The STAT3 targeted gene, Pim1, is significantly lost in ATL tumors established from mice. (A) Real-time PCR analysis in ED-40515(–) pTRIPZ or miR-124a TET-On tumors. Each line represents the expression of the target gene (-ΔCt) between pTRIPZ and miR-124a in the corresponding mice. P values are calculated using a 2-tailed Student t test between the -ΔCt values for pTRIPZ vs miR-124a (n = 10). (B) Correlation graph plotting Pim1 expression (-ΔCt; y-axis) vs miR-124a expression (-ΔCt; x-axis) in ED-40515(–) tumors. The Spearman correlation coefficient is indicated along with corresponding P value. (C-D) Pim1 expression is decreased by immunohistochemistry staining (C) or western blot (D) in ED-40515(–) pTRIPZ or miR-124a TET-On tumors. Images were taken at room temperature on a Nikon Eclipse 80i microscope (Nikon Instruments, Inc.; Melville, NY) and a Nikon DSFI1 camera, with a 40× objective lens. Pim1 (E) and Pim1 target genes SOCS3, p-Bcl2 (Ser70), p-4EBP1 (Thr37/46), and p-p70S6K (Thr389) (F) were analyzed by western blot in miR-124a TET-On, ED-40515(–), and Tl-Om1 induced with 2 µg/mL Dox for 72 hours. (G) Illustration of the miR-124a binding sites in the 3′-UTR of Pim1. Pim1 UTR luciferase (H) or Pim1 western blot (I) was performed in 293T cells. pCDNA and miR-124a/pCDNA (left) or pCDNA and miR-33a/pCDNA or miR-214/pCDNA (right) were transfected into 293T cells, along with the RL-TK plasmid. Forty-eight hours after transfection, cell lysates were measured for firefly (Pim1 UTR) and renilla (RL-TK, internal control) activity. All luciferase was performed at least twice. (J) Exogenous STAT3 expression rescues miR-124a–mediated inhibition of Pim1. pCDNA (control) or STAT3-HA–tagged lines were established under puromycin selection in 293T cells, transefected with miR-124a or negative oligos, and analyzed several days later for Pim1 expression. Stable STAT3 expression was detected by HA antibodies.