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Fig. 1 | Skeletal Muscle

Fig. 1

From: miR-1/206 downregulates splicing factor Srsf9 to promote C2C12 differentiation

Fig. 1

Srsf9 is a direct target of miR-1 and miR-206. a miR-206 and miR-1 target 3′ UTRs with cognate MREs in C2C12 myoblasts. Cells were transfected with reporter constructs and expression constructs for miR-206, miR-1, or the control miRNA, miR-450a-1. Renilla luciferase linked to two tandem copies of the reverse complement of miR-206 or miR-1 (2x206 and 2x1, respectively) or to the 3′ UTR of known miR-1/206 target Ccnd1 were positive controls. A 3′ UTR with no MREs in the empty reporter construct (miRless) was the negative control. Cells were harvested 24 h after transfection. The graph presents fold changes (FC) in firefly-normalized Renilla luciferase activity for miR-206- or miR-1-transfected vs. miR-450a-1-transfected cells. The dashed line at y = 1 represents miR-450a-1-transfected levels. **p ≤ 0.01; ***p ≤ 0.001 vs. miR-450a-1. b 3′ UTRs from Srsf9 and Tra2b but not Srsf3 are sensitive to miR-206 and miR-1 activity. C2C12 myoblasts were transfected with reporter constructs containing the Srsf3, Srsf9, or Tra2b 3′ UTRs along with miRNA expression constructs. Data were collected and analyzed as in (a). c Srsf9 is a direct target of miR-1/206. The predicted MRE in the Srsf9 3′ UTR was reversed in the reporter construct to abolish miR-1/206 binding. C2C12 myoblasts were transfected and analyzed as in panel (a). d The Srsf9 3′ UTRs are sensitive to endogenous myogenic cues. 3′ UTR reporter constructs were transfected into C2C12 myoblasts which were then differentiated for 0, 1, 2, or 4 days. Firefly-normalized Renilla luciferase activity was measured. Fold changes for differentiating vs. day 0 myoblasts were calculated and normalized to the miRless control. The dashed line at y = 1 represents day 0 levels. *p ≤ 0.05; **p ≤ 0.01 vs. D0 For all panels, N = 3 independent cultures

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