Journal: Scientific Reports

Article Title: Foxn1 expression in keratinocytes is stimulated by hypoxia: further evidence of its role in skin wound healing

doi: 10.1038/s41598-018-23794-5

Figure Lengend Snippet: Mmp-9 is expressed in mice injured skin tissues ( a–d ) and in keratinocytes ( e–j ). Mmp-9 mRNA expression in the skin of nude (Hsd:Athymic Nude-Foxn1 nu ) and BALB/c mice at post-wounding days 1, 3, and 5 ( a ). Immunofluorescent detection of Foxn1-eGFP ( b ) and Mmp-9 ( c ) and co-localization of Foxn1-eGFP and Mmp-9 signals ( d ) in the skin of Foxn1::Egfp mice at day 2 after injury. Mmp-9 mRNA ( e , g ) and corresponding Mmp-9 protein ( f , h ) expression in keratinocytes isolated from B6 (E-H, J), BALB/c ( i ) and nude ( i ) mice and co-cultured with dermal fibroblasts (DFs). Keratinocytes were transfected with Foxn1-containing plasmid ( e , f ) or adenovirus (Ad-Foxn1; g–j ) and cultured for 48 h. Control cultures were transfected with a vector expressing GFP alone. Representative Western blot analysis of phospho-PKC (pan) protein expression in Ad-Foxn1- or Ad-GFP-transfected keratinocytes co-cultured with DFs ( j ). Full-length blots and densitometric analysis are presented in Supplementary Fig. . ( b–d ) Scale bar 50 µm. Values are the mean ± SD, ****p < 0.0001. ( a ) Modified from: Scarless skin wound healing in FOXN1-deficient (nude) mice is associated with distinctive matrix metalloproteinase expression. Gawronska-Kozak B., Matrix Biol 2011;30(4):290–300 , with permission of Elsevier.

Article Snippet: Foxn1-GFP (MG226744) and Foxn1-Ddk (MR226744) plasmid vectors were purchased from OriGene.

Techniques: Expressing, Isolation, Cell Culture, Transfection, Plasmid Preparation, Western Blot, Modification

Journal: Scientific Reports

Article Title: Foxn1 expression in keratinocytes is stimulated by hypoxia: further evidence of its role in skin wound healing

doi: 10.1038/s41598-018-23794-5

Figure Lengend Snippet: Foxn1 regulates keratinocyte differentiation and migration. Flow cytometric analysis of keratinocytes: non-transfected and transfected with Ad-Foxn1 or Ad-GFP, showing the percentage expressing keratin 14 (K14) only, keratin 10/keratin 14 (K10/K14) and K10 only ( a ). Representative Western blots of keratin 16 and involucrin protein expression in keratinocytes transfected with Ad-Foxn1 or Ad-GFP (control) ( b ). Full-length blots are presented in Supplementary Figure . Migratory abilities after pre-treatment with mitomycin C and wounding in monolayer cultures were analysed in non-transfected B6 keratinocytes and in keratinocytes transfected with Ad-Foxn1 or Ad-GFP ( c , d ). Representative images were taken at 0, 20, 32 and 68 h ( d ). Dotted lines indicate the distance between migrating keratinocytes ( d ). Migration is expressed as the percentage of the distance between the unclosed edges ( c ). Each well was prepared in duplicate, and the experiment was repeated two times (n = 11 animals). Values are the mean ± SD. Asterisks indicate significant differences (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Article Snippet: Foxn1-GFP (MG226744) and Foxn1-Ddk (MR226744) plasmid vectors were purchased from OriGene.

Techniques: Migration, Transfection, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Foxn1 expression in keratinocytes is stimulated by hypoxia: further evidence of its role in skin wound healing

doi: 10.1038/s41598-018-23794-5

Figure Lengend Snippet: Simplified list of proteins with differential abundance in keratinocytes transfected with Ad-Foxn1 and control keratinocytes (transfected with Ad-GFP).

