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Fig. 4 | Journal of Hematology & Oncology

Fig. 4

From: JAG1 overexpression contributes to Notch1 signaling and the migration of HTLV-1-transformed ATL cells

Fig. 4

miR-124a inhibits JAG1 expression directly and indirectly through STAT3 and NFATc1. a Schematic representation of the JAG1 transcript and its interplay with NFAT, STAT3, and miR-124. Solid black marks represent miR-124a binding sites within the 3′UTR of JAG1 (2 sites), STAT3 (1 site), and NFATc1 (3 sites). b, c pCDNA, miR-124a/pCDNA, or mutant miR-124a/pCDNA (b) (mutated miR-124a sequence) were transfected into 293T cells along with wild-type or mutant (c) NFATC1-UTR-pGL3 and the RL-TK plasmid. For mutant NFATC1-UTR, mutations were made at single miR-124a binding sites (#352-359, #565-571, or #1355-1362) or at all three mir-124a binding sites (Mut-UTR SDM#3). Forty-eight hours after transfection, cell lysates were measured for firefly (NFATC1 3′UTR) and renilla (RL-TK, internal control) activity. All luciferase was performed at least twice, and standard deviation is shown. Fold change was calculated compared to cells transfected with empty vector. d Detection of NFATC1 in stable 293T-pTRIPZ or –miR-124a cells induced 72 h with 2 μg/ml Dox. e Pre-miR-124a expression was detected by RT-PCR on ED-40515(−)-, Tl-Om1-, and ATLT-pTRIPZ and miR-124-pTRIPZ Tet-On inducible lines. f STAT3, NFATc1, and JAG1 expression were detected by RT-PCR on ED-40515(−)-, Tl-Om1-, and ATLT-pTRIPZ and miR-124-pTRIPZ Tet-On inducible lines. For e and f, 2 μg/ml Dox was added every day for 72 h. Post-induction, RT-PCR was performed and samples were normalized to GAPDH expression. Results are plotted as the average fold change from pTRIPZ-induced lines from at least two independent experiments. g 293T cells were transfected with pCDNA control or miR-124a/pCDNA, along with NFAT and STAT3 reporter luciferase vectors. Results are represented as a fold change compared to pCDNA transfected cells

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