normal human epidermal primary melanocytes nhems  (ATCC)

Journal: Scientific Reports

Article Title: Delphinidin upregulates microRNA-let-7b expression through family with sequence similarity 222 member B

doi: 10.1038/s41598-025-15588-3

Figure Lengend Snippet: Identification of molecules that regulate the growth inhibitory effects of melanoma cells by delphinidin. ( a ) Schematic of genetic screening for molecules that mediate the inhibition of melanoma cell proliferation by delphinidin. ( b ) Fam222B protein levels in melanoma cell lines and NHEMs were assessed using Western blot analysis ( n = 1). ( c and d ) Fam222B mRNA expression and protein levels were evaluated in B16 cells treated with delphinidin for 48 h ( n = 3). Data are presented as mean ± SEM. Statistical analysis was conducted using one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: Mouse melanoma B16 cells, human melanoma cell lines (A375, Hs294t, and MeWo), and normal human epidermal melanocytes (NHEMs) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Inhibition, Western Blot, Expressing, Comparison

Journal: Cells

Article Title: The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin

doi: 10.3390/cells13161336

Figure Lengend Snippet: Analysis of purity and size distribution of melanocyte- and melanoma-derived ectosomes. ( A ) TEM imaging. ( B ) Nanoparticle Tracking Analysis (NTA). The means of five independent measurements (black lines) are presented on histograms. The colored area depicts ± standard deviation. Ectosomes were derived from NHEM—normal human epidermal melanocytes; WM115 (primary) and WM266-4 (metastatic)—melanoma cell lines originating from the same individual, radial/vertical growth phase and lymph node metastasis, respectively; primary WM793 cell line—representing the vertical growth phase; WM1205Lu cells—a metastatic variant of WM793 cells obtained from lung metastasis.

Article Snippet: As a reference, primary normal human epidermal melanocytes (NHEM cell line) (PromoCell GmbH, Heidelberg, Germany, cat. C-12400) isolated from the epidermis of juvenile foreskin were used.

Techniques: Derivative Assay, Imaging, Standard Deviation, Variant Assay

Journal: Cells

Article Title: The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin

doi: 10.3390/cells13161336

Figure Lengend Snippet: Analysis of the incorporation of melanoma- and melanocyte-derived ectosomes by recipient endothelial cells. Ectosomes were labeled with the fluorescent dye PKH67. Following 18 h of coincubation with ectosomes, endothelial cell fluorescence was analyzed using a flow cytometer, multi-well plate reader and confocal microscope. ( A ) Representative histograms from flow cytometry analysis. ( B ) Endothelial cell lysate fluorescence was measured in a multi-well plate reader (490/502 nm). ( C ) Confocal microscope imaging of the incorporation of melanoma- and melanocyte-derived ectosomes by recipient endothelial cells. Isolated ectosomes were labeled with the green fluorescent dye PKH67. Following an 18 h incubation period with ectosomes, recipient endothelial HUVEC and HDMEC cells were subsequently stained with Alexa Fluor™ 633 Phalloidin to visualize the cytoskeleton (red) and DAPI to visualize the nucleus (blue). All experiments were performed in triplicate. “*” indicates statistically significant differences compared to the control (Tukey’s post-hoc test, p < 0.05).

Article Snippet: As a reference, primary normal human epidermal melanocytes (NHEM cell line) (PromoCell GmbH, Heidelberg, Germany, cat. C-12400) isolated from the epidermis of juvenile foreskin were used.

