Figure 2.

Effect of knockdown of MELK by small-interfering RNA (siRNA) on cell viability and proliferation. Four psiH1 promoter-based siRNA constructs (si-#1, si-#2, si-#3 and si-#4) were introduced into (a) T47D and (b) MCF-7 cell lines. SC refers to scramble used as a control for siRNA experiments. Gene silencing was evaluated by semi-quantitative RT-PCR and western blot analyses at four and five days after neomycin selection, respectively. β2-microglobulin (β2MG) was used as a control for normalization of semi-quantitative RT-PCR, and β-actin was used as a control in western blot analysis. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were performed to evaluate cell viability at 10 days after neomycin selection, and graphed after standardization using the scramble control (SC) as 1.0 (T47D, P = 0.0003, P = 0.0013; MCF-7, P = 0.0001, P = 0.0001; unpaired t-test). Colony formation assays were carried out three weeks after neomycin selection (see Materials and methods). Two siRNA constructs (si-#3 and -#4) showed significant knockdown effects against internal MELK expression and inhibited cell growth in both T47D (a) and MCF-7 (b) cell lines. Values represent the average from triplicate experiments. Error bars indicate standard deviation.