Journal: International Journal of Molecular Sciences

Article Title: Genotype-Encoded UV Sensitivity in iPSC-Derived Human Melanocytes Reveals MX2 as a Physiological Amplifier of p53/p38-Mediated DNA Damage Signaling

doi: 10.3390/ijms27062617

Figure Lengend Snippet: Molecular, morphological, and phenotypic characterization of patient-derived melanocyte lines with distinct sun-sensitivity profiles. ( A ) Schematic representation of the experimental workflow. Skin biopsies from patients were reprogrammed into induced pluripotent stem cells (iPSCs) using OCT3/4, SOX2, KLF4, and c-MYC. iPSCs were subsequently differentiated into melanocytes through exposure to SCF, EDN3, and bFGF. ( B ) Representative phase-contrast micrographs showing the morphology of embryoid bodies (EBs) and differentiated melanocytes derived from iPSCs. Differentiated melanocytes exhibit characteristic dendritic morphology. Immunofluorescence staining of melanocyte-specific markers MelanA and HMB45 in Leeds3, Leeds4, H1m, and NHM160 cell lines. Nuclei were counterstained with DAPI. Scale bar: 50 μm. ( C ) Relative mRNA expression levels of melanocyte lineage genes MITF, PMEL, and TYR in Leeds3, Leeds4, H1m, and NHM160, as determined by quantitative RT-PCR. Expression values are normalized to housekeeping genes. ( D ) Quantification of intracellular melanin content (pg/cell) across the same cell lines, revealing differences in pigment production capacity. ( E ) Summary of MC1R gene variants identified in each cell line, along with corresponding sun-sensitivity scores and phenotypic classification. Lines Leeds3, Leeds4, Leeds5, Leeds7, Leeds8, and Leeds9 exhibit distinct MC1R polymorphisms and sun-sensitivity profiles, which correlate with pigmentation phenotypes. Notably, total melanin content does not correlate strictly with UV resistance, consistent with the known effects of MC1R variants on melanocyte signaling and redox balance rather than pigment quantity alone. H1m and NHM160 cells were included as standardized stem cell-derived and primary melanocyte reference controls, respectively.

Article Snippet: Quantitative real-time Reverse-Transcription PCR: RT-qPCR reactions were performed as previously described [ ] using TaqMan Gene Expression Assays: MITF (Hs01117294_m1), TYR (Hs00165976_m1), and PMEL (Hs00173854_m1).

Techniques: Derivative Assay, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR

Journal: Cosmetics

Article Title: Ethanolic Extract of Hippocampus abdominalis Exerts Anti-Melanogenic Effects in B16F10 Melanoma Cells and Zebrafish Larvae by Activating the ERK Signaling Pathway

doi: 10.3390/cosmetics7010001

Figure Lengend Snippet: Figure 4. EEHA inhibits the cAMP–CREB–MITF-tyrosinase axis in α-MSH-stimulated B16F10 melanoma cells. (A) B16F10 melanoma cells were seeded at a density of 5 × 104 cells/mL and pretreated with 1 mM IBMX for 10 min. The intracellular cAMP levels were subsequently measured 15 min after treatment with EEHA in the presence or absence of 500 ng/mL α-MSH (B). (C,D) In a parallel experiment, B16F10 melanoma cells were treated with EEHA at the indicated concentrations in the presence or absence of α-MSH. The expression of MITF and tyrosinase was detected by RT-PCR at 48 h (C), and Western blotting at 72 h (D). GAPDH and β-actin were used as the controls for normalizing the expression of MITF and tyrosinase and the relative density was calculated using ImageJ software. (E) The B16F10 melanoma cells were treated with EEHA at the indicated concentrations in the presence or absence of α-MSH for 72 h, following which the cell lysates were mixed with 5 mM l-DOPA in a 96-well microplate. After incubation at 37 ◦C for 30 min, the absorbance was measured at a wavelength of 405 nm. The results represent the average of data obtained from three independent experiments and are expressed as the mean ± SEM (*** p < 0.001 vs. the untreated group; ### p < 0.001, ## p < 0.01, and # p < 0.05 vs. the α-MSH-treated group).

