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. 2020 Jan 16;5(1):e121781. doi: 10.1172/jci.insight.121781

Figure 5. TET1 and TET2 are direct targets of miR-543.

Figure 5

(A) Top: Western blot analysis of TET1 and TET2 proteins in BM samples from patients with MF positive for the JAK2V617F mutation who responded to ruxolitinib treatment (complete elimination of palpable splenomegaly, low miR-543) or did not respond to ruxolitinib treatment (0%–24% spleen size reduction, high miR-543). GAPDH (for TET1) or β-actin (for TET2) protein levels were used as normalizers. Bottom: Expression levels of miR-543 in the peripheral blood (PB) samples derived from the same patients. Relative expression of miR-543 was measured by RT-qPCR and normalized to U6 and RNU48 levels. (B) Representative Western blot analysis of TET1 and TET2 proteins in K562 cells stably transduced with empty vector (control) or p–miR-543 expression vector. GAPDH or β-actin protein levels were used as normalizers. Relative expression of miR-543 measured by RT-qPCR and normalized to U6 and RNU48 levels (n = 3; bottom). (C) Representative Western blot analysis of TET1 and TET2 proteins in OCI-AML3 cells stably transduced with empty vector (control) or p–miR-543 expression vector. Vinculin or β-actin protein levels were used as normalizers. Relative expression of miR-543 measured by RT-qPCR and normalized to U6 and RNU48 levels (n = 3; bottom). (D) Expression of TET1 or TET2 in K562 cells stably transduced with STAT3 shRNA or control shRNA treated with ruxolitinib after being transiently cotransfected with the miR-543 mimic or control miRNA (scramble mimic). Results presented as relative mRNA levels relative to the geometric mean of the 2 normalizers: β-actin and GAPDH. (E) Levels of TET1 and TET2 mRNAs bound to the Ago2 complex were pulled down with IgG or anti-Ago2 antibody from HEK293 cells transiently transfected with empty vector (control) or with p–miR-543 expression vector. Two-tailed t test was used to assess statistical significance for B and C. Mann-Whitney-Wilcoxon nonparametric test was used for D. All experiments were performed independently 2 or 3 times, and representative blots are shown. Ago2, protein argonaute-2; RIP, RNA immunoprecipitation; NS, not significant.