cells were treated with 50 μM of PGE2 for 5 h. Then, the extracts were either prepared for immunoprecipitation experiments or cell migration and invasion measurements in transwell and matrigel assays, respectively. ( A ) CAV1 was immunoprecipitated from protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via Western blotting. ( B ) Representative Western blot of immunoprecipitates obtained in three independent experiments. E-cad and CAV1 protein levels were quantified in several experiments via densitometry and normalized to actin (mean ± SD, n = 3; ** p < 0.01). A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (E-cad/CAV1) cells were serum-deprived for 5 h and treated with PGE2 (50 μM) for 2 h. Then, CAV1 was immunoprecipitated from the protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via Western blotting. ( B ) Graph showing the CAV1 as normalized pixels (mean ± SEM) for each condition; n = 3; and ** p < 0.01 with respect to A375 (mock). ( C ) Graph showing E-cad as normalized pixels (mean ± SEM) for each condition; n = 3; ** p < 0.01 with respect to A375 (mock). ( D ) CAV1/E-cad as normalized pixels (mean ± SEM) for each condition; n = 3; and * p < 0.05 or ** p < 0.01 with respect to A375 (mock). ( E ) A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (E-cad/CAV1) cells were treated with 50 μM of PGE2 for 5 h. Then, 150.000 cells were seeded into Boyden chambers (transwells) in the presence of 50 μM PGE2. Migration was measured after 2 h. Significant differences are shown (* p < 0.05; n = 3). ( F ) Panels showing representative images of results obtained for cells migrating either without treatment (basal condition, upper panel ) or when treated with 50 μM of PGE2 for 5 h ( lower panel ), in both cases stained with crystal violet. ( H ) Cells cultured in the presence or absence of 50 µM of PGE2 for 5 h were used for the invasion assays. Cells (150.000) were seeded into matrigel Boyden chambers in the presence of 50 μM of PGE2. Invasion was measured after 48 h. Graphs show averages (mean ± SEM; n = 3). Significant differences are indicated (* p < 0.05). ( G ) Panel showing representative images obtained in invasion assays for cells cultured in the absence (basal condition, upper panel ) or presence ( lower panel ) of 50 μM of PGE2, in both cases stained with DAPI Bar = 50 μm. (* p < 0.05; ** p < 0.01; *** p < 0.001; n = 3).

    Article Snippet: B16F10 metastatic murine melanoma cells (ATCC, #CRL6475, provided by Laurence Zitvogel, Institut Gustav Roussy, Villejuif, France) and the human melanoma cell line A375M (ATCC, #CRL1619), also employed previously [ ], were maintained in RPMI 1640 medium.

    Techniques: Migration, Cell Culture, Transfection, Immunoprecipitation, Western Blot, Staining

    Figure 2. In vitro antitumor activity of EV20‑based ADCs. (A) The cytotoxic response of LC cells to EV20‑based ADCs treatment was evaluated by MTT after 120 h of treatment with increasing doses ranging between 0.006 nM and 100 nM. The IC50 values were calculated with GraphPad Prism 5.0 software and reported. (B) A375m and HepG2 cells were maintained for 30 min on ice in the presence of 10 µg/ml EV20 and placed again in the incubator at 37˚C for 1 h. The internalization rate of the antibody was evaluated by flow cytometry and by confocal microscopy imaging. The histogram represents the percentage of MFI referred to control (cells maintained on ice). Plotted results are an average ± SD of three independent experiments. For confocal microscopy imaging, EV20 and nuclei were visualized on green and blue channels, respectively. (C) A375m and HepG2 cells were incubated for 72 h with eight increasing concentrations of free MMAF, diluted from 10 µM to 10 pM, in 1:10 dilution increments. Proliferation was evaluated by MTT assay (left panel). HepG2 cells were incubated for 72 h with the same increasing doses of free MMAF used in (C) in absence or presence of MK571 (25 µM), PSC833 (3 µM) and Reversan (15 µM) and proliferation was evaluated by MTT assay (right panel). (D) EV20‑sss‑vc/MMAF characterization. HIC was used for DAR calculation; melanoma A375m HER3+ cells were used for cell binding by flow cytometry. ELISA was performed for in vitro binding with naked EV20‑sss mAb used as control. (E) PLC/PRF/5 LC cells were incubated for 2 h or not with naked or conjugated EV20 mAb, at a dose of 10 µg/ml, before NRG‑1β stimulation (10 min, 10 ng/ml). Total and phosphorylated HER‑3 receptor was analysed by western blotting and bands were quantified using actin as loading control. Histograms represent densitometric analysis of a single experiment, expressed as arbitrary units. MFI, mean fluorescence intensity; ADCs, antibody‑drug conjugates; LC, liver cancer; MTT, 3‑(4,5‑dimethyldiazol‑2‑yl)‑2,5‑diphenyl tetrazolium bromide; MMAF, monomethyl auristatin F; vc, valine‑citrulline; HIC, hydrophobic chromatography; DAR, drug antibody ratio.