Techniques: Derivative Assay, Labeling, Fluorescence, Flow Cytometry, Microscopy, Imaging, Isolation, Incubation, Staining, Control

Journal: Cells

Article Title: The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin

doi: 10.3390/cells13161336

Figure Lengend Snippet: Western blot analysis of total αvβ3 and αvβ5 integrin expression in melanocytes and melanoma cell lines, and the corresponding derivative ectosome samples. Thirty micrograms of proteins from whole-cell protein extracts (lines C) and ectosome samples (lines E) were separated by 10% SDS-PAGE and transferred to the PVDF membrane. Membranes were then probed with rabbit monoclonal primary antibodies anti-αvβ3 (1:1000 dilution) and anti-αvβ5 (1:2000), and goat anti-rabbit HRP-conjugated secondary antibody (1:5000). Mouse monoclonal anti-β-actin antibody (1:10,000) was used as loading control. ( A ) Representative Western blots. ( B , C ) Densitometric analysis of αvβ3 and αvβ5 integrin expression relative to β-actin. All experiments were performed in triplicate. “*” indicates statistically significant differences (Tukey’s post-hoc test, p < 0.05).

Article Snippet: As a reference, primary normal human epidermal melanocytes (NHEM cell line) (PromoCell GmbH, Heidelberg, Germany, cat. C-12400) isolated from the epidermis of juvenile foreskin were used.

Techniques: Western Blot, Expressing, SDS Page, Membrane, Control

Journal: Cells

Article Title: The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin

doi: 10.3390/cells13161336

Figure Lengend Snippet: Analysis of αvβ3 and αvβ5 integrin total protein and gene expression in endothelial cells after 18-h of incubation with melanocyte- and melanoma-derived ectosomes. ( A ) Representative Western blots. Thirty micrograms of proteins from whole-cell protein extracts were separated by 10% SDS-PAGE and transferred to the PVDF membrane. Membranes were then probed with rabbit monoclonal primary antibodies anti-αvβ3 (and anti-αvβ5), and goat anti-rabbit HRP-conjugated secondary antibody. Mouse monoclonal anti-β-actin antibody was used as a loading control. ( B , C ) Densitometric analysis of αvβ3 and αvβ5 expression relative to β-actin. ( D ) RT-qPCR analysis of gene expression for αv, β3 and β5 integrin subunits in endothelial HUVEC and HDMEC cells after 18 h incubation with melanocyte- and melanoma-derived ectosomes. Housekeeping ( YWHZ ) and target ( ITGAV , ITGB3 , ITGB5 ) gene-specific mRNAs were amplified from 250 ng of cDNA with the use of TaqMan™ Gene Expression Assays. Analysis of relative gene expression was performed using the 2 −ΔΔCt method. All experiments were performed in triplicate. “*” denotes statistically significant differences (Tukey’s post-hoc test, p < 0.05).

Article Snippet: As a reference, primary normal human epidermal melanocytes (NHEM cell line) (PromoCell GmbH, Heidelberg, Germany, cat. C-12400) isolated from the epidermis of juvenile foreskin were used.

Techniques: Gene Expression, Incubation, Derivative Assay, Western Blot, SDS Page, Membrane, Control, Expressing, Quantitative RT-PCR, Amplification

Journal: Cells

Article Title: The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin

doi: 10.3390/cells13161336

Figure Lengend Snippet: Flow cytometry analysis of αvβ3 and αvβ5 integrin surface expression in HUVEC cells following 18 h of incubation with melanocyte- and melanoma-derived ectosomes. After the incubation, 5 × 10 4 cells were collected, indirectly labeled with rabbit monoclonal primary anti-αvβ3 integrin antibody and secondary FITC-conjugated goat anti-rabbit IgG, and subsequently analyzed by flow cytometry. ( A ) Representative histograms are depicted, where the gray-shaded histograms represent background signals acquired from secondary antibody staining. Based on these signals, the histogram markers were set, delineating the histogram sections corresponding to αvβ3 integrin- or αvβ5 integrin-positive HUVEC cells. ( B ) Surface expression of αvβ3 integrin on HUVEC cells presented as the percentage of positive cells and ( C ) relative fluorescence intensity of specific staining. All experiments were performed in triplicate. “*” indicates statistically significant differences compared to the control (Tukey’s post-hoc test, p < 0.05).

Article Snippet: As a reference, primary normal human epidermal melanocytes (NHEM cell line) (PromoCell GmbH, Heidelberg, Germany, cat. C-12400) isolated from the epidermis of juvenile foreskin were used.

