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: Nature communications

Article Title: Persister state-directed transitioning and vulnerability in melanoma.

doi: 10.1038/s41467-022-30641-9

Figure Lengend Snippet: Fig. 6 Enforced KDM5B expression facilitates melanocytic lineage-directed elimination by TMECG. a Quantitation of mRNA after 24 h, 48 h, 72 h and 7 days of Cpd1 treatment of MaMel63a cells as assessed by qPCR. Mean ± SD. Shown is one representative example. b Regulation of differentiation, cytokinesis, and mitotic spindle assembly genes as detected by cDNA microarray analysis after KDM5B shRNA knockdown in WM3734 cells (n = 1). c, d Immunoblotting of melanocytic lineage and (de-)differentiation markers after 24 h of KDM5B induction in WM3734Tet3G-KDM5B cells (c) and after 72 h of Cpd1 treatment in MaMel63a cells (d). Shown are representative data (n = 2). e Anti-MITF immunostaining (upper panel) and Fontana-Masson staining (lower panels) of CM melanoma tumor grafts from Cpd1-treated vs. control mice. f MTT cell viability assay of WM3734 cells. Representative example is shown left (mean ± SD, n = 2) and corresponding IC50 values on the right. TMECG was either concurrently given together with Cpd1 (“con”) or added 3 days after Cpd1 pre-treatment (“pre”). Readout was performed after 72 h of TMECG treatment. g Persister-state-directed therapy model in vivo. Left: schematic representation of treatment dosing and timing in immunodeficient NMRI-(nu/nu)-nude mice. Right: tumor volumes of WM3734 xenografts (endpoint at day 30). TMECG was either concurrently given together with Cpd1 (“con”) or added one week after Cpd1 pre-treatment (“pre”). Mean ±SEM (6 mice in TMECG and Cpd1 control group, five mice in “con” and seven mice in “pre” group). Significance was determined by two-sided Mann–Whitney test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies (Axl (8661), CDH2 (13116), tubulin (2148), ZEB1/TCF8 (3396), Smad1 (9743), and TJP1 (8193; all diluted 1:1000, all Cell Signaling, Cambridge, UK), FKBP12 (ab24373), histone H3 (ab1791, diluted 1:5000), histone H3K4me3 (ab8580, diluted 1:2000), MITF (ab80651, diluted 1:500), and Notch1 (ab52627, all diluted 1:1000, all Abcam, Cambridge, UK), KDM5B (NB100-97821, diluted 1:2000, Novus Biologicals, St. Louis, MO, USA) and GAPDH (SC-510, diluted 1:5000, Santa Cruz, Dallas, TX, USA)) were incubated overnight at 4 °C either in PBS containing 0.1% Tween-20 and 5% milk or in 1× Net-G buffer (10× Net-G contains 1.5 M NaCl, 50 mM EDTA, 500 mM Tris 0.5% Tween-20, and 0.4% gelatine).

Techniques: Expressing, Quantitation Assay, Microarray, shRNA, Knockdown, Western Blot, Immunostaining, Staining, Control, Viability Assay, In Vivo, MANN-WHITNEY