Article Snippet: Antibodies against phospho (p)-CREB, MITF, tyrosinase, p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, p38, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Software, Incubation

Journal: International journal of cancer

Article Title: Effects on proliferation and melanogenesis by inhibition of mutant BRAF and expression of wild-type INK4A in melanoma cells.

doi: 10.1002/ijc.20865

Figure Lengend Snippet: FIGURE 4 – Effect on melanogenesis. (a) Color of cell pellets. 624Mel controls (5 x 106) and cells expressing T1796ABRAF RNAi or INK4A were pelleted, and a visible color difference was noted in cells expressing T1796ABRAF RNAi. (b) Melanin contents; 5 x 106 cells were used. Data are means 6 SE from 3 experiments performed in triplicate (p < 0.001, t-test). (c) Representative electron micrographs of cultured 624Mel controls and cells expressing T1796ABRAF RNAi or INK4A. Note the increased numbers of mature melanosomes in RNAi-expressing cells (arrows). Magni- fication x2,000. Similar changes were obtained using dissected xenograft tumors. (d) Expression of tyrosinase (TYR) and TRP-1 proteins. Western blot was probed with TYR, TRP-1 and a-tubulin antibodies. (e) Increased MITF in cells expressing T1796ABRAF RNAi, shown by Western blot- ting. (f) Model of separate regulation of proliferation and differentiation by mutant BRAF and p16INK4A in melanoma cells.

Article Snippet: Western blotting was performed as described.26 The following antibodies were used: BRAF (Upstate Biotechnology, Lake Placid, NY); p16INK4A, tyrosinase and cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA); phospho-MEK, MEK-1 and phospho-pRB (Cell Signaling, Beverly, MA); TRP-1 and cyclin D3 (BD Biosciences, San Diego, CA); MITF (Novus Biologicals, Littleton, CO); tubulin (Sigma-Aldrich, St. Louis, MO); and horseradish peroxidase–conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA).

Techniques: Expressing, Cell Culture, Western Blot, Mutagenesis

Journal: STAR Protocols

Article Title: An improved protocol for generation and characterization of human-induced pluripotent stem cell-derived retinal pigment epithelium cells

doi: 10.1016/j.xpro.2022.101803

Figure Lengend Snippet: Characterization of RPE (A–E) Immunostaining of iPSC derived RPE cultures for the marker expression during stages of retinal differentiation. Images showing the expression of RPE commitment marker MITF (A), melanocyte protein- PMEL17 (B), RPE maturation markers TYRP1, RPE65 and TYR (C-E)and tight junction marker ZO-1 (D and E). (F) Flow cytometry analysis of iPSC derived RPE cells for positive (MITF, RPE65, TYRP1) and negative (OCT4) markers. (G and H) Gene expression analysis using qPCR for selected markers represented as fold change compared to iPSC. All the positive markers (G) have higher expression and the negative markers (H) show a negligible expression in iPSC derived RPE. (I) ELISA based quantification of secreted PEDF from the in vivo culture supernatant from apical, basal and 2D RPE cultures. Images and graphs are representative of minimum three independent experiments. Scale bars 100 μm.

Article Snippet: MiTF Recombinant Rabbit Monoclonal Antibody (JF100-01) (1:150) , Novus Biologicals , MA5-32554.

Techniques: Immunostaining, Derivative Assay, Marker, Expressing, Flow Cytometry, Gene Expression, Enzyme-linked Immunosorbent Assay, In Vivo

Journal: STAR Protocols

Article Title: An improved protocol for generation and characterization of human-induced pluripotent stem cell-derived retinal pigment epithelium cells

doi: 10.1016/j.xpro.2022.101803

Figure Lengend Snippet:

Article Snippet: MiTF Recombinant Rabbit Monoclonal Antibody (JF100-01) (1:150) , Novus Biologicals , MA5-32554.

Techniques: Recombinant, Modification, Gentle, Saline, Knock-Out, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, cDNA Synthesis, Sequencing, Generated, Software, Suspension, Real-time Polymerase Chain Reaction, Microscopy