PTPN9 is a target of miR-126 and can partially rescue the suppression of erythropoiesis. (A) Predicted binding site for the seed sequence of miR-126 in the 3′-UTR of PTPN9 mRNA. The seed region of miR-126 (top) matches the 3′-UTR of PTPN9. Mutated (mu) and deleted (de) forms of the 3′-UTR of PTPN9 used for creating the luciferase reporter construct are shown. (B) miR-126 inhibited wild-type, but not mutated or deleted PTPN9–3′-UTR reporter activity. The 293T cells were transfected with miR-126 (or control nontargeting oligonucleotide) and luciferase construct containing different forms of PTPN9–3′-UTR as indicated. After 36 hours of incubation, cells were subjected to luciferase assay. (C) Western blot analysis of PTPN9 expression in day 15 EB cells with or without Dox treatment; ERK2 levels are shown as an internal control. (D) Western blot analysis of PTPN9 expression in parental hESCs (I-miRs-126/126*) and 2 PTPN9 overexpressing hESC lines. (E) Restored expression of PTPN9 attenuated the suppressed erythropoiesis. I-miRs-126/126* and PTPN9 overexpressing hESCs were differentiated with or without Dox for 15 days. Dissociated single EB cells were then subjected to CFU assay. The number of erythroid colonies under both conditions was counted and the ratio was determined. Data are mean ± SD (n = 5). *P < .05. **P < .01.