Journal: Genes to Cells

Article Title: Derivation of human differential photoreceptor cells from adult human dermal fibroblasts by defined combinations of CRX, RAX , OTX2 and NEUROD

doi: 10.1111/gtc.12127

Figure Lengend Snippet: Induction of retina-specific genes in human dermal fibroblasts by the retroviral infection of genes for defined transcription factors. (A) RT-PCR analysis for photoreceptor-specific genes in cultured human dermal fibroblasts (NHDF) obtained from Lonza after gene transfer of several kinds of transcription factors. Recoverin, blue opsin and PDE6c genes were up-regulated by CRN transduction. ‘Negative control’: amplified water as a negative control. ‘GFP’: cultured fibroblasts after retroviral gene transfer of the GFP gene as another negative control. ‘w/o’: cultured fibroblasts without gene transfer as the other negative control. ‘SPPO’: SOX2, POU1F1, PAX6 and OTX2 . ‘SPO’: SOX2, POU1F1 and OTX2 . ‘CRN’: CRX, RAX and NEUROD . ‘Human retina’: human retinal tissue as a positive control. The amount of cDNA as a template was a half in the positive control. (B) RT-PCR analysis for photoreceptor-specific genes in cultured human dermal fibroblasts (NHDF) obtained from Promo Cell after gene transfer of several transcription factors. Recoverin, blue opsin and PDE6c genes were up-regulated by CRN or CRNO transduction. ‘w/o’: cultured fibroblasts without gene transfer as a negative control. ‘GFP’: cultured fibroblasts after retroviral gene transfer of the GFP gene as another negative control. ‘CRNO’: CRX, RAX, NEUROD and OTX2 . (C) RT-PCR analysis for photoreceptor-specific genes in cultured human dermal fibroblasts (HDF-a) obtained from ScienCell after gene transfer of several transcription factors. Recoverin, blue opsin and PDE6C genes were up-regulated by CRN or CRNO transduction. Expression levels of blue opsin were increased by additional OTX2 gene transduction. ‘w/o’: cultured fibroblasts without gene transfer as a negative control. ‘GFP’: cultured fibroblasts after retroviral gene transfer of the GFP gene as another negative control. ‘CRNO’: CRX , RAX , NEUROD and OTX2 . ‘1’, ‘2’ and ‘3’ mean independently cultured, transfected and harvested cells by the same combination of CRN genes. (D) Immunocytochemistry using antibodies to rhodopsin and blue opsin (green). Nuclei were stained with DAPI (blue). Experiments were carried out at 2 weeks after infection. The cells in the left panel and the right panel are CRN-infected Fib#2 and Fib#1, respectively. Scale bars represent 10 μm.

Article Snippet: Three strains of cultured human dermal fibroblasts were used: one was obtained from Lonza (NHDF), another was from Promo Cell (NHDF) and the other was from ScienCell (HDF-a).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Transduction, Negative Control, Amplification, Positive Control, Expressing, Transfection, Immunocytochemistry, Staining

Journal:

Article Title: ?-Melanocyte-Stimulating Hormone Counteracts the Suppressive Effect of UVB on Nrf2 and Nrf-Dependent Gene Expression in Human Skin

doi: 10.1210/en.2008-1315

Figure Lengend Snippet: Expression analysis of Nrf1-3 in human skin cells in vitro and in situ. A, Detection of Nrf1-3 in various human skin cell types in vitro as shown by conventional RT-PCR analysis. NC, Negative control, H2O as template; 1, NHK; 2, HaCaT; 3, NHM; 4, HDF; 5, HMEC-1. B, Protein expression of Nrf2 in human skin cell types. Total cell lysates (50 μg/lane) were subjected to Western immunoblot analysis using an anti-Nrf2 antibody. The same blot was reprobed with an anti-α-tubulin antibody. C, Expression of Nrf2 in healthy human skin. Nrf2 was detected by immunohistochemistry using the immunoperoxidase technique (a). Note specific cytoplasmic Nrf2 immunoreactivity within the basal layer of epidermal keratinocytes (red cells, arrows) but not in the IgG control (c). Double immunostaining of Nrf2 and melanocytes by the immunoperoxidase (Nrf2) and immunogold-silver enhancement technique (melanocytes) using a pan-Mel antibody (b). Note gray-blackish precipitate within a melanocyte but lack of Nrf2 staining within (arrow).

Article Snippet: Cell culture Normal human keratinocytes (NHK) and human dermal fibroblasts (HDF) were all derived from newborn foreskin and purchased from PromoCell (Heidelberg, Germany).

Techniques: Expressing, In Vitro, In Situ, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot, Immunohistochemistry, Double Immunostaining, Staining