Figure 2. Inhibitory activity of miR-34a in cultured lung cancer cells.
(A) Transient effect of synthetic miR-34a. Cancer cell lines were transfected in triplicate with 30 nM miR-34a or miR-NC. Proliferation was assessed 3–7 d post transfection using Alamar Blue™ (Invitrogen). Percent (%) change in proliferation was plotted relative to proliferation of miR-NC transfected cells. Averages and standard deviations of three replicates are shown. (B) Colony formation assay using SW-900 lung cancer cells. Cells were transfected with synthetic miR-34a, miR-NC and siEG5 siRNA and seeded at 3000 cells per 100-mm dish. After 32 days, cells were stained with 2% crystal violet. Colonies containing >50 cells were counted. (C) Long-term effects of miR-34a. H226 cells were transfected in triplicate with miR-34a and miR-NC, seeded and propagated in regular growth medium. When the control cells reached confluence, cells were harvested, counted and transfected again with the respective miRNAs. The population doublings were calculated, and cell counts were extrapolated and plotted on a linear scale. Arrows represent transfection events. Average cell counts and standard deviations of three replicates are shown. Cell lines carrying mutated p53 include Calu-3, H596, H1299, H522 and SW-900 (21, 22). A549 and H460 carry wild-type p53 (21, 22).