Fig. 3

UNC5B and SIRT4 are the direct downstream targets of miR-424. a Potential downstream targets of miR-424 implicated in apoptotic cell death. b Luciferase reporter assay. The putative miR-424-binding sequence within 3′-UTR of UNC5B or SIRT4 are shown, and the mutated nucleotides are indicated in red (upper). For luciferase reporter assays, T24 cells were co-transfected with the indicated luciferase reporter constructs together with miR-424 mimics or with the scramble miRNA. Forty-eight hours after transfection, their luciferase activities were measured (lower). c miR-424 down-regulates UNC5B and SIRT4. Five thousand six hundred thirty-seven and T24 cells were transfected with miR-424 mimics or with the scramble miRNA. Forty-eight hours after transfection, total RNA was prepared and analyzed for UNC5B (left panel) and SIRT4 (right panel) by qRT-PCR. d miR-424 down-regulates UNC5B and SIRT4 at protein level. Five thousand six hundred thirty-seven and T24 cells were transfected as in (c). Forty-eight hours after transfection, cell lysates were extracted and subjected to Western blotting with the indicated antibodies. β-actin was used as a loading control. e Inhibition of miR-424 promotes UNC5B and SIRT4 transcription. Five thousand six hundred thirty-seven and T24 cells were transfected with the negative control or with miR-424 inhibitor. Forty-eight hours after transfection, total RNA was prepared and analyzed for UNC5B (left panel) and SIRT4 (right panel) by qRT-PCR. f Inhibition of miR-424 increases UNC5B and SIRT4 at protein level. Five thousand six hundred thirty-seven and T24 cells were transfected as in (e). Forty-eight hours after transfection, cell lysates were extracted and subjected to Western blotting with the indicated antibodies. β-actin was used as a loading control. *P < 0.05, **P < 0.01