Figure 1.

(a) Overview of protocol and outcomes in this study using normal human dermal fibroblast (NHDF) cells transfected with hsa-miR-520d-5p. ① UV (302 nm) irradiation (17 min by 0.5 J cm⁻²; 4 min is lethal to NHDF cells). ② Lentiviral transfection by 520d-5p 3 days after UVB irradiation. Both the scramble/NHDF and NHDF cells did not survive, and clonal expansion could not be observed later. ③ Approximately 2 weeks later, we observed the emergence of several spheroid colonies with weak green fluorscent protein (GFP) expression (average 10 colonies per 10 cm² dish). At this time, we transferred the colonies to six-well plates for further culture. ④ More than a month after transfection, each spheroid colony generated fibroblast-like cells. GFP expression was strongly observed in the spheroid colonies and weakly observed in the fibroblast-like cells. ⑤ After trypsinization for passaging, numerous spheroid cells with strong GFP expression emerged. ⑥ Cells exhibited senescence an average of 75 days after irradiation, after which they died. The left-most column is the phenotype of the NHDF cells before both irradiation and transfection. The second column from the left (>2 weeks) is the phenotype (top) of a surviving colony and its GFP expression (bottom). The second column from the right (more than a month) shows representative spheroid colonies with strong GFP expression (top) and an instance in which fibroblast-like cells were produced from a colony (bottom). The right-most column shows that fibroblast-like cells grew until ~80 days post-transfection (top), with GFP-expressing small round cells scattered between the fibroblast-like cells. Scram., transfection with scramble sequence; 520d, transfection with hsa-miR-520d-5p. (b) Confirmation of hsa-miR-520d-5p expression in NHDF cells. Hsa-miR-520d-5p expression was maintained in NHDF cells received transfection after UV irradiation. Average relative hsa-miR-520d-5p levels in NHDF cells transfected after UVB irradiation are shown, with the parental NHDF cells standardized as 1. T and arrowhead, the transfection by hsa-miR-520d-5p. NHDF cells were used as a control. 520d-5p was not detected the third day after irradiation. Thirty-five days later, transfected cells had 10-fold higher GFP expression compared with the baseline expression in NHDF cells.