Journal: PLoS Genetics

Article Title: TFAP2 paralogs regulate melanocyte differentiation in parallel with MITF

doi: 10.1371/journal.pgen.1006636

Figure Lengend Snippet: (A-C) Density-based clustering of H3K27ac signal at (A) TFAP2A peaks, (B) MITF peaks, and (C) TFAP2A peaks that overlap MITF peaks in human melanocytes (H3K27ac data from GSM1127072 ), MITF peaks from ). (D) Overlap between genes associated with active TFAP2A peaks and genes associated with active MITF peaks in human melanocytes. (E) Typical enhancers (gray) and super-enhancers (colored) in human melanocytes that overlap neither TFAP2A nor MITF peaks, TFAP2A peaks only, MITF peaks only, or both TFAP2A and MITF peaks. Labels identify melanocyte genes of interest. (F) Diagram of the TRPM1 -700 bp promoter element depicting the positions of four TFAP2A binding sites (A1–A4) and the previously reported E-box 1 MITF binding site (E1). (G) Luciferase assays in M21 melanoma cells. Deletion of all four TFAP2A binding sites (ΔAP2) significantly reduced reporter activity compared to the intact TRPM1 -700bp element (Student’s t-test, **p = 0.01).

Article Snippet: Human foreskin melanocyte H3K27ac ChIP-seq alignment results were obtained from the Roadmap Epigenomics Project (GEO accession number, GSM1127072) [ ].

Techniques: Binding Assay, Luciferase, Activity Assay

Journal: PLoS Genetics

Article Title: TFAP2 paralogs regulate melanocyte differentiation in parallel with MITF

doi: 10.1371/journal.pgen.1006636

Figure Lengend Snippet: Mouse coat color genes associated with peaks of TFAP2A and MITF that overlap active enhancer marks (H3K27ac).

Article Snippet: Human foreskin melanocyte H3K27ac ChIP-seq alignment results were obtained from the Roadmap Epigenomics Project (GEO accession number, GSM1127072) [ ].

Techniques:

Journal: Current biology : CB

Article Title: Unmasking Transcriptional Heterogeneity in Senescent Cells

doi: 10.1016/j.cub.2017.07.033

Figure Lengend Snippet: (A) Experimental design. RNA-seq datasets obtained from the indicated studies of melanocytes, keratinocytes, and astrocytes were compared to the senescence signature of fibroblasts. The intersection of genes differentially expressed (p ≤ 0.01) in all the datasets are shown in the flower plot. D.E., differential expression.

Article Snippet: Cell strains and culture Human foreskin fibroblasts HCA2 (male) were obtained from the laboratory of O. Pereira-Smith (University of Texas Health Science Center, San Antonio); human foreskin fibroblasts BJ were purchased from ATCC (Cat: CRL-2522); Human neonatal foreskin epidermal melanocytes and keratinocytes (male) were purchased from ATCC (Cat: PCS-200-012 and PCS-200-010, respectively).

Techniques: RNA Sequencing, Quantitative Proteomics

Journal: Current biology : CB

Article Title: Unmasking Transcriptional Heterogeneity in Senescent Cells

doi: 10.1016/j.cub.2017.07.033

Figure Lengend Snippet: (A) Experimental design. Fibroblasts (HCA-2; yellow), melanocytes (red), and keratinocytes (magenta) were exposed to ionizing radiation (IR), and RNA was harvested 4, 10, or 20 days later. Transcriptomes of the different cell types and intervals after senescence induction were obtained by RNA-seq. A time-point signature with genes differentially expressed (p ≤ 0.01) in all three cell types and a shared IR-induced senescence (IRIS) signature with genes shared by all cell types and time points (p ≤ 0.01) were generated.

Article Snippet: Cell strains and culture Human foreskin fibroblasts HCA2 (male) were obtained from the laboratory of O. Pereira-Smith (University of Texas Health Science Center, San Antonio); human foreskin fibroblasts BJ were purchased from ATCC (Cat: CRL-2522); Human neonatal foreskin epidermal melanocytes and keratinocytes (male) were purchased from ATCC (Cat: PCS-200-012 and PCS-200-010, respectively).

Techniques: RNA Sequencing, Generated

Journal: Current biology : CB

Article Title: Unmasking Transcriptional Heterogeneity in Senescent Cells

doi: 10.1016/j.cub.2017.07.033

Figure Lengend Snippet: Each panel shows one of the 55 genes in the core signature at the indicated points before and after irradiation. All genes show a dynamic temporal behavior at the time points tested: day 0 (proliferation), day 4, day 10, and day 20 after irradiation. Notably, all genes show a similar trend in the three cell types tested: fibroblasts (yellow), keratinocytes (red), and melanocytes (magenta). Genes in red correspond to those that reached significance (p ≤ 0.01) at all time points tested. N = 6.

