Figure 2.

Overexpression of miR-137 or miR-668 causes senescence in NHK. (A) Rapidly replicating NHK were transduced by lentiviruses that express miR-137 (LV-miR137), miR-668 (LV-miR668), or GFP alone (LV-GFP) for 3 h. Three days post-transduction, phase contrast photographs and GFP epifluorescence images were obtained. GFP fluorescence revealed those cells that were transduced by the lentiviruses. Original magnification x100. (B) Three days post-transduction of NHK, the levels of miR-137 and miR-668 expression were measured by quantitative real-time RT-PCR. The value for miRNA from NHK transduced with LV-GFP was set at 1, and the relative amounts of miRNA from NHK transduced with LV-miR137 or LV-miR668 were plotted as fold induction. (C) SA β-gal staining of each culture was obtained 3 days post-transduction. Original magnification x100. (D) SA β-gal positive cells were scored by counting 1,000 cells with normal light microscopy. (E) Proliferation kinetics of NHK transduced with LV-GFP, LV-miR137, or LV-miR668 was determined. One day post-transduction, 2×10⁴ cells were plated, and the number of cells was counted after 2 and 4 days of incubation. Results were expressed as the average number of triplicate cultures.