Figure 4. Inducible expression of miR-497 impairs colony formation and proliferation in vitro and tumor growth in vivo .

(A) Representative images of a colony-formation assay in pTRIPZ-Control or pTRIPZ-miR-497 stable SK-N-BE(2) cells treated with or without doxycycline (0.5 μg/mL) and allowed to grow for 13 days (n = 3). Expression of the TurboRFP permits the visual marking of miRNA-expressing cells. (B) Colonies were stained with crystal violet, photographed and the number of colonies was quantified. (C) Proliferation time course comparing pTRIPZ-Control or pTRIPZ-miR-497 stable SK-N-BE(2) cells with or without 0.5 μg/mL of doxycycline. Data represent mean ± SEM of three independent experiments (3 replicates per condition for each experiment). ** or *** indicated significant difference comparing cells with or without doxycycline at p < 0.01 or p < 0.001, respectively. (D) Mouse model design. 5 · 10⁶ of SK-N-BE(2) stably transduced cells with pTRIPZ-miR-497 (n = 20) were injected into the right flank of NMRI-nude mice. Once tumors were detected, mice were randomized in two groups (n = 10/group). MiR-497 expression was then induced with the administration of 1 mg/mL doxycycline with 2% sucrose in drinking water ad libitum. Tumor measurements were taken every 2–3 days for three weeks. Tumor growth (E) and weight (F) of NB xenografts derived from SK-N-BE(2)-pTRIPZ-miR497 cells. * means p < 0.05.