Fig. 6

STAT5A is upregulated in CML cells and directly activates miR-202-5p transcription by binding to the pre-miR-202 promoter. (a) K562 cells were transfected with si-STAT5A or si-NC. miR-202-5p level was detected by qRT-PCR. ** P < 0.01 vs si-NC. (b) K562 cells were transfected with pGEX-STAT5 or pGEX-vector. miR-202-5p level was detected by qRT-PCR. *** P < 0.001 vs pGEX-vector. (c) ChIP-qPCR was used to detect STAT5A binding to the pre-miR-202 promoter regions in K562 cells. Three primers of indicate positions was used for ChIP-PCR analysis.**P < 0.01, ***P < 0.001 vs. IgG. (d) Pre-miR-202 promoter-luciferase reporters were co-transfected with pGEX-STAT5A expression vector into K562 cells, and luciferase reporter assays were performed. *** P < 0.001 vs. pGEX-vector. (e) K562 cells were transfected with pGEX-STAT5A, pGEX-vector, si-STAT5A and si-NC, respectively. Protein levels of STAT5A, pSTAT5A, USP15 and Caspase-6 were detected by Western blot analysis. (f) K562 cells were transfected with pGEX-STAT5A or miR-202-5p inhibitor respectively, or co-transfected with them together. USP15 and caspase6 protein level was detected by Western blot analysis. (g) qRT-PCR detected STAT5A mRNA level in PBMCs of CML-CP patients (n = 30) and PBMCs of healthy donors (n = 30). **P < 0.01 vs. normal. (h) Western blot analysis was used to measure protein levels of STAT5A and p-STAT5A in PBMCs of CML-CP patients (n = 30) and PBMCs of healthy donors (n = 30). The representative experiments were present. (I) qRT-PCR detected STAT5A mRNA level in CML cell lines (K562 and KCL22) and PBMCs of healthy donors. **P < 0.01 vs. normal. (j) Western blot analysis was used to measure protein levels of STAT5A and p-STAT5A in CML cell lines (K562 and KCL22) and PBMCs of healthy donors