normal human epidermal melanocytes line  (PromoCell)

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: Expressing, Incubation, Derivative Assay, Western Blot, SDS Page, Membrane, Control, 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, Amplification, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Unbiased functional genetic screens reveal essential RNA modifications in human cancer and drug resistance

doi: 10.1101/2024.07.13.603368

Figure Lengend Snippet: (A) Principal component analysis (PCA) using log 2 fold change (FC) of 159 gRNAs in 18 cell lines originating from blood, plasma cells, epithelial cells or skin melanocytes at day 22 of the experiment. ( B ) Number of significantly (FDR<0.5; purple) depleted gRNAs in at least one cell line. ( C, D ) Total number (C) and list of RNA modifying proteins (RMPs) (D) corresponding to significantly (FDR<0.05) depleted gRNAs in the indicated cell lines. ( E ) Proportion of all (left) (n=150), essential (middle) (n=50) or non-essential (right) (n=100) RMPs showing weak or strong tissue-specific expression (Human Protein Atlas). ( F,G ) PCA using RMP expression (n=143) (F) or whole transcriptomes (G) (normalized log 2 FPKM) in 18 cell lines (n=3 sequencing reactions per cell line). ( H ) Heatmap of RMP-expression shown in (F) average over tissue or showing primary normal cells (NHEK, NHEM). ( I ) Averaged correlation distance of RMP-expression shown in (H) in cancer and normal cells. ( J ) Protein level of RMPs in the indicated cell lines grouped by gRNAs significantly (FDR<0.05) depleted in the indicated cell line (purple) or in any tested cell line (orange) or never significantly (FDR>0.05) depleted in any cell line (grey). Shown are 114 out of 159 proteins present in all cell lines. Box plots show minimum, first quartile, median, third quartile, and maximum (I).

Article Snippet: Primary melanocytes (NHEM) were kept in melanocyte growth medium M3 (PromoCell) with 1% PS.

Techniques: Expressing, Sequencing

Journal: bioRxiv

Article Title: Unbiased functional genetic screens reveal essential RNA modifications in human cancer and drug resistance

doi: 10.1101/2024.07.13.603368

Figure Lengend Snippet: (A) Principal component analysis (PCA) using log 2 fold change (FC) of 159 gRNAs in 18 cell lines originating from blood, plasma cells, epithelial cells or skin melanocytes at day 22 of the experiment. ( B ) Number of significantly (FDR<0.5; purple) depleted gRNAs in at least one cell line. ( C, D ) Total number (C) and list of RNA modifying proteins (RMPs) (D) corresponding to significantly (FDR<0.05) depleted gRNAs in the indicated cell lines. ( E ) Proportion of all (left) (n=150), essential (middle) (n=50) or non-essential (right) (n=100) RMPs showing weak or strong tissue-specific expression (Human Protein Atlas). ( F,G ) PCA using RMP expression (n=143) (F) or whole transcriptomes (G) (normalized log 2 FPKM) in 18 cell lines (n=3 sequencing reactions per cell line). ( H ) Heatmap of RMP-expression shown in (F) average over tissue or showing primary normal cells (NHEK, NHEM). ( I ) Averaged correlation distance of RMP-expression shown in (H) in cancer and normal cells. ( J ) Protein level of RMPs in the indicated cell lines grouped by gRNAs significantly (FDR<0.05) depleted in the indicated cell line (purple) or in any tested cell line (orange) or never significantly (FDR>0.05) depleted in any cell line (grey). Shown are 114 out of 159 proteins present in all cell lines. Box plots show minimum, first quartile, median, third quartile, and maximum (I).

Article Snippet: Primary cell lines normal human epidermal keratinocytes (NHEK) and normal human epidermal melanocytes (NHEM) were purchased from PromoCell.

Techniques: Expressing, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Exploring the Influence of Cold Plasma on Epidermal Melanogenesis In Situ and In Vitro

doi: 10.3390/ijms25105186

Figure Lengend Snippet: Representative microscopic images of cultured melanocytes and cell viability after treatment with CAP, IBMX and UV-B. ( a ) Light NHEMs (left panel) and dark NHEMs (right panel) maintain their normal cell morphology. Magnification 10×, scale bar 100 µm, phase contrast microscopy. ( b ) Cell viability was assessed by resazurin assay and normalized to the untreated control ( n = 6). Treatment with CAP, IBMX and UV-B did not affect cell viability.

Article Snippet: Normal human epidermal melanocytes (NHEMs) derived from juvenile foreskin of donors with light and dark skin pigmentation, respectively, were purchased from PromoCell (Heidelberg, Germany).

Techniques: Cell Culture, Microscopy, Resazurin Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Exploring the Influence of Cold Plasma on Epidermal Melanogenesis In Situ and In Vitro

doi: 10.3390/ijms25105186

Figure Lengend Snippet: Detection of melanin content by autofluorescence after excitation at 808 nm (NIR) in light and dark melanocytes. NHEMs were analyzed at two emission wavelengths, 840 nm and 885 nm, after treatment with either CAP, IBMX, UV-B or no treatment (control). Increased melanin content results in an increase in MFI. Treatment with IBMX and UV-B revealed higher MFI values than untreated NHEMs. Bars represent mean ± SD. ns: not significant, *: p ≤ 0.05, **: p ≤ 0.01.

Article Snippet: Normal human epidermal melanocytes (NHEMs) derived from juvenile foreskin of donors with light and dark skin pigmentation, respectively, were purchased from PromoCell (Heidelberg, Germany).

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Exploring the Influence of Cold Plasma on Epidermal Melanogenesis In Situ and In Vitro

doi: 10.3390/ijms25105186

Figure Lengend Snippet: HPLC-MS analysis of eumelanin and pheomelanin in cultured melanocytes. Relative amounts of oxidation products of eumelanin (PDCA and PTCA) and pheomelanin (TDCA and TTCA) in ( a ) lightly pigmented normal human epidermal melanocytes (NHEMs) and ( b ) darkly pigmented NHEMs after treatment with CAP, IBMX and UV-B in comparison to untreated NHEMs (control). The AUC was detected by HPLC-MS using 10 4 cells in six independent runs and normalized to all cell counts. Statistical analysis was performed using an unpaired t -test with p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***). ns: not significant.

Article Snippet: Normal human epidermal melanocytes (NHEMs) derived from juvenile foreskin of donors with light and dark skin pigmentation, respectively, were purchased from PromoCell (Heidelberg, Germany).

Techniques: Cell Culture, Comparison, Control