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. 2014 Jan 30;105(3):297–307. doi: 10.1111/cas.12339

Figure 2.

Figure 2

Forced expression of miR-217 sensitizes dasatinib-resistant K562 (K562DR) cells to dasatinib. (a) The sequence of miR-217 and its potential matching site in the DNMT3A 3′-UTR. DNMT3A 3′-UTR mutant vector with deletion of 4 bp (CAUG) in the binding site of miR-217 was generated by using the PrimeSTAR Mutagenesis Basal Kit. (b) Reporter gene assay. miR-217 overexpressed K562DR cells were transfected with either DNMT3A 3′-UTR WT or mutant luciferase reporter vector. The pRL-SV40-Luciferase (Renilla luciferase) vector was co-transfected for normalization. After 48 h, cells were harvested and subjected to the reporter assay. (c) Real-time RT-PCR. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated gene. Results represent the mean ± SD of three experiments performed in triplicate. The statistical significance was assessed using a paired t-test. **P < 0.01; *P < 0.05. (d) Western blot analysis. K562DR cells were transfected with control or miR-217 expression vector. These cells were subjected to western blot analysis to monitor the levels of the indicated proteins. Each lane was loaded with 30 μg of whole protein lysate. Band intensities were quantified using ImageJ software (Wayne Rasband, NIH). (e) MTT assay. K562DR cells were transfected with control or miR-217 expression vector. These cells were plated in 96-well plates and cultured with dasatinib (1, 5 or 10 nM). After 96, their proliferation was measured by MTT assay. Results represent the mean ± SD of three experiments performed in triplicate. Downregulation of DNMT3A in K562DR cells sensitizes these cells to dasatinib. (f) Real-time RT-PCR. K562DR cells were transiently transfected with either scrambled control or DNMT3A siRNA. RNA was extracted from these cells. cDNA were synthesized and subjected to real-time RT-PCR to measure the levels of the indicated gene. Results represent the mean ± SD of three experiments performed in triplicate. The statistical significance was assessed using a paired t-test. **P < 0.01; *P < 0.05. (g) MTT assay. K562DR cells were transiently transfected with either scrambled control or DNMT3A siRNA. These cells were plated in 96-well plates and cultured with dasatinib (10 nM). At the indicated time point, their proliferation was measured by MTT assay. Results represent the mean ± SD of three experiments performed in triplicate. Mut, mutation; WT, wild type.