Article Snippet: Cell strains and culture Human foreskin fibroblasts HCA2 (male) were obtained from the laboratory of O. Pereira-Smith (University of Texas Health Science Center, San Antonio); human foreskin fibroblasts BJ were purchased from ATCC (Cat: CRL-2522); Human neonatal foreskin epidermal melanocytes and keratinocytes (male) were purchased from ATCC (Cat: PCS-200-012 and PCS-200-010, respectively).

Techniques: Irradiation

Journal: Current biology : CB

Article Title: Unmasking Transcriptional Heterogeneity in Senescent Cells

doi: 10.1016/j.cub.2017.07.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell strains and culture Human foreskin fibroblasts HCA2 (male) were obtained from the laboratory of O. Pereira-Smith (University of Texas Health Science Center, San Antonio); human foreskin fibroblasts BJ were purchased from ATCC (Cat: CRL-2522); Human neonatal foreskin epidermal melanocytes and keratinocytes (male) were purchased from ATCC (Cat: PCS-200-012 and PCS-200-010, respectively).

Techniques: Recombinant, Staining, Imaging, Reverse Transcription, Sample Prep, Software

Journal: Molecular Cancer

Article Title: Potent anti-tumor effects of a dual specific oncolytic adenovirus expressing apoptin in vitro and in vivo

doi: 10.1186/1476-4598-9-10

Figure Lengend Snippet: Schematic of the recombinant adenoviruses and the adenovirus-mediated transgene expression . (A) Schematic diagram depicting the organization elements in the recombinant adenoviruses. Polyadenylation signal sequence is designated as pA. The indicated cells were infected with Ad-mock (control) or indicated recombinant viruses at a MOI of 100 for 48 h. Western blot analysis was performed to detect (B) Apoptin or E1a protein from A375 cells, (C) Apoptin or E1a expression in B16 cells, or (D) Apoptin or E1a expression in HEM cells. Infection of normal HEM human epidermal melanocytes (D) with Ad-CMV-E1a or Ad-CMV-E1a-Apoptin, but not Ad-hTERT-E1a or Ad-hTERT-E1a-Apoptin, resulted in production of E1a proteins, whereas in A375 (B) and B16 (C) melanoma cells, infection with all these replication-competent recombinant adenoviruses generated E1a proteins. In HEM cells (D), infection with Ad-CMV-E1a-Apoptin and Ad-CMV-Apoptin resulted in Apoptin production, whereas infection with Ad-hTERT-Apoptin or Ad-hTERT-E1a-Apoptin resulted in barely detectable of Apoptin production. In A375 (B) and B16 (C) cells, infection with Ad-CMV-Apoptin, Ad-hTERT-Apoptin, Ad-CMV-E1a-Apoptin, or Ad-hTERT-E1a-Apoptin generated significant Apoptin production. 1. Ad-mock; 2. Ad-CMV-Apoptin; 3. Ad-hTERT-Apoptin; 4. Ad-CMV-E1a; 5. Ad-hTERT-E1a; 6. Ad-CMV-E1a-Apoptin; 7. Ad-hTERT-E1a-Apoptin.

Article Snippet: HEM human epidermal melanocytes (primary cells isolated from normal human neonatal foreskin) were obtained from ScienCell Research Laboratories (San Diego, CA) and cultured in MelM medium (ScienCell, Carlsbad, CA) supplemented with 0.5% FBS and 100 units/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Recombinant, Expressing, Sequencing, Infection, Control, Western Blot, Generated

Journal: Molecular Cancer

Article Title: Potent anti-tumor effects of a dual specific oncolytic adenovirus expressing apoptin in vitro and in vivo

doi: 10.1186/1476-4598-9-10

Figure Lengend Snippet: Assessment of the selective inhibition effect of Ad-hTERT-E1a-Apoptin on melanoma cells . Effects of the different MOIs and infection times on (A), A375 cell viability, (B) B16 cell viability, and (C), on HEM cell viability. Cells were seeded in 96-well plates (1 × 10 4 cells/well) one day before cells were infected with various concentrations (1 MOI, 10 MOI, and 100 MOI) of the indicated adenoviruses. Tumor viability was measured every day over a 4 days period by MTT colorimetric assay and all measurements were performed in triplicate. Data are presented as mean ± SD. In normal HEM human epidermal melanocytes (C), infection with Ad-CMV-E1a or Ad-CMV-E1a-Apoptin, but not Ad-CMV-Apoptin, Ad-hTERT-Apoptin, Ad-hTERT-E1a, or Ad-hTERT-E1a-Apoptin, induced growth inhibition. In contrast, in A375 (A) and B16 (B) melanoma cells, Ad-hTERT-E1a-Apoptin, Ad-hTERT-E1a-Apoptin, Ad-CMV-E1a, and Ad-hTERT-E1a infection resulted in significant growth inhibition. 1. Ad-mock; 2. Ad-CMV-Apoptin; 3. Ad-hTERT-Apoptin; 4. Ad-CMV-E1a; 5. Ad-hTERT-E1a; 6. Ad-CMV-E1a-Apoptin; 7. Ad-hTERT-E1a-Apoptin.