    Journal: Oncology reports

    Article Title: EV20‑sss‑vc/MMAF, an HER‑3 targeting antibody‑drug conjugate displays antitumor activity in liver cancer.

    doi: 10.3892/or.2020.7893

    Figure Lengend Snippet: Figure 2. In vitro antitumor activity of EV20‑based ADCs. (A) The cytotoxic response of LC cells to EV20‑based ADCs treatment was evaluated by MTT after 120 h of treatment with increasing doses ranging between 0.006 nM and 100 nM. The IC50 values were calculated with GraphPad Prism 5.0 software and reported. (B) A375m and HepG2 cells were maintained for 30 min on ice in the presence of 10 µg/ml EV20 and placed again in the incubator at 37˚C for 1 h. The internalization rate of the antibody was evaluated by flow cytometry and by confocal microscopy imaging. The histogram represents the percentage of MFI referred to control (cells maintained on ice). Plotted results are an average ± SD of three independent experiments. For confocal microscopy imaging, EV20 and nuclei were visualized on green and blue channels, respectively. (C) A375m and HepG2 cells were incubated for 72 h with eight increasing concentrations of free MMAF, diluted from 10 µM to 10 pM, in 1:10 dilution increments. Proliferation was evaluated by MTT assay (left panel). HepG2 cells were incubated for 72 h with the same increasing doses of free MMAF used in (C) in absence or presence of MK571 (25 µM), PSC833 (3 µM) and Reversan (15 µM) and proliferation was evaluated by MTT assay (right panel). (D) EV20‑sss‑vc/MMAF characterization. HIC was used for DAR calculation; melanoma A375m HER3+ cells were used for cell binding by flow cytometry. ELISA was performed for in vitro binding with naked EV20‑sss mAb used as control. (E) PLC/PRF/5 LC cells were incubated for 2 h or not with naked or conjugated EV20 mAb, at a dose of 10 µg/ml, before NRG‑1β stimulation (10 min, 10 ng/ml). Total and phosphorylated HER‑3 receptor was analysed by western blotting and bands were quantified using actin as loading control. Histograms represent densitometric analysis of a single experiment, expressed as arbitrary units. MFI, mean fluorescence intensity; ADCs, antibody‑drug conjugates; LC, liver cancer; MTT, 3‑(4,5‑dimethyldiazol‑2‑yl)‑2,5‑diphenyl tetrazolium bromide; MMAF, monomethyl auristatin F; vc, valine‑citrulline; HIC, hydrophobic chromatography; DAR, drug antibody ratio.

    Article Snippet: A375m human melanoma cell line (CRL3223) and SJSA‐1 human osteosarcoma cell line (CRL2098) were purchased from ATCC.

    Techniques: In Vitro, Activity Assay, Software, Flow Cytometry, Confocal Microscopy, Imaging, Control, Incubation, MTT Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Fluorescence, Chromatography

    Figure 3. EV20‑sss‑vc/MMAF cell killing activity is target‑dependent and superior to T‑DM1. (A) A375m cells were incubated for 120 h with increasing doses of EV20‑sss‑vc/MMAF or EV20/MMAF ranging between 0.006 nM and 100 nM, and proliferation was evaluated by MTT assay. (B) Cytotoxic response of HER‑3 negative SJSA‑1 cells to EV20‑sss‑vc/MMAF treatment (with doses ranging between 0.006 and 100 nM) was evaluated by MTT after 120 h of drug exposure. HER‑3 expression by flow cytometric analysis is presented as inset. (C) A375m cells were incubated for 120 h with increasing doses of EV20‑sss‑vc/MMAF (ranging between 0.006 and 100 nM) alone or with 500‑fold molar excess of naked EV20 and proliferation was evaluated by MTT assay. (D) LC cells were exposed for 120 h to increasing doses of T‑DM1 or EV20‑sss‑vc/MMAF (ranging between 0.006 and 100 nM) and proliferation was evaluated by MTT assay as aforementioned. vc, valine‑citrulline; MMAF, monomethyl auristatin F; MTT, 3‑(4,5‑dimethyldiazol‑2‑yl)‑2,5‑diphenyl tetrazolium bromide; LC, liver cancer.