Article Snippet: Foxn1-GFP (MG226744) and Foxn1-Ddk (MR226744) plasmid vectors were purchased from OriGene.

Techniques: Transfection

Journal: Scientific Reports

Article Title: Foxn1 expression in keratinocytes is stimulated by hypoxia: further evidence of its role in skin wound healing

doi: 10.1038/s41598-018-23794-5

Figure Lengend Snippet: Proteins identified both in proteomic analysis as differentially abundant in keratinocytes transfected with Ad-Foxn1 and keratinocytes transfected with Ad-GFP and among genes selected from sequencing data (skin samples of Foxn1-deficient nude mice vs Foxn1-active control mice).

Article Snippet: Foxn1-GFP (MG226744) and Foxn1-Ddk (MR226744) plasmid vectors were purchased from OriGene.

Techniques: Transfection, Sequencing

Journal: Scientific Reports

Article Title: Foxn1 expression in keratinocytes is stimulated by hypoxia: further evidence of its role in skin wound healing

doi: 10.1038/s41598-018-23794-5

Figure Lengend Snippet: Foxn1 overexpression in keratinocytes affects proteins that regulate the hypoxia/normoxia response. Schematic representation of molecular cross-talk between proteins exhibiting hypoxic or normoxic characteristics, according to proteome profiling results, and induced in keratinocytes transfected with Ad-Foxn1 (black colour) or Ad-GFP (control; white colour) ( a ). Grey colour indicates proteins that were not detected in the analysis but link identified proteins ( a ). Foxn1 mRNA expression in primary cultures of B6 keratinocytes ( b ) and Ad-Foxn1 or Ad-GFP (control)-transfected nude keratinocytes ( c ) cultured under normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions. Corresponding Mmp-9 mRNA ( d ) and Mmp-9 protein ( f ) expression in keratinocytes isolated from B6 mice and Mmp-9 mRNA ( e ) and Mmp-9 protein ( g ) expression in nude keratinocytes transfected with Ad-Foxn1 or Ad-GFP (control) cultured under normoxic (21% O 2 ) or hypoxic (1% O 2 ) conditions. Values are the mean ± SD; **p < 0.01, ***p < 0.001, ****p < 0.0001; m – molecular size marker.

Article Snippet: Foxn1-GFP (MG226744) and Foxn1-Ddk (MR226744) plasmid vectors were purchased from OriGene.

Techniques: Over Expression, Transfection, Expressing, Cell Culture, Isolation, Marker

Journal: Scientific Reports

Article Title: Foxn1 expression in keratinocytes is stimulated by hypoxia: further evidence of its role in skin wound healing

doi: 10.1038/s41598-018-23794-5

Figure Lengend Snippet: Foxn1 overexpression in keratinocytes affects pro-survival and pro-apoptotic protein profiles. Schematic representation of molecular cross-talk between proteins exhibiting pro- or anti-apoptotic characteristics, according to proteome profiling results, and induced in keratinocytes transfected with Ad-Foxn1 (black colour) or Ad-GFP controls (white colour) ( a ). Grey colour indicates proteins that were not detected in the analysis but link identified proteins ( a ). Representative Western blot ( b ) and densitometric analysis ( c ) (n = 4) of Vdac1 protein expression in keratinocytes transfected with Ad-GFP (control) or Ad-Foxn1. Full-length blots are presented in Supplementary Fig. . Flow cytometric analysis of keratinocytes transfected with Ad-Foxn1 or Ad-GFP (control) showing the percentages of necrotic, viable and apoptotic cells and cells in G0/G1, S or G2/M phase ( d ). The values are the mean ± SD; *p < 0.05, ***p < 0.001; ****p < 0.0001.

Article Snippet: Foxn1-GFP (MG226744) and Foxn1-Ddk (MR226744) plasmid vectors were purchased from OriGene.

Techniques: Over Expression, Transfection, Western Blot, Expressing