Article Snippet: HEM human epidermal melanocytes (primary cells isolated from normal human neonatal foreskin) were obtained from ScienCell Research Laboratories (San Diego, CA) and cultured in MelM medium (ScienCell, Carlsbad, CA) supplemented with 0.5% FBS and 100 units/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Inhibition, Infection, Colorimetric Assay

Journal: Molecular Cancer

Article Title: Potent anti-tumor effects of a dual specific oncolytic adenovirus expressing apoptin in vitro and in vivo

doi: 10.1186/1476-4598-9-10

Figure Lengend Snippet: Induction of apoptosis selectively in melanoma cells by Ad-hTERT-E1a-Apoptin . (A) Flow cytometry analysis of A375 cells infected with the recombinant adenoviruses. (B) Fluorescence images of the adenovirus-infected A375 cells stained with Annexin V/PI. (C) Flow cytometry analysis of B16 cells infected with recombinant adenoviruses. (D) Fluorescence images of the adenovirus-infected B16 cells stained with Annexin V/PI. (E) Flow cytometry analysis of HEM cells infected with recombinant adenoviruses. (F) Fluorescence images of the adenovirus-infected HEM cells stained with Annexin V/PI. Representative images of three independent experiments at 100× magnification were used to show Annexin V binding. Infection with only Ad-CMV-E1a and Ad-CMV-E1A-Apoptin elevated the percentage of apoptotic normal HEM human epidermal melanocytes (E and F). However, all of the recombinant adenoviruses, except for Ad-mock, resulted in significant apoptosis in A375 (A and B) and B16 (C and D) melanoma cells. 1. Control; 2. Ad-mock; 3. Ad-CMV-Apoptin; 4. Ad-hTERT-Apoptin; 5. Ad-CMV-E1a; 6. Ad-hTERT-E1a; 7. Ad-CMV-E1a-Apoptin; 8. Ad-hTERT-E1a-Apoptin.

Article Snippet: HEM human epidermal melanocytes (primary cells isolated from normal human neonatal foreskin) were obtained from ScienCell Research Laboratories (San Diego, CA) and cultured in MelM medium (ScienCell, Carlsbad, CA) supplemented with 0.5% FBS and 100 units/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Flow Cytometry, Infection, Recombinant, Fluorescence, Staining, Binding Assay, Control

Journal: PLoS ONE

Article Title: Generation of Human Melanocytes from Induced Pluripotent Stem Cells

doi: 10.1371/journal.pone.0016182

Figure Lengend Snippet: Either 3F- (3F-iPS MEL, A–E) or 4F- (4F-iPS MEL, F–J) iPS cells derived melanocytes were positive for an array of melanocyte markers: SILV (silver protein), TYRP1 (tyrosinase-related protein-1), TYR (tyrosinase), MITF (microphthalmia-associated transcription factor) , and S100. Normal human epidermal melanocytes (NHEM) were also stained with the same antibodies (K–O). Transmission electron microscopy images of either 3F (P), 4F (Q)-iPS cells derived melanocytes, or NHEM (R) showed that those melanocytes have many melanosomes in the cytoplasm. Scale bar, 20 µm (A–O), 0.5 µm (P, Q, and R). (S) Quantitative real-time PCR analysis of MITF-M and c-Kit in 3F-iPS, 3F-iPS MEL, and NHEM. The data are normalized to GAPDH and represented as fold change relative to RNA levels in 3F-iPS. The graphs show the average of two independent experiments. Error bar indicates mean±S.E.M. (T) Quantitation of SILV-positive cells induced from 3F and 4F-iPS cells. Percentage of SILV positive cells upon total differentiation of EBs cells 7 weeks after differentiation under melanocyte differentiation medium. Error bar indicates mean±S.E.M (3F-iPS, n = 3; 4F-iPS, n = 2). (U) The global gene-expression patterns were compared between human 3F-iPS cells and 3F-iPS derived-MEL (3F-iPS MEL), and between NHEM and 3F-iPS MEL. The lines indicate the linear equivalent and 5-fold differences on either side in gene expression levels between the two samples.

Article Snippet: Cultured normal human foreskin-derived epidermal melanocyte (NHEM) cells were obtained from Kurabo (Osaka, Japan).

Techniques: Derivative Assay, Staining, Transmission Assay, Electron Microscopy, Real-time Polymerase Chain Reaction, Quantitation Assay, Gene Expression