    Journal: Oncology reports

    Article Title: EV20‑sss‑vc/MMAF, an HER‑3 targeting antibody‑drug conjugate displays antitumor activity in liver cancer.

    doi: 10.3892/or.2020.7893

    Figure Lengend Snippet: Figure 3. EV20‑sss‑vc/MMAF cell killing activity is target‑dependent and superior to T‑DM1. (A) A375m cells were incubated for 120 h with increasing doses of EV20‑sss‑vc/MMAF or EV20/MMAF ranging between 0.006 nM and 100 nM, and proliferation was evaluated by MTT assay. (B) Cytotoxic response of HER‑3 negative SJSA‑1 cells to EV20‑sss‑vc/MMAF treatment (with doses ranging between 0.006 and 100 nM) was evaluated by MTT after 120 h of drug exposure. HER‑3 expression by flow cytometric analysis is presented as inset. (C) A375m cells were incubated for 120 h with increasing doses of EV20‑sss‑vc/MMAF (ranging between 0.006 and 100 nM) alone or with 500‑fold molar excess of naked EV20 and proliferation was evaluated by MTT assay. (D) LC cells were exposed for 120 h to increasing doses of T‑DM1 or EV20‑sss‑vc/MMAF (ranging between 0.006 and 100 nM) and proliferation was evaluated by MTT assay as aforementioned. vc, valine‑citrulline; MMAF, monomethyl auristatin F; MTT, 3‑(4,5‑dimethyldiazol‑2‑yl)‑2,5‑diphenyl tetrazolium bromide; LC, liver cancer.

    Article Snippet: A375m human melanoma cell line (CRL3223) and SJSA‐1 human osteosarcoma cell line (CRL2098) were purchased from ATCC.

    Techniques: Activity Assay, Incubation, MTT Assay, Expressing

    (A) Representative western blot analysis of COX-2 expression by VEGF-treated B16M cells . Cultured B16M cells were given 10 or 100 ng/ml murine recombinant VEGF for 4 h. Cell lysates were collected and assayed for COX-2 and β-tubulin levels by western immunoblot. (B) Effect of celecoxib on the proadhesive response of VEGF-treated B16M cells on immobilized VCAM-1 . B16M cells received 1 μg/ml celecoxib for 30 min and then incubated with 100 ng/ml rmVEGF for 4 h. In other experiments B16M cells were given 10 or 100 ng/ml of PGE2 for 2 h. Then, cell adhesion assay to rhVCAM-1-coated plate was performed. Data are expressed as mean percent of added labeled-cells binding to quadruplicate wells ± SD. Statistical significance by ANOVA and Bonferroni's post- hoc test: * P < 0.01 as compared with basal medium-treated B16M cells; ** P < .001 as compared with VEGF-treated B16M cells. C) Effect of celecoxib and anti-VEGF on the proadhesive response of A375M cells to bone marrow-conditioned media on immobilized VCAM-1 . Human A375M cells received 1 μg/ml celecoxib for 30 min and then incubated in the presence of basal medium, hBMSC-CM, LPS-treated hBMSC-CM or rhVEGF (10 ng/ml) for 4 h. Then, cell adhesion assay to a rhVCAM-1-coated plate was performed. Data are expressed as mean percent of added labeled-cells binding to quadruplicate wells ± SD. Statistical significance by ANOVA and Bonferroni's post- hoc test: * P < 0.01 as compared with basal medium-treated A375M cells; ** P < 0.01 as compared with BMSC-CM-; + P < 0.01 as compared with LPS-treated BMSC-CM-treated A375M cells; # P < 0.01 as compared with rhVEGF-treated A375M cells.

