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. 2017 May 8;8(30):49451–49469. doi: 10.18632/oncotarget.17706

Figure 3. Effects of TET2 silencing on K562 cells' response to IM.

Figure 3

(A) qRT-PCR analysis of miR-29a-3p predicted targets performed 24 h after miR-29a-3p transfection. Results were normalized to the NegCTR mimic sample (n = 3). (B) Representation of the four miR-29a-3p predicted binding sites in TET2 3′UTR sequence as reported by TargetScanHuman v7.0. The seed region of the miRNA is highlighted. (C) Normalized luciferase activity of K562 cells co-nucleofected with miR-29a-3p miRNA mimic and TET2 3′UTR reporter vector. Each bar represents the luciferase activity upon miRNA overexpression normalized on the value of the same 3′UTR luciferase vector upon Neg-mimic transfection (set to 1). Values are reported as mean ± SEM (n = 3). (D) Western blot analysis of TET2 protein levels in whole cell lysates from K562 cells overexpressing miR-29a-3p 48 hours after mimic nucleofection. TET2 protein level in miR-29a-3p overexpressing cells was compared with control sample nucleofected with mimic Negative Control (Neg CTR). β-actin was included as loading control. (E) TET2 mRNA expression levels 24 hours after the last nucleofection as evaluated by qRT-PCR. Data are reported as RQ mean ± S.E.M of 3 independent experiments. Results of Annexin V/PI staining on K562 cells after 24 h (F) and 48 h (G) of IM treatment (mean ± SEM; n = 3) *p < 0.05, **p < 0.01. Representative dot plots for flow cytometry detection of Annexin V and PI staining at 24 h and 48 h after treatment are shown (H), i: NT siRNA, ii: NT siRNA IM 1 μM, iii: TET2 siRNA NT, iv: TET2 siRNA IM 1 μM, v: NT siRNA NT, vi: NT siRNA IM 0.4 μM, vii: TET2 siRNA NT, viii: TET2 siRNA IM 0.4 μM. Abbreviations: RQ, Relative Quantity; NT siRNA, Non-targeting siRNA; siRNA, small interfering RNA; IM, Imatinib Mesylate; NT, Not Treated; 24 h, 24 hours; 36 h, 36 hours; 48 h, 48 hours; PI, Propidium Iodide.