Journal: The American Journal of Pathology

Article Title: Slug Expression during Melanoma Progression

doi: 10.1016/j.ajpath.2012.02.014

Figure Lengend Snippet: Endogenous Slug expression is higher at both mRNA and protein levels in NHM than in primary (WM115) or metastatic (WC62) melanoma cell lines. A: RT-qPCR revealed significantly higher expression of Slug and E-cadherin mRNAs in NHM than in primary or metastatic melanoma cells, whereas Snail and N-cadherin expression were significantly lower. Error bars indicate SDs. *P < 0.01 compared to both primary and metastatic melanoma cells. B: Western blots confirmed that differences in mRNA levels were reflected in differences in protein expression in NHM versus primary and metastatic melanoma cell lines. Both mRNA and protein were extracted from confluent cell cultures to eliminate density-dependent variations in expression of Slug and its putative target genes. MITF isoforms include both melanocyte-specific (M) and ubiquitous (A) isoforms.

Article Snippet: Cell Culture, Adenoviral Transduction, and Immunofluorescence Normal human melanocytes (NHM) of neonatal origin were obtained from the ATCC (Manassas, VA) and were maintained in Dermal Basal Medium supplemented with a melanocyte growth kit (ATCC).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: The American Journal of Pathology

Article Title: Slug Expression during Melanoma Progression

doi: 10.1016/j.ajpath.2012.02.014

Figure Lengend Snippet: NHM and melanoma cells transduced with Slug-expressing adenovirus (Ad-Slug) express less E-cadherin and more N-cadherin mRNA and protein than cells transduced with a control adenovirus (Ad-null). A: RT-qPCR revealed significantly lower E-cadherin expression in NHM transduced with Ad-Slug compared to Ad-Null-transduced cells. Neither Ad-Slug nor Ad-Null-transfected WC62 cells expressed detectable E-cadherin mRNA. N-cadherin levels were significantly increased in Ad-Slug-transduced NHM and melanoma cells compared to Ad-Null-transduced cells, although basal levels of expression were low and increases were modest. Note the difference in scale for the two graphs. Error bars indicate SEMs. *P < 0.03. B: Western blot results confirmed that Ad-Slug enhanced Slug protein expression in NHM and melanoma cells and that the differences seen in mRNA levels for E-cadherin in NHM and N-cadherin in melanoma cells were reflected in differences in protein expression. In addition, Ad-Slug transduction also enhanced expression of melanocyte-specific MITF (M) in NHM but not in melanoma cells. Melanoma cells expressed higher levels of the MITF isoform constitutively expressed in many tissues (A) and expression of this isoform was not altered by Ad-Slug transduction. Ad-Slug transduction did not alter levels of Snail expression in either cell type.

Article Snippet: Cell Culture, Adenoviral Transduction, and Immunofluorescence Normal human melanocytes (NHM) of neonatal origin were obtained from the ATCC (Manassas, VA) and were maintained in Dermal Basal Medium supplemented with a melanocyte growth kit (ATCC).

Techniques: Transduction, Expressing, Control, Quantitative RT-PCR, Transfection, Western Blot

Journal: bioRxiv

Article Title: Overcoming efflux of fluorescent probes for actin imaging in living cells

doi: 10.1101/2020.02.17.951525

Figure Lengend Snippet: Confocal and nanoscopy images of living cells and tissues stained with the new actin probes. (a) Comparison of confocal and STED images of human fibroblasts stained with 6-610CP-JAS. (b) Human melanocytes co-stained with 6-580CP-JAS and 5-SiR-Hoechst. (c) live-cell imaging of body wall muscle of dissected D. melanogaster larva costained with 6-580CP-JAS and 6-SiR-CTX. (d) Max intensity projection of a frog erythrocyte stained with 6-610CP-JAS. (e) Rat primary neuron culture co-stained with 6-610CP-JAS and neurofascin (AlexaFluor 488). Scale bars 10 μm (a-d), 5 μm (e).

Article Snippet: Normal neonatal human melanocytes (MatTek Corporation, Cat. NHM-CRY-NEO) were cultured in Normal human melanocyte cells growth medium (MatTek Corporation, Cat. NHM-GM) in a humidified 5% CO2 incubator at 37 °C.

Techniques: Staining, Comparison, Live Cell Imaging

Journal: Journal of Immunology Research

Article Title: Transcriptome Analysis of Large to Giant Congenital Melanocytic Nevus Reveals Cell Cycle Arrest and Immune Evasion: Identifying Potential Targets for Treatment

doi: 10.1155/2021/8512200

Figure Lengend Snippet: Cell cycle arrest and significantly upregulated P16 expression in lgCMN. (a) Relative gene expression of cell cycle inhibitors in lgCMN and normal skin tissues. Red bars represent upregulation, and blue bars represent downregulation. (b) Morphology of A375, NHM, and lgCMN cells with different shapes and number of dendrites. (c) Cell cycle assays. P2 cells of each cell line were cultured for the experiment. (d) Immunohistochemical staining for SOX10 (upper panels), P16 (middle panels), and Ki67 (lower panels) of normal skin ( n = 5; patient 2 + patient 4 + patient 5 + patient 9 + patient 10), lgCMN ( n = 5; patient 2 + patient 4 + patient 5 + patient 9 + patient 10), and lgCMN-developed melanoma ( n = 1; a supplementary lgCMN patient with malignant transformation at 4 years of age). SOX10, P16, and Ki67 were stained using the AEC chromogen. (e) Histograms show the number of P16+ or Ki67+ cells per SOX10+ cells in the epidermis and dermis. (f) Immunofluorescent staining of SOX10, P16, and Ki67 in P2 NHM ( n = 3; patient 2 + patient 9 + patient 10) and P2 lgCMN cells ( n = 3; 3 foreskin samples from different children). P16+ and Ki67+ cells were stained using FITC-conjugated antibodies, and SOX10+ cells were stained using Cy3-conjugated antibodies. (g) Histograms show the number of P16+ or Ki67+ cells per DAPI and SOX10+ cells. ∗∗∗∗ P < 0.0001 represents differences between NHM and lgCMN in the t -test. Error bars represent the mean ± SD. lgCMN: large to giant congenital melanocytic nevus; NHM: normal human melanocytes; P2: passage 2; ND: not detected; SD: standard deviation. Scale bar = 50 μ m.

Article Snippet: Both NHM and lgCMN cells were cultured in melanocyte medium (ScienCell, Carlsbad, CA, USA) according to the manufacturer's instructions.

Techniques: Expressing, Gene Expression, Cell Culture, Immunohistochemical staining, Staining, Transformation Assay, Standard Deviation