Journal: Molecular therapy : the journal of the American Society of Gene Therapy
Article Title: Telomerase mRNA therapy protects human skin against radiation-induced DNA damage
doi: 10.1016/j.ymthe.2025.09.029
Figure Lengend Snippet: (A) Human epidermal fibroblasts and keratinocytes were treated with vehicle control (PBS) or GFP mRNA encapsulated by cationic lipid nanoparticle DOTAP or mixed with commercial transfection reagents (Lipofectamine MAX or jetMESSENGER) to assess transfection efficiency. Representative images of green fluorescence from live cells (left panel) and DAPI-stained nuclei are shown ( n = 3 independent experiments). Quantification GFP+ cells/DAPI are presented as mean percentage ± SD ( n = 3). (B) Schematic outline of the experimental setup. (C–E) Representative flow cytometry plots and quantitative analysis of early and late apoptosis in human epidermal keratinocytes, assessed 24 h after ionizing radiation (5 Gy) or no irradiation (No IR control). (F and G) Representative flow cytometry plots and quantitative analysis of Annexin V-positive cells in indicated groups of human epidermal keratinocytes. (H and I) Representative flow cytometry plots and quantitative analysis of Annexin V-positive cells in human dermal microvascular endothelial cells treated with indicated groups. Primary cells were transfected with 1 μg/mL DOTAP LNP encapsulated GFP or TERT mRNA or treated with vehicle control (PBS). Flow cytometry analysis was performed 24 h following 5 Gy irradiation or No IR control. Early apoptosis was identified as Zombie Aqua (−)/Annexin V (+), while late apoptosis was identified as Zombie Aqua (+)/Annexin V (+). Data are shown as mean ± SD ( n = 3). ns, p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. p values were calculated using two-way ANOVA. KTNs, epidermal keratinocytes; MVECs, dermal microvascular endothelial cells; US, unstained.
Article Snippet: Skin explants were cultured in keratinocyte growth medium (ATCC, no. PCS-200–040) at 37°C, 5% CO 2 , maintaining an air-liquid interphase as described.
Techniques: Control, Transfection, Fluorescence, Staining, Flow Cytometry, Irradiation
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