Techniques: Flow Cytometry, Expressing, Incubation, Derivative Assay, Labeling, Staining, Fluorescence, Control

Journal: Cells

Article Title: The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin

doi: 10.3390/cells13161336

Figure Lengend Snippet: Alamar Blue cell viability assay performed on endothelial cells after 18 h of incubation with ectosomes derived from melanocytes and melanoma cells. The isolated ectosomes were pre-incubated with anti-αvβ3 and anti-αvβ5 integrin antibodies or the RGD mimetics (cilengitide and echistatin) and then added to 5 × 10 4 of HUVEC ( A ) or HDMEC ( B ) cells. After 18 h of incubation with ectosomes, Alamar Blue reagent was added to each well and fluorescence intensity was measured at 560/595 nm in a multi-well plate reader. Results were normalized against the untreated control. All experiments were performed in triplicate. “*” indicates statistically significant differences compared to the control (Tukey’s post-hoc test, p < 0.05).

Article Snippet: As a reference, primary normal human epidermal melanocytes (NHEM cell line) (PromoCell GmbH, Heidelberg, Germany, cat. C-12400) isolated from the epidermis of juvenile foreskin were used.

Techniques: Viability Assay, Incubation, Derivative Assay, Isolation, Fluorescence, Control

Journal: Cells

Article Title: The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin

doi: 10.3390/cells13161336

Figure Lengend Snippet: Wound healing assay performed on HUVEC ( A ) and HDMEC ( B ) cells after 18 h of incubation with ectosomes derived from melanocytes and melanoma cells. The isolated ectosomes were pre-incubated with anti-αvβ3 and anti-αvβ5 integrin antibodies or RGD mimetics (cilengitide and echistatin). Wounds (1 mm wide) were created on HUVEC ( A ) and HDMEC ( B ) monolayers and allowed to heal for 18 h without or in the presence of ectosomes. Each wound was photographed immediately after scraping (0 h) and after 18 h. Red dashed lines mark wound borders. Scale bar: 0.5 mm.

Article Snippet: As a reference, primary normal human epidermal melanocytes (NHEM cell line) (PromoCell GmbH, Heidelberg, Germany, cat. C-12400) isolated from the epidermis of juvenile foreskin were used.

Techniques: Wound Healing Assay, Incubation, Derivative Assay, Isolation

Journal: Cells

Article Title: The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin

doi: 10.3390/cells13161336

Figure Lengend Snippet: Quantitative analysis of results from wound healing assay carried out on HUVEC ( A ) and HDMEC ( B ) cells after 18 h incubation with melanocyte- and melanoma-derived ectosomes. Isolated ectosomes were pre-incubated with anti-αvβ3 and anti-αvβ5 integrin antibodies or RGD mimetics–cilengitide and echistatin. Wounds (1 mm in width) were created on HUVEC monolayers and allowed to heal for 18 h without or in the presence of ectosomes. Each wound was photographed in 10 separate fields immediately after scraping (0 h) and after 18 h. The average wound closure rate was evaluated by multiple measurements of the wound width on each image. Results were standardized in relation to the untreated control (taken as 1). All experiments were performed in triplicate. “*” indicates statistically significant differences compared to the control (Tukey’s post-hoc test, p < 0.05).

Article Snippet: As a reference, primary normal human epidermal melanocytes (NHEM cell line) (PromoCell GmbH, Heidelberg, Germany, cat. C-12400) isolated from the epidermis of juvenile foreskin were used.

Techniques: Wound Healing Assay, Incubation, Derivative Assay, Isolation, Control

Journal: Cells

Article Title: The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin

doi: 10.3390/cells13161336

Figure Lengend Snippet: Tube formation assay carried out on HUVEC ( A ) and HDMEC ( B ) cells after 18 h of incubation with melanocyte- and melanoma-derived ectosomes. Isolated ectosomes were pre-incubated with anti-αvβ3 and anti-αvβ5 integrin antibodies or RGD mimetics–cilengitide and echistatin and then added to HUVEC and HDMEC endothelial cells seeded previously on a Geltrex matrix-coated plates. After incubation, HUVEC and HDMEC cell cultures were photographed at 10 separate fields per well. Scale bar: 0.5 mm.