    Journal: Journal of Translational Medicine

    Article Title: Vascular endothelial growth factor regulates melanoma cell adhesion and growth in the bone marrow microenvironment via tumor cyclooxygenase-2

    doi: 10.1186/1479-5876-9-142

    Figure Lengend Snippet: (A) Representative western blot analysis of COX-2 expression by VEGF-treated B16M cells . Cultured B16M cells were given 10 or 100 ng/ml murine recombinant VEGF for 4 h. Cell lysates were collected and assayed for COX-2 and β-tubulin levels by western immunoblot. (B) Effect of celecoxib on the proadhesive response of VEGF-treated B16M cells on immobilized VCAM-1 . B16M cells received 1 μg/ml celecoxib for 30 min and then incubated with 100 ng/ml rmVEGF for 4 h. In other experiments B16M cells were given 10 or 100 ng/ml of PGE2 for 2 h. Then, cell adhesion assay to rhVCAM-1-coated plate was performed. Data are expressed as mean percent of added labeled-cells binding to quadruplicate wells ± SD. Statistical significance by ANOVA and Bonferroni's post- hoc test: * P < 0.01 as compared with basal medium-treated B16M cells; ** P < .001 as compared with VEGF-treated B16M cells. C) Effect of celecoxib and anti-VEGF on the proadhesive response of A375M cells to bone marrow-conditioned media on immobilized VCAM-1 . Human A375M cells received 1 μg/ml celecoxib for 30 min and then incubated in the presence of basal medium, hBMSC-CM, LPS-treated hBMSC-CM or rhVEGF (10 ng/ml) for 4 h. Then, cell adhesion assay to a rhVCAM-1-coated plate was performed. Data are expressed as mean percent of added labeled-cells binding to quadruplicate wells ± SD. Statistical significance by ANOVA and Bonferroni's post- hoc test: * P < 0.01 as compared with basal medium-treated A375M cells; ** P < 0.01 as compared with BMSC-CM-; + P < 0.01 as compared with LPS-treated BMSC-CM-treated A375M cells; # P < 0.01 as compared with rhVEGF-treated A375M cells.

    Article Snippet: Murine B16 melanoma (B16M) cells from the B16F10 subline, and human A375 melanoma (A375M) cell lines were obtained from ATCC (Manassas, VA) and utilized in the present study.

    Techniques: Western Blot, Expressing, Cell Culture, Recombinant, Incubation, Cell Adhesion Assay, Labeling, Binding Assay

    Effect of celecoxib and anti-VEGF on the proliferation rate of BMSC-CM-treated B16M (A) and A375M (B) cells . Murine B16M (A) or A375M (B) cells were plated onto 96-well plates at a density of 2,500 cells per well. Some cells received BMSC-CM, LPS-treated BMSC-CM or 10 ng/ml rmVEGF in the presence or absence of 1 μg/ml anti-VEGF monoclonal antibody or 1 μg/ml celecoxib. Control melanoma cells were cultured in the presence of basal medium (DMEM). After 48 h incubation, the number of cells was determined by microscopic counting in 5 different fields per well and by sulforhodamine-101-based fluorimetry as described in Methods. Every assay was done in quadruplicate and repeated three times. Data represent average values ± SD. Differences were statistically significant cells ( P < 0.01) with respect to (*) basal medium- or (**) BMSC-CM- or (#) LPS-treated BMSC-CM or (##) rmVEGF-treated melanoma cells according by ANOVA and Bonferroni's post- hoc test.

    Journal: Journal of Translational Medicine

    Article Title: Vascular endothelial growth factor regulates melanoma cell adhesion and growth in the bone marrow microenvironment via tumor cyclooxygenase-2

    doi: 10.1186/1479-5876-9-142

    Figure Lengend Snippet: Effect of celecoxib and anti-VEGF on the proliferation rate of BMSC-CM-treated B16M (A) and A375M (B) cells . Murine B16M (A) or A375M (B) cells were plated onto 96-well plates at a density of 2,500 cells per well. Some cells received BMSC-CM, LPS-treated BMSC-CM or 10 ng/ml rmVEGF in the presence or absence of 1 μg/ml anti-VEGF monoclonal antibody or 1 μg/ml celecoxib. Control melanoma cells were cultured in the presence of basal medium (DMEM). After 48 h incubation, the number of cells was determined by microscopic counting in 5 different fields per well and by sulforhodamine-101-based fluorimetry as described in Methods. Every assay was done in quadruplicate and repeated three times. Data represent average values ± SD. Differences were statistically significant cells ( P < 0.01) with respect to (*) basal medium- or (**) BMSC-CM- or (#) LPS-treated BMSC-CM or (##) rmVEGF-treated melanoma cells according by ANOVA and Bonferroni's post- hoc test.

    Article Snippet: Murine B16 melanoma (B16M) cells from the B16F10 subline, and human A375 melanoma (A375M) cell lines were obtained from ATCC (Manassas, VA) and utilized in the present study.

    Techniques: Control, Cell Culture, Incubation