Figure 2
miR-29b-enforced expression triggers anti-proliferative and pro-apoptotic effects in MM cells. Cell growth curves of (a) RPMI-8226, (b) SKMM1, (c) OPM1, (d) INA-6, and (e) NCI-H929 transfected with synthetic miR-29b (miR-29b) or scrambled oligonucleotides (NC). Averaged values of three independent experiments are plotted including±S.D. P-values were obtained using two-tailed t-test *P<0.01; •P<0.05. (f) Immunoblot of p21WAF1 and p27KIP1 in SKMM1 and NCI-H929 cells 24 h after transfection with synthetic miR-29b or scrambled oligonucleotides (NC). Colony formation assay of SKMM1 (g) and NCI-H929 (h) after transfection with synthetic miR-29b or scrambled oligonucleotides (NC). Six hours after transfection, cells were counted and seeded in triplicate at 200 cells/ml in 18-well plates containing methylcellulose-based medium. After 2 weeks, colony formation was scored by counting colonies including >100 cells. Data indicate the percentage of colonies in the miR-29b methylcellulose cultures with respect to control±S.E. for three independent experiments. *P<0.01. (i) Annexin V-staining of SKMM1 and NCI-H929 cells 48 h after transfection with synthetic miR-29b or scrambled oligonucleotides (NC). The percentage of Annexin-V-positive cells is reported. Data are the average of three independent experiments±S.D. *P<0.01; •P<0.05. (j) Caspase 3/7 activity assay was performed in SKMM1 and NCI-H929 cells 48 h after transfection with synthetic miR-29b or scrambled oligonucleotides (NC). Transfected cells were grown in 96-well microplates and luminescence was measured using the Caspase 3/7 Glo assay (Promega). Results are expressed as fold increase of caspase activity with respect to NC-transfected cells, of at least three independent experiments. *P<0.01. (k) Immunoblot of cleaved caspase 3 and survivin, 48 h after transfection of SKMM1 and NCI-H929 cells with synthetic miR-29b or scrambled oligonucleotides (NC). Loading control was performed using GAPDH
