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