Article Snippet: As a reference, primary normal human epidermal melanocytes (NHEM cell line) (PromoCell GmbH, Heidelberg, Germany, cat. C-12400) isolated from the epidermis of juvenile foreskin were used.

Techniques: Tube Formation Assay, Incubation, Derivative Assay, Isolation

Journal: Cells

Article Title: The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin

doi: 10.3390/cells13161336

Figure Lengend Snippet: Qualitative analysis of results from tube formation assay carried out on HUVEC and HDMEC cells after 18 h of incubation with melanocyte- and melanoma-derived ectosomes. Isolated ectosomes were pre-incubated with anti-αvβ3 and anti-αvβ5 integrin antibodies or RGD mimetics–cilengitide and echistatin and then added to HUVEC and HDMEC endothelial cells seeded previously on a Geltrex matrix-coated plates. After incubation, HUVEC and HDMEC cell cultures were photographed at 10 separate fields per well. Obtained images were binarized and analyzed in ImageJ with the Angiogenesis Analyzer plug-in. Quantitative image analysis included ( A ) total tube length, ( B ) the number of closed tubes, and ( C ) the number of branches. Results were standardized in relation to the untreated control (taken as 1). All experiments were performed in triplicate. “*” indicates statistically significant differences compared to the control (Tukey’s post-hoc test, p < 0.05).

Article Snippet: As a reference, primary normal human epidermal melanocytes (NHEM cell line) (PromoCell GmbH, Heidelberg, Germany, cat. C-12400) isolated from the epidermis of juvenile foreskin were used.

Techniques: Tube Formation Assay, Incubation, Derivative Assay, Isolation, Control

Journal: Cells

Article Title: The Proangiogenic Effects of Melanoma-Derived Ectosomes Are Mediated by αvβ5 Integrin Rather than αvβ3 Integrin

doi: 10.3390/cells13161336

Figure Lengend Snippet: Analysis of changes in gene and protein expression/secretion of TNF-α and VEGF in endothelial cells after 18 h incubation with integrin-bearing ectosomes derived from melanocytes and melanoma cells. ( A ) Representative Western blots from analysis of total VEGF-A and TNF-α protein expression in HUVEC and HDMEC cells after incubation with ectosomes. Thirty micrograms of proteins from whole-cell protein extracts were separated by 10% SDS-PAGE and transferred to the PVDF membrane. Membranes were then probed with rabbit monoclonal primary antibodies, anti-VEGF-A and anti-TNF-α, and goat anti-mouse HRP-conjugated secondary antibodies. Mouse monoclonal anti-β-actin antibody was used as a loading control. ( B ) Densitometric analysis of total VEGF-A and TNF-α protein expression relative to β-actin. ( C ) RT-qPCR analysis of gene expression for VEGFA and TNFA . Housekeeping ( YWHZ ) and target ( VEGFA , TNFA ) gene-specific mRNAs were amplified from 250 ng of cDNA with the use of TaqMan™ Gene Expression Assays. Analysis of relative gene expression was performed using the 2 −ΔΔCt method. ( D ) The results of ELISA tests for both proteins were performed on the conditioned medium. All experiments were performed in triplicate. “*” indicates statistically significant differences compared to the control (Tukey’s post-hoc test, p < 0.05).

Article Snippet: As a reference, primary normal human epidermal melanocytes (NHEM cell line) (PromoCell GmbH, Heidelberg, Germany, cat. C-12400) isolated from the epidermis of juvenile foreskin were used.

Techniques: Expressing, Incubation, Derivative Assay, Western Blot, SDS Page, Membrane, Control, Quantitative RT-PCR, Gene Expression, Amplification, Enzyme-linked Immunosorbent Assay