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: Mitf, but not GNAS, and phospho-ERK1/2 were uniformly increased in heterozygous hSASH1(Y551D) gene knock-in BABL/cJ mice compared to wild-type mice. (A) Downregulation of SASH1 was induced by the Y551D SASH1 mutation in homozygous mice of the F2 generation compared to that of wild-type mice. GNAS and Mitf expression was attenuated by the downregulation of SASH1; however, phospho-ERK1/2 expression was increased (left panel). The total densitometry values of these proteins were also compared collectively (lower right panel). Upper right panel: # P<0.01 vs. all 3 wild-type mice, ** P<0.001 vs. all 3 wild-type mice; ns, not significant vs. all 3 wild-type mice. Lower right panel: # P<0.01, homozygous mice vs. wild-type mice, ** P<0.001 vs. all 3 wild-type mice. (B) SASH1, GNAS and Mitf expression was not attenuated by the Y551D-SASH1 mutation in the F3 generation, as indicated by western blot and statistical analyses. ns: No significance vs. all four wild-type mice. Mitf, but not GNAS, and phospho-ERK1/2 were uniformly increased in heterozygous hSASH1(Y551D) gene knock-in BABL/cJ mice compared to wild-type mice. (C-E) Tail biopsies of wild-type mice and heterozygous mice of the F2 generation were lysed, ultrasonicated and subjected to western blot analysis. The results of western blot analysis indicated that the expression of SASH1, GNAS, phospho-ERK1/2 and Mitf was enhanced in the heterozygous hSASH1(Y551D) gene knock-in BABL/cJ mice of the F2 generation compared to wild-type mice. (D) The densitometry values of SASH1, GNAS, phospho-ERK1/2 and Mitf of 11 heterozygous and 3 wild-type mice were compared, and (E) the total densitometry values of these proteins were also compared collectively. (D) * P<0.05 vs. all 3 wild-type mice, # P<0.01 vs. all 3 wild-type mice, ** P<0.001 vs. all 3 wild-type mice. (E) * P<0.05 vs. wild-type mice; # P<0.01 vs. wild-type mice, ** P<0.001 vs. wild-type mice. Mitf, but not GNAS, and phospho-ERK1/2 were uniformly increased in heterozygous hSASH1(Y551D) gene knock-in BABL/cJ mice compared to wild-type mice. (F-H) Western blot analysis revealed that the expression of Mitf was enhanced in heterozygous human Y551D SASH1 gene knock-in mice of the F3 generation compared with wild-type mice. (G) The densitometry values of SASH1, GNAS, phospho-ERK1/2 and Mitf of 10 heterozygous and 4 wild-type mice were compared, and (H) the total densitometry values of these proteins were also compared collectively. (G) * P<0.05 vs. all 3 wild-type mice, # P<0.01 vs. all 3 wild-type mice; ** P<0.001 vs. all 3 wild-type mice. (H) ** P<0.001 vs. wild-type mice. For all panels, ns, not significant.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Gene Knock-In, Mutagenesis, Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: Mitf, but not GNAS, and phospho-ERK1/2 expression is increased in heterozygous human Y551D SASH1 knock-in mice compared to homozygous mice. (A) Western blot analysis indicated that SASH1 was downregulated in heterozygous human Y551D-SASH1- knock-in mice of the F2 generation compared to homozygous mice. However, the protein levels of phospho-ERK1/2 and Mitf were upregulated in heterozygous mice compared to those of homozygous mice. (B and C) The densitometry values of SASH1, GNAS, phospho-ERK1/2 and Mitf of 11 heterozygous and 2 homozygous mice of the F2 generation were compared, and the total densitometry values of these proteins were also compared collectively, respectively. (B) * P<0.05 vs. both homozygous mice, # P<0.01 vs. both homozygous mice; ** P<0.001 vs. both homozygous mice. (C) # P<0.01 vs. homozygous mice, ** P<0.001 vs. homozygous mice, * P<0.05 vs. homozygous mice. (D) Western blot analysis demonstrated that the expression of Mitf and GNAS was enhanced in heterozygous human Y551D SASH1 knock-in mice of the F3 generation compared to homozygous mice. Mitf, but not GNAS, and phospho-ERK1/2 expression is increased in heterozygous human Y551D SASH1 knock-in mice compared to homozygous mice. (E and F) The densitometry values of SASH1, GNAS, phospho-ERK1/2 and Mitf of 11 heterozygous and 1 homozygous F3 generation mice were compared. (F) The total gray values of these proteins were also compared collectively. (E) * P<0.05 vs. one homozygous mouse, respectively, # P<0.01 vs. one homozygous mouse, respectively, ** P<0.001 vs. one homozygous mouse, respectively. (F) ** P<0.001 vs. homozygous mice. (G) The densitometry values of SASH1, Mitf, GNAS and the ratio of p-ERK/ERK in 11 heterozygous mice of the F2 generation and 10 heterozygous mice of the F3 generation were compared. For all panels, ns, not significant.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Expressing, Knock-In, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: In the F2 generation, the number of Mitf- and Mitf-positive epithelial cells was enhanced in heterozygous human Y551D SASH1 knock-in mice. (A) Representative images (magnification, ×10) of Mitf in 3 wild-type mice, 2 homozygous mice and 11 heterozygous mice. (B) A total of 5 random visual fields in each section of 16 mice, including wild-type, homozygous and heterozygous mice, were photographed. The Mitf-positive epithelial cells in the tail tissues of wild-type, homozygous and heterozygous mice were counted and analyzed statistically. Representative Mitf-positive cells, which were stained dark brown in the nucleus, are indicated by red arrows. * P<0.01. (C) The staining intensity and percentage of Mitf-positive cells per mouse were calculated, scored and analyzed statistically. * P<0.05, *** P<0.001; ns, not significant.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Knock-In, Staining

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: In the F3 generation, the number of Mitf- and Mitf-positive epithelial cells was augmented in heterozygous human Y551D SASH1 knock-in mice. (A) Representative images (magnification, ×10) of Mitf in 4 wild-type mice, 1 homozygous mouse and 11 heterozygous mice. (B) A total of 51 visual fields in each section of 15 mice, including wild-type, homozygous and heterozygous mice, were photographed. The Mitf-positively stained epithelial cells in the tail tissues of wild-type, homozygous and heterozygous mice were calculated in 3 visual fields and analyzed statistically. Representative Mitf-positive cells, which were stained yellowish-brown in the nucleus, are indicated by red arrows. * P<0.01. (C) Staining intensity and percentage of Mitf-positive cells per mouse were calculated, scored and analyzed statistically. ** P<0.01 *** P<0.001; ns, not significant.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Knock-In, Staining

Journal: International Journal of Molecular Medicine

Article Title: Mutated SASH1 promotes Mitf expression in a heterozygous mutated SASH1 knock-in mouse model

doi: 10.3892/ijmm.2020.4652

Figure Lengend Snippet: Mitf expression is promoted by mutated SASH1 in vitro and in epithelial tissues affected by the Y551D-SASH1 mutation. (A) Exogenous SASH1 is not associated with endogenous Mitf in 293T cells. GFP-SASH1 was transfected into 293T cells. At 48 h following transfection, transfected cells were lysed, GFP-SASH1 was immunoprecipitated, and the associated endogenous Mitf was analyzed by IP-WB analyses. (B) Expression of endogenous Mitf was induced by Y551D SASH1. Exogenous Y551D SASH1 and a wild-type SASH1 were introduced into 293T cells. Following transfection, transfected cells were lysed and subjected to western blot analyses. ** P<0.001 vs. wild-type SASH1 and *** P<0.001 vs. blank control. (C) In the lesional epithelial tissues of Y551D SASH1 -affected individuals, SASH1- and Mitf-positive cells were demonstrated in different epithelial layers of the affected epithelial tissues and calculated and analyzed statistically. ** P<0.01, *** P<0.001. Upregulation of SASH1 and enhanced Mitf were also induced in the affected tissues. Magnification, ×40. Mitf-positive cells, which were stained dark brown in the nucleus, are indicated by red arrows. (D) More melanin was synthesized and present in different epithelial layers of the affected skin epithelial tissues. Magnification, ×40.

Article Snippet: The sections were then incubated with the rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC), rabbit anti-SASH1 polyclonal antibody (1:200 dilution, cat. no. NBP-26650, Novus Biologicals, LLC), rabbit anti-Mitf (C5) monoclonal antibody (1:200 dilution, cat. no. NB110-10872, Novus Biologicals, LLC) at 37°C for 1 h and then at 4°C for at least 8 h. After washing with PBS 3 times, the sections were incubated with the horseradish peroxidase-linked anti-rabbit and anti-mouse universal secondary antibodies provided by the Immunochromogenic Kit (KIT-5006, MXB Biotechnologies) for 1 h at 37°C.

Techniques: Expressing, In Vitro, Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Control, Staining